Under pathophysiologic conditions such as Alzheimer’s disease and cancer, the endolysosomal cysteine protease legumain was found to translocate to the cytosol, the nucleus, and the extracellular space. These noncanonical localizations demand for a tight regulation of legumain activity, which is in part conferred by protein inhibitors. While there is a significant body of knowledge on the interaction of human legumain with endogenous cystatins, only little is known on its regulation by fungal mycocypins. Mycocypins are characterized by (i) versatile, plastic surface loops allowing them to inhibit different classes of enzymes and (ii) a high resistance toward extremes of pH and temperature. These properties make mycocypins attractive starting points for biotechnological and medical applications. In this study, we show that mycocypins utilize an adaptable reactive center loop to target the active site of legumain in a substrate-like manner. The interaction was further stabilized by variable, isoform-specific exosites, converting the substrate recognition into inhibition. Additionally, we found that selected mycocypins were capable of covalent complex formation with legumain by forming a disulfide bond to the active site cysteine. Furthermore, our inhibition studies with other clan CD proteases suggested that mycocypins may serve as broad-spectrum inhibitors of clan CD proteases. Our studies uncovered the potential of mycocypins as a new scaffold for drug development, providing the basis for the design of specific legumain inhibitors. While the proenzyme is stable at neutral pH, the isolated AEP domain is stable only at acidic pH  and macrocypin (isolated from the parasol Macrolepiota procera)   antigens for presentation on the MHCII complex . Its marcidic pH , 10. In cidic pH , 12. Bescidic pH , 15, 16.cidic pH , 18. Itscidic pH , 21, 22.families , 23, 24.procera) , 26. Undprocera) . The tre21.5 nM) . In agre21.5 nM) , 28. The21.5 nM) , 30. Becotatoes) , 32, forM. procera macrocypin 1a (Mcp1a) and C. nebularis clitocypin 2 (Clt2), representatives of the macrocypin and clitocypin families, respectively. We selected the human legumain variant as a target enzyme because it is especially interesting for drug development. Mcp1a and Clt2 were recombinantly expressed in Escherichia coli and purified from nonclassical inclusion bodies or as soluble protein, respectively. To prevent undesired proteolytic processing, we covalently blocked the active site cysteine Cys189 of legumain with S-methyl methanethiosulfonate (MMTS), before cocrystallizing legumain in complex with Mcp1a or Clt2. We solved the crystal structure of legumain in complex with Mcp1a to a resolution of 2.2 Å in space group C2221, with two legumain–Mcp1a complexes in the asymmetric unit (A and S1A). Overall, the structures of both legumain and Mcp1a within the complex looked similar as compared to their free forms. No major conformational changes had occurred upon complex formation. The structure furthermore showed that the interaction of Mcp1a with legumain was mediated via three major regions on the inhibitor (D). We identified (i) the RCL (loop β5-β6) that is interacting with the active site of legumain, (ii) exosite 1 (EX1Mcp) that is mediating ionic interactions to the prime substrate interaction sites on legumain, and (iii) exosite 2 (EX2Mcp) that is mediating hydrophobic interactions to the area south of the nonprime substrate-binding site. Furthermore, we resolved the crystal structure of legumain in complex with Clt2 to a resolution of 1.8 Å in space group P21 and with one enzyme-inhibitor assembly in the asymmetric unit (B and S1B). Similarly, the overall fold of legumain and Clt2 remained largely unchanged in the complex. No major conformational changes were observed within the complex. Clt2 was binding to legumain via three primary interaction sites (E): (i) A reactive center loop which is establishing the interaction to the legumain active site, (ii) exosite 1 (EX1Clt) that is mediating further interactions to the nonprime substrate-interaction sites, and (iii) exosite 2 (EX2Clt) which is mediating interactions to the area north of the nonprime substrate-binding cleft. Overall, both inhibitors show the same β-trefoil fold that is reminding of a tree (F). In this picture, the tree is built up by a short trunk formed by a six-stranded β-barrel and a crown harboring the interaction site for papain-like enzymes (loops β1-β2 and β3-β4) and the legumain RCL (β5-β6). Interestingly, even though Mcp1a and Clt2 shared a high structural similarity  and exploited a similar mode of interaction with legumain dominated by an RCL, they differed dramatically in their exosite interactions. While the Mcp1a exosites predominantly interacted with the area south of the substrate-binding cleft and the prime side, the Clt2 exosite interaction was localized to the north of the substrate-binding cleft and the nonprime-interaction sites on legumain. Consistent with these largely disjunct Mcp1a and Clt2 exosites, they also mapped to different areas on the inhibitors. While exosites 1 and 2 on Mcp1a localized to its trunk and crown, both Clt2 exosites localized to the crown. Consequently, the trunk regions of Mcp1a and Clt2 were shifted by approximately 15 Å relative to each other and did not superpose onto each other when complexed with legumain (C).To get a detailed understanding of how mycocypins interact with legumain, we solved the crystal structure of human legumain in complex with ric unit A and S1Anhibitor D. We ideric unit B and S1Bon sites E: (i) A f a tree F. In thilegumain C.Table 14aw9) confirmed a substrate-like binding mode of the RCL’s of both inhibitors (A). We identified Asn74Mcp (Mcp denotes Mcp1a numbering) and Asn70Clt (Clt denotes Clt2 numbering) as P1 residues on the Mcp1a and Clt2 RCLs, respectively . Similarly, the RCL of Clt2 provides interactions to the S4–S1 substrate-binding sites. Specifically, Gln67Clt served as P4 residue, the side chain of Tyr66Clt and the main chain of Gly68Clt as P3 residue, and Leu69Clt as P2 residue in Clt2 (C). Our structural analysis revealed that both Mcp1a and Clt2 bound to legumain in a substrate-like manner, using a conserved P1-Asn on the RCL. This observation further suggested that the inhibitors would get hydrolyzed upon binding to legumain. To test whether mycocypins are substrates to the legumain protease activity, we coincubated them with legumain at pH 4.0 to 7.0 in a 1:2 molar ratio (enzyme:inhibitor) and analyzed the reaction for cleavage products using SDS-PAGE and mass spectrometry assays. Indeed, we found that Clt2 was efficiently cleaved by legumain at pH 4.0 to 6.0 (D). When the samples analyzed by SDS-PAGE were not heated before loading on the gel, the cleavage product was visible as a band migrating slightly faster than the intact inhibitor. If the samples were heated before loading them on the gel, the band corresponding to the intact inhibitor was shifted downward and the cleavage products migrated as two distinct bands, corresponding to the N-terminal Met1Clt–Asn70Clt and C-terminal Thr71Clt–His160Clt2  fragments (A and C). These differences in migration behavior suggested that without heating, Clt2 would remain (partly) folded on SDS-PAGE. Mass spectrometry analysis confirmed Asn70Clt on the RCL as the predominant processing site (E), providing further evidence that Asn70Clt acts as a P1-residue. Interestingly, when we repeated the experiment using Mcp1a, we observed cleavage of the inhibitor only at pH ≤ 4.0 (E). Similarly, when we analyzed the samples by SDS-PAGE without heating them, the cleavage product was visible as a single band migrating below the intact inhibitor. Heating of the samples led to complete denaturation, associated with a shift of intact Mcp1a to an apparent higher molecular weight and migration of the cleavage products as two distinct bands. These bands corresponded to the N-terminal Met1Mcp–Asn74Mcp and C-terminal Ser75Mcp–His177Mcp  fragments (B and C). Mass spectrometry experiments confirmed that the predominant cleavage site was the P1-residue Asn74Mcp on the RCL (F). In a next step, we were wondering whether the inhibitors remained bound to legumain after they were processed. To answer this question, we subjected the cleavage reactions to size-exclusion chromatography (SEC) experiments. Indeed, we found comigration of the cleavage products with legumain on SEC and subsequent SDS-PAGE analysis (A and B). Given these results, we hypothesized that inhibitor processing was critical for the inhibition mechanism exploited by mycocypins. To further investigate the relevance of processing for inhibition, we tested the inhibitory activity of intact Clt2 and processed Clt2 (Clt2∗) toward legumain. To that end, we incubated legumain with a 4-fold excess of Clt2 at pH 5.5 for 4 h. Subsequently, we separated the processed, excess Clt2∗ from the legumain-Clt2 complex by SEC. When we tested inhibition of legumain by processed Clt2∗ and intact Clt2 at pH 4.0 and pH 6.0, we found that while both variants showed similar inhibition at pH 6, the cleaved inhibitor was more potent at acidic pH (F). This observation further supported our hypothesis that processing is indeed important for the inhibitory mechanism. It is turning the inhibitor even more potent at acidic pH.Superposition of the complex structures with the Ac-Tyr-Val-Ala-Asp-chloromethylketone (YVAD-cmk)-bound legumain structure . Interestingly, we did not observe conversion of the processed inhibitors to the intact precursors at the investigated conditions, which suggested that the processed mycocypins are unfavorable substrates to the legumain ligase activity.Along the same line, we were wondering whether the processed inhibitor was also a substrate to the legumain ligase activity. In a previous study, we observed that cystatin E, an endogenous inhibitor of human legumain, was processed and religated by legumain in a pH-dependent manner . To testFig. S5Mcp and P6-Glu69Mcp to P4-Arg71Mcp and Ser75Mcp to Asp73Mcp (A). These interactions are conserved throughout the known macrocypin sequences (3C). Contrasting the situation in Mcp1a, the RCL structure of Clt2 was missing an internal, stabilizing electrostatic clamp (B). This lacking clamp likely renders the Clt2 RCL conformation more flexible in solution, unlike the Mcp1a RCL. This conclusion is further supported by the absence of electron density of the RCL in the crystal structure of isolated Clt2 (pdb 3h6r). The RCL of Clt2 is, however, well defined when bound to legumain (B). At acidic pH, where we observed processing of the Mcp1a RCL, the internal electrostatic clamp will be disturbed due to protonation of Asp73Mcp and Glu69Mcp, which could provide an explanation for the pH-dependent differences in processing we observed for Mcp1a and Clt2. Protonation at pH 4.0 may lead to an increase in Mcp1a RCL flexibility, similar as we find it in the RCL of Clt2. Together, this led us to the hypothesis that the increase in RCL flexibility at acidic pH would turn Mcp1a into a better protease substrate. To test the relevance of the RCL conformation for inhibition, we prepared Mcp1a-D73A and Mcp1a-R71E RCL-mutants. In agreement with our hypothesis, these mutants showed a decrease in inhibition and an increase in RCL hydrolysis . While Mcp1a harbors a Ser75Mcp in position P1′, Mcp3 harbors a cysteine at the equivalent position. Looking at the crystal structure of the legumain–Mcp1a complex, we observed that the P1′ side chain was in close proximity to the Sγ atom of the catalytic Cys189 residue on legumain (B). Based on this observation, we hypothesized that macrocypin 3a (Mcp3a) could potentially be a covalent-reversible legumain inhibitor, mediated by disulfide formation between the P1′-Cys75 of Mcp3a and Cys189 on legumain. To test this hypothesis, we recombinantly expressed Mcp3a, coincubated it with legumain at pH 4.0 to 7.0 and analyzed the reactions on SDS-PAGE using nonreducing and reducing sample loading buffer. Indeed, in the absence of DTT, we observed a band corresponding in size to the covalent legumain–Mcp3a complex  (A). The band was resolved to AEP and Mcp3a bands, when DTT was added, which confirmed that the complex was stabilized by disulfide formation. Additionally, we observed that Mcp3a formed a covalent, disulfide mediated dimer, which was similarly resolved in the presence of DTT . Furthermore, monomeric Mcp3a migrated as a double band on SDS-PAGE, most likely due to incomplete unfolding in the SDS-PAGE loading buffer (C and D). Formation of the covalent AEP–Mcp3a complex was further confirmed using SEC experiments (C). The covalent complex eluted as a separate peak. SDS-PAGE analysis of the peak fractions showed that under nonreducing conditions, it contained a band at the expected height of the covalent complex, which was resolved when DTT was added to the SDS-PAGE sample loading buffer. Since covalent complex formation is typically associated with a long-lived interaction, we expected that Mcp3a would be a more potent legumain inhibitor than Mcp1a. Interestingly, when we compared the inhibition of legumain by Mcp1a and Mcp3a in the presence or absence of DTT, we observed similar inhibition for both inhibitors (B). In addition to the sequence variation in P1′ position, Mcp3 also harbors P3-Ile72Asp and P5-Phe70Ile variations relative to Mcp1a (C). Both residues are localized on the nonprime-binding side of the RCL. To test whether these additional sequence variations on Mcp3a cause additional differences in inhibition, we set up control reactions using an Mcp1a-S75C variant. This mutant only differs at P1′ position and therefore allows to specifically test the effect of the P1′-Cys on inhibition. Interestingly, the Mcp1a-S75C mutant showed an approximately 2-fold increase in inhibition compared to WT Mcp1a (D), suggesting that cysteine in P1′ position indeed strengthens the enzyme–inhibitor complex. This effect was however dampened in Mcp3a most likely due to additional, less favorable sequence variations in the P3 and P5 positions on the RCL.Even though the RCL sequences of different macrocypin isoforms were highly conserved in sequence alignments, we noticed that there were specific sequence variations at critical positions C. While legumain B. Based  56 kDa) A. The bag buffer , C and Deriments C. The cohibitors B. In addto Mcp1a C. Both rWT Mcp1a D, suggesClt at P1′ position was replaced by cysteine, incubated it with legumain, and subsequently analyzed the reactions by SDS-PAGE in the absence (E) or presence (F) of the reducing agent DTT. Indeed, in the absence of reducing agent, we observed a band at the expected height of the covalent legumain–Clt2-T71C complex . This band was resolved when DTT was added to the samples. Similarly, Clt2-T71C was additionally prone to intramolecular disulfide formation, which led to the formation of a covalent dimer. The dimer migrated as a single band, when the samples were not heated prior to loading them on SDS-PAGE (C). When the samples were heated, the dimer converted to a double band, composed of (partly) folded and unfolded protein (E). Addition of DTT to the samples resolved the dimer bands to monomeric Clt2-T71C (F and S4D). Consistent with the observation of the covalent complex on SDS-PAGE, we also observed inhibition of legumain by the Clt2-T71C mutant using the fluorogenic Z-Ala-Ala-Asn-7-amino-4-methylcoumarin (AAN-AMC) substrate (G). Remarkably, the inhibitory potency of the P1′ cysteine variant Clt2-T71C was lower than that of the WT Clt2. P1′ cysteine-harboring mycocypins are partly present in a dimeric state and thereby engage the RCLs in the dimer interface. Consequently, the dimeric state is inhibitory inactive. Depending on the equilibrium of active monomer and inactive dimer states, the P1′ cysteine contribution to inhibition will strengthen or weaken the inhibition. In Clt2-T71C, the dimer band is much more dominant as in Mcp3a (A and S7E), explaining the weaker inhibition in Clt2-T71C. Unlike Mcp3a, disulfide-mediated covalent complex formation was most favorable at around pH 6.0 for the Clt2-T71C mutant (E). Importantly, for Mcp3a, Mcp1a-S75C, and Clt2-T71C, we observed covalent complex formation only with the intact, unprocessed inhibitor. In none of them, we observed a covalent complex composed of legumain and processed inhibitor , togethnhibitor .H).To further investigate the relevance of the P1′ residue, we additionally prepared an Mcp1-S75A mutant and tested its inhibition of legumain. The S75A mutation showed a slight reduction in the inhibition of legumain by Mcp1a H.66QGLNTP72) and two head-to-tail cyclized peptides based on the RCL sequence of Mcp1a (Mcp1a-RCL1: T68EFRIDNSIPGQ79 and Mcp1a-RCL2: T68EFRIDNSIPGQ79G). Cyclization should further stabilize the electrostatic clamp on the Mcp1a-RCL peptides. Additionally, we designed a cyclic peptide based on the RCL sequence of Mcp1a but harboring a Ser to Cys variation at position P1’ (Mcp1a-RCL2-P1′Cys: T68EFRIDNC75IPGQ79G). Indeed, we observed inhibition of legumain by all four peptides (A). Interestingly, the cyclic Mcp1a-derived peptides bound with higher apparent affinity than the Clt2-derived peptide. This observation is in agreement with the generally higher affinity of Mcp1a to legumain than Clt2. And it is further highlighting the importance of the RCL conformation for the interaction with legumain. Importantly, the cyclic Mcp1a-RCL2-P1′Cys peptide showed strong legumain inhibition  even at 250 μM concentration, where the WT peptide would still leave ∼80% residual activity. Furthermore, the interaction of the Mcp1-RCL2-P1′Cys peptide was sensitive to the presence of a reducing agent (B), providing evidence that the peptide was similarly establishing a covalent interaction to the catalytic Cys189 residue. However, overall, the affinity of all tested peptides was approximately three orders of magnitude lower (IC50 in the μM range) than the Mcp1a and Clt2 proteins . Exosite 1 (EX1Mcp) is built up by a conserved Arg96Mcp residue on Mcp1a, which is establishing ionic interactions to Asp160 on legumain. Asp160 forms the eastern wall of the S2′ substrate-binding site on legumain. Thereby, EX1Mcp is mediating prime-side interactions and is extending the substrate-like interaction of the macrocypin RCL. Additionally, we identified Arg51Mcp which is hydrogen bonding to Tyr221 on the legumain S4/5 site and thereby also extending interactions to the nonprime substrate-binding site. Exosite 2 (EX2Mcp) is formed by Phe7Mcp and Trp53Mcp, which mediate hydrophobic interactions to Tyr41 in the south of the nonprime substrate-binding cleft of legumain. While EX1Mcp is localized to the crown of Mcp1a, EX2Mcp is localized to the trunk of the inhibitor (F). Since both exosites are sequentially distant to the RCL, they could potentially stabilize the enzyme–inhibitor complex, also after processing after the P1-Asn74Mcp residue by legumain. To test this hypothesis, we prepared an EX1Mcp Mcp1a-R96A mutant and tested its interaction with legumain. Indeed, the EX1Mcp mutant showed an approximately 100-fold reduction in legumain inhibition (B), which suggested that if the exosite interaction was removed, Mcp1a was rather a substrate to legumain than an inhibitor. In a previous study, we observed a similar exosite interaction also in cystatins (C). This effect was abolished when the EX1Mcp Mcp1a-R96A variant was coincubated with legumain, which further confirmed the importance of this residue for the interaction with legumain. In line with this observation, the Mcp1a-RCL mutants (Mcp1a-D73A and Mcp1a-R71E), where the RCL conformation was disturbed, were similarly unable to stabilize legumain at near neutral pH (E).Closely analyzing the crystal structure of legumain in complex with Mcp1a, we found in addition to the RCL, two exosites that mediate binding to legumain A and S8Anhibitor F. Since hibition . Additiohibition B, which ystatins . Exositeystatins C. This eutral pH E.Figure B and S8F). Specifically, exosite 1 (EX1Clt) interactions are mediated by residues Glu48Clt, Gln49Clt, and Ile51Clt on Clt2, which are interacting by hydrogen bonding and hydrophobic interactions with the c341-loop on legumain. The c341-loop serves as a template for nonprime substrate binding. EX1Clt interaction involves Tyr220, Tyr221, and Tyr228 on legumain. Exosite 2 (EX2Clt) is mediating interactions to the north of the nonprime substrate-binding cleft via residues Gly32Clt–Arg35Clt. Similarly, EX2Clt is establishing interactions to the c341-loop via residues Arg213, Ser215, and Tyr217 on legumain. Both exosites are localized to the crown of the inhibitor (F). To test the relevance of these residues for the interaction with legumain, we prepared an EX1Clt Clt2-E48A-Q49A-I51A triple mutant (Clt2-3x) which should disrupt the interaction to the S4/5 site on legumain. As expected, the triple mutant showed reduced inhibition of legumain (D). This finding clearly showed that similar to the Mcp1a-R96A mutant, the Clt2-3x mutant behaved more like a substrate to legumain than an inhibitor. Thereby we could confirm that the Clt2 exosite interaction is indeed important for the inhibition of legumain. Interestingly and unlike Mcp1a, Clt2 did not have a significant pH-stabilizing effect on legumain in a thermal shift assay (E). This observation further confirmed that the pH-stabilizing effect was directly linked to the electrostatic stability switch, which is localized mainly to the prime substrate-binding sites and south of the substrate-binding cleft on legumain, which is addressed by Mcp1a but not by Clt2. Together, these results clearly show that it is the exosite interaction that turns the mycocypins into inhibitors rather than substrates.In stark contrast to Mcp1a, the Clt2 exosites are mediating interactions to the nonprime substrate-binding sites and the north of the nonprime substrate-binding cleft on legumain B and S8Fnhibitor F. To teslegumain  and an ilegumain D. This fft assay E. This o4fgu) and the AEP–Mcp1a and AEP–Clt2 complexes (A and B). These models suggested that the binding mode exploited by Clt2 is compatible with binding both to AEP and ACP. We did not observe major sterical clashes between Clt2 and the LSAM domain within this model (B). However, we did observe clashes between the LSAM domain and Mcp1a in the model of the ACP–Mcp1a complex (A), which suggested that Clt2 would inhibit AEP and ACP equally well, but Mcp1a would be a better inhibitor of AEP than ACP. To test this assumption, we tested the inhibition of AEP and ACP (C) by Mcp1a, Mcp3a, and Clt2 using the AAN-AMC fluorescence substrate (D and E). In line with our hypothesis, we found that Clt2 was inhibiting AEP and ACP equally well, leading to a residual activity of approximately 60% – 70% (D and E). Importantly, Mcp1a and Mcp3a were inhibiting AEP approximately three times better than ACP, consistent with our finding that macrocypins need to establish prime side exosite interactions for inhibition, which are sterically not easily accessible in ACP.Depending on the pH-environment, prolegumain will activate to specific AEP and asparaginyl carboxypeptidase (ACP) forms. AEP is generated upon incubation of prolegumain at pH ≤ 4.0, which results in the complete autocatalytic removal of the activation peptide and the LSAM domain. ACP is generated upon incubation of prolegumain at pH > 4.0 to ≤ 5.0. Under these conditions, the activation peptide is removed but the LSAM domain remains electrostatically bound to the catalytic AEP domain . Importaomplexes , A and Bis model B. Howeve complex A, which  and ACP C by Mcp1ubstrate , D and E0% – 70% , D and EArabidopsis thaliana legumain isoforms β and γ . Overall, all three investigated legumain variants were inhibited by the tested mycocypins (A and 8A). Interestingly, however, all of them were most potently inhibited by the macrocypins and less potent by Clt2, in agreement also with previously published data . Both residues are more bulky and do not allow ionic interactions to EX1Mcp Arg96Mcp. Additionally, His182 on AtLEGβ even has an opposing charge, which will cause electrostatic repulsion of EX1Mcp. Looking at EX2Mcp, we found that Tyr41 on human legumain is replaced by Gly63 in AtLEGβ and Trp71 in AtLEGγ. While Trp71 is still capable of mediating hydrophobic interactions to Trp53Mcp, Gly63 is not. Together, these differences indicated that the interaction of Mcp1a to the southern rim on AtLEGβ is weaker, which can explain why Mcp’s are better inhibitors of AtLEGγ than AtLEGβ. Additionally, we found that the cyclic protein recognition motif insertion on the nonprime substrate-binding cleft of AtLEGβ and γ is in steric conflict with EX1Clt on Clt2 (B), which can provide an explanation why Clt2 is an even worse inhibitor of plant legumains than human legumain. Together, these results clearly show that mycocypins will inhibit different legumain variants with different affinities.When we analyzed sequences of legumain inhibitory mycocypins, we observed specific variations of the RCL but also of the exosite sequences. Based on this observation, we were wondering whether such isoform-specific sequence variations might encode for specific inhibitory activities toward specific legumain isoforms. To test this hypothesis, we compared the inhibitory potential of Mcp1a, Mcp3a, and Clt2 toward human legumain and cocypins A and 8A.hed data . Furthern AtLEGγ A. Both r on Clt2 B, which Porphyromonas Gingivalis gingipain K (Kgp) (B). While we determined a Ki of 1.47 ± 0.04 μM for Mcp4a, Mcp2a showed a 10-fold increase in affinity with a Ki of 0.18 ± 0.02 μM. Even though both Mcp variants showed only minor differences in the RCL sequences, they showed significant differences in inhibition. Analyzing the sequence variations more closely, we found that Mcp2a and Mcp4a were differing at position P3, which is a valine in Mcp2a and alanine in Mcp4a (D). To understand the differences in affinity, we prepared a model of a Kgp–Mcp2a complex by superposing the crystal structure of legumain in complex with Mcp1a onto the crystal structure of Kgp (A). The model suggested that a P3-Val (Mcp2) or P3-Ile residue (Mcp1a) would fit better into the Kgp substrate-binding cleft than a P3-Ala, as found in Mcp4. To test the relevance of the P3 residue for the inhibition of Kgp, we prepared Mcp1a-I72A-N74K, Mcp1a-I72V-N74K, and Mcp2a-V72I mutants. Indeed, we found that mutation of Ile72Mcp to Ala had a negative effect on the propensity of Mcp1a-N74K to inhibit Kgp (B). Mutations of P3-Ile to P3-Val or vice versa had no negative impact on inhibition. Using mass spectrometry experiments, we could furthermore show that Mcp1a-N74K was cleaved by Kgp after the P1-Lys74 residue, confirming a similar, substrate-like binding mode. Additionally, introducing a cysteine residue at the P1′ position of Mcp2a (Mcp2a-S75C) had a positive effect on the inhibition of Kgp, suggesting a similar covalent bond formation with the catalytic cysteine residue (B).When we were looking at the RCL sequences of so far identified mycocypin sequences, we noticed variations not only at the P5, P3, and P1′ positions but also at the P1 position. Interestingly, Mcp2 and Mcp4 sequences showed a lysine residue at P1 position, which suggested that they might be inhibiting other clan CD proteases with specificity for lysine. Potential candidates included paracaspases and the bacterial  K (Kgp) , 36. To  K (Kgp) B. While in Mcp4a D. To unde of Kgp A. The moibit Kgp B. Mutati residue B.C). Since caspases have a strong preference for small residues at position P1′, we prepared a Mcp1a-N74A mutant to further test the relevance of the P1′ position. Indeed, the Mcp1a-N74A variant showed inhibition of caspase-9, which confirmed that the P2-Asp73 on the Mcp1a RCL may serve as a P1 residue for the inhibition of caspases.Further analysis of the macrocypin RCL sequences revealed a conserved aspartic acid residue in position P2. Knowing that caspases specifically cleave substrates after aspartic acid residues, we were in a next step wondering if macrocypins would also be suited as caspase inhibitors. To test the propensity of macrocypins as caspase inhibitors, we recombinantly expressed active caspase-9 and used it as a test enzyme. Interestingly, WT Mcp1a showed only little inhibition of caspase-9 C. Since A). In both inhibitors, the RCL binds to the legumain active site in a substrate-like manner, with the asparagine residue functioning as P1-residue. By this binding mode, both Clt’s and Mcp’s represent legumain substrates. Indeed, even though the RCL–protease interaction is necessary for mycocypin recognition, it is not sufficient for inhibition. Instead, both inhibitors additionally encode exosite interactions, which are required to stabilize the enzyme–substrate complex and turn the substrates into inhibitors. Importantly, the exosite interactions encoded by clitocypins and macrocypins are localized to different regions on the inhibitors, even though their overall fold is highly similar. Clitocypin 2 utilizes loops on the crown of the tree to interact with the nonprime substrate-binding side and the area north to the active site of legumain. Mcp1a utilizes amino acids localized to the crown and the trunk to interact with the prime-side and the area south to the active site of legumain (A). Taken together, our structural and functional data suggest a two-step mode of inhibition. In a first step, the inhibitors are recognized like substrates, utilizing the P1-Asn residue on the RCL. In parallel, substrate recognition goes along with binding of the exosites to legumain, thereby stabilizing the enzyme–inhibitor complex. A second, regulatory step occurs only once the inhibitor is stably and productively bound to the enzyme. In this step, pH- and conformation-dependent cleavage after the P1-Asn residue on the RCL may occur, which goes along with a pH-dependent increase in affinity in Clt2. While the affinity of legumain toward processed Clt2 remained similar to intact inhibitor at pH 6.0, it increased approximately three fold at pH 4.0. This increase in affinity may be partly attributed to protonation of residues like His148, which will be positively charged at pH 4.0 and might form a salt bridge with the neo C-terminus of the cleaved inhibitor or to a pH-dependent increase in flexibility of the cleavage products. Furthermore, the regulatory phase may also include the formation of a covalent complex, if the inhibitor harbors a cysteine in P1′ position. Disulfide formation may be mediated by a P1′-Cys on the mycocypin and the catalytic Cys189 on legumain. Importantly, disulfide formation is in general unfavorable at acidic pH due to protonation of the Cys Sγ . Howeve4n6o), Clt2, and Mcp1a, we found that the scissile peptide bond was slightly shifted in Mcp1a and significantly shifted in Clt2, relative to cystatin E (2), only few ordered water molecules are to be expected.A mechanistically similar two-step mode of inhibition also evolved in the family 2 cystatins, even though they structurally belong to a completely different protein family. Cystatins harbor a substrate-like RCL that serves as a bait and an exosite that is stabilizing the enzyme–substrate complex and turns the substrate into an inhibitor. The mechanistic similarity suggests that this mode of inhibition may serve as a universal strategy to target legumain. Interestingly, even though macrocypins and clitocypins similarly show a substrate-like processing of the RCL by legumain, unlike cystatins, we did not observe ligation of the cleaved inhibitors. This may indicate that the newly generated N-terminus of the primed cleavage product is flexible and quickly released from the active site, which consequently prevents religation of the RCL. Even though the RCL is in principle internally stabilized in Mcp’s, the newly generated N-terminus may still be flexible after cleavage. When we superposed the crystal structures of legumain with cystatin E , which implies that they are bispecific, janus-faced inhibitors. Interestingly, EX1Clt on Clt2 is sequentially and sterically close to the papain-binding site. However, the two sites evolved such, that they are not overlapping each other. Consequently, mushrooms might use mycocypins to defend themselves against predators, which utilize papain-like enzymes and legumains as digestive enzymes as, for example, found in schistosome or tick , Mcp2a (B9V977), Mcp3a (B9V979), Mcp4a (B9V982), and C. nebularis Clt2 (Q3Y9I4) full-length cDNA sequences were purchased from Eurofins Genomics.6-tag for Ni2+-affinity purification. Various point mutations were introduced using round the horn mutagenesis, which is based on the inverse PCR method, as described earlier (via DNA-sequencing by Eurofins Genomics.Expression constructs were subcloned into the pET-22b(+) expression vector using NdeI and XhoI restriction enzyme cleavage sites, which were added directly 5′ or 3′ of the cDNA sequence. The expression constructs contained a C-terminal His earlier , 40. Cor2. Briefly, the human prolegumain expression construct, which harbored a C-terminal His6-tag for purification, was stably transfected into the LEXSY P10 host . Positive clones were selected using nourseothricin (Jena Bioscience). Cells were grown in brain heart infusion medium (Carl Roth) containing Hemin (Applichem), Pen-Strep , and nourseothricin, and incubated shaking (140 rpm) at 26 °C until an A600 ∼3 was reached. Cells were pelleted via centrifugation, and His6-tagged prolegumain was harvested from the supernatant by batch incubation with Ni2+-NTA Superflow resin (Qiagen). Following Ni2+-purification, elutions were concentrated using Amicon Ultra centrifugal filter units  and buffer exchanged via PD-10 columns (Cytiva) to get the protein in the final buffer 20 mM Tris pH 7.0, 50 mM NaCl, and 2 mM DTT. For subsequent assays, prolegumain was activated to the AEP at pH 3.5 as described previously. Activation to the ACP form was achieved upon incubation of prolegumain at pH 4.5. Progress of autocatalytic activation was monitored on SDS-PAGE. Subsequently, the activated proteins were concentrated using Amicon Ultra centrifugal filter units  and further purified using SEC utilizing the Äkta FPLC system equipped with a Superdex 75 10/300 Gl column (Cytiva) equilibrated in a buffer composed of 20 mM citric acid pH 4.0, 50 mM NaCl, and 2 mM DTT in case of AEP and 20 mM citric acid pH 5.0 and 50 mM NaCl in case of ACP. Similarly, A. thaliana prolegumain isoforms β and γ were expressed in LEXSY and purified as described previously  cells. Expression plasmids were chemically transformed into the E. coli cells and positive clones were selected using ampicillin. Expression cultures were grown in LB medium, supplemented with 100 μg/ml ampicillin, at 37 °C and shaking at 220 rpm, until an A600 ∼ 0.8 was reached. Subsequently, cultures were transferred to 25 °C and expression was induced upon addition of 1 mM IPTG. Following overnight incubation, cells were harvested by centrifugation , and the cell pellet was resuspended in a buffer composed of 50 mM Tris pH 7.5 and 300 mM NaCl  or 50 mM Tris pH 7.5 and 2 mM EDTA (Mcp1a). Subsequently, cells were lysed by sonication on ice at 40% power, 4 times for 45 s. The lysed cells were centrifuged at 17,500g for 30 min at 4 °C to separate the soluble and insoluble fractions. While Clt2, Mcp2a, and Mcp4a were expressed as soluble forms, Mcp1a was expressed as nonclassical inclusion bodies and Mcp3a was expressed in soluble form and as nonclassical inclusion bodies. For Mcp1a, the pellet containing the nonclassical inclusion bodies was solubilized in a buffer composed of 50 mM Tris pH 7.5, 2 mM EDTA, and 3 M urea, for 2 to 4 h at 4 °C  and placed in 2 l dialysis buffer (50 mM Tris pH 7.5 and 300 mM NaCl) overnight at 4 °C. On the next day, the tube was transferred into 2 l of fresh dialysis buffer and stirred at room temperature (21 °C) for another 2 h, then centrifuged at 17,500g for 30 min and 4 °C. The soluble fraction containing correctly folded Mcp1a was further subjected to Ni2+-affinity purification and protein was eluted using a buffer composed of 50 mM Tris pH 7.5, 300 mM NaCl, and 250 mM imidazole. The elutions were concentrated using Amicon Ultra centrifugal filter units  and subjected to SEC utilizing the Äkta FPLC system equipped with a Superdex 75 10/300 Gl column (Cytiva) preequilibrated in buffer composed of 20 mM Tris pH 7.5 and 50 mM NaCl. Clt2, Mcp2a, Mcp3a, and Mcp4a, which were found in the soluble fraction after cell lysis, were directly subjected to Ni2+-purification and SEC, as described for Mcp1a.Mycocypins were expressed in ΔCARD-caspase-9 was expressed and purified as described previously .68EFRIDNSIPGQ79, Mcp1a-RCL2: T68EFRIDNSIPGQ79G, and Mcp1a-RCL2-P1′Cys: T68EFRIDNC75IPGQ79G) and Clt2 (Clt2-RCL: Y66QGLNTP72) were purchased from JPT Peptide Technologies and dissolved in DMSO to a final concentration of 100 mM.Peptides derived from the RCL sequences of Mcp1a  substrate. Activity was measured at 460 nm after excitation at 380 nm in an Infinite M200 Plate Reader (Tecan) at 37 °C and an enzyme concentration of 1 μM. Inhibition of caspase-9 by Mcp1a and Mcp1a-N74A was tested after supplementing the assay buffer with 25 μM of the respective inhibitor. Full-length P. gingivalis Kgp was kindly provided by Prof. Jan Potempa. Kgp activity was assayed in a buffer composed of 200 mM Tris pH 7.5, 150 mM NaCl, 5 mM CaCl2, 0.03% NaN3, and 4 mM L-cysteine hydrochloride monohydrate supplemented with 830 μM of the chromogenic substrate Tos-Gly-Pro-Lys-pNA (Bachem). The reaction was started by the addition of 0.25 μM enzyme . Subsequently, the increase in absorbance at 405 nm was measured at 37 °C. To test the inhibition of Kgp by Mcp1a, Mcp2a, Mcp4a, Mcp1a-N74K, Mcp1a-I72A-N74K, Mcp1a-I72V-N74, and Mcp2a-V72I, the enzyme was preincubated with the respective inhibitor in assay buffer lacking the chromogenic substrate, for 10 min at 25 °C. Subsequently, activity was tested at 0.2 μM enzyme and 5 μM inhibitor concentration. For all activity measurements, activity was calculated as velocity in nmole per second, if not specified differently. For Ki determination, Kgp was preincubated with increasing concentrations of Mcp2a or Mcp4a in assay buffer lacking the chromogenic substrate, for 10 min at 25 °C. Subsequently, the enzyme-inhibitor mix was added to the reaction buffer containing the substrate and activity was measured as an increase in absorption at 405 nm for 10 min at 37 °C. The final concentration of the enzyme in the assay was 0.25 μM, and the final concentration of the inhibitors was 0.4 to 53 μM (Mcp2a) or 0.8 to 105 μM (Mcp4a). The velocity of substrate turnover was calculated as fluorescence units/s, and the data points were fitted to the Morrison equation using the GraphPad Prism program (version 5.0). All experiments were carried out at least in triplicate.Legumain protease activity was monitored using the fluorogenic peptide substrate Z-Ala-Ala-Ans-AMC  at 460 nm after excitation at 380 nm in an Infinite M200 Plate Reader (Tecan) at 37 °C. The assay buffer contained 50 mM citric acid pH 5.5, 100 mM NaCl, 0.05% Tween-20, and 50 μM Z-AAN-AMC substrate. The reaction was started by the addition of 2 nM legumain. Inhibition of legumain was tested using assay buffer supplemented with 10 nM macrocypins, 2 μM Clt2, or 1 mM or 250 μM RCL peptide, respectively. If indicated, the assay buffer was additionally supplemented with 2 mM of the reducing agent DTT. To test the inhibition of To test if legumain processes mycocypins, it was incubated with mycocypins in a 1:2 molar ratio for 1 h at 37 °C in a buffer composed of 50 mM NaCl and 20 mM citric acid pH 4.0, 5.0, or 6.0 or 50 mM NaCl and 20 mM Tris pH 7.0. For the Clt2-T71C mutant, a molar ratio of 1:3 was used and incubation was done on ice. Progress of inhibitor hydrolysis was monitored on SDS-PAGE with or without preheating of the samples at 95 °C for 20 min.To test whether mycocypins were also a substrate to legumain’s ligase activity, we incubated Mcp1a or Clt2 with legumain in a 1:2 molar ratio at pH 4, for 1 h at 37 °C. Subsequently we shifted pH to neutral by adding 100 mM of Tris pH 7.0 or we added 2 mM of MMTS to the sample and further incubated the reactions for 1 h at 37 °C. Progress of cleavage and religation was monitored on SDS-PAGE.The thermal stability of legumain upon incubation with different mycocypins was assayed using differential scanning fluorimetry. Briefly, legumain was mixed with different mycocypin variants in a 1:1 molar ratio at 0.1 mg/ml final protein concentration, in an assay buffer composed of 100 mM citric acid pH 6.0, 100 mM NaCl, and 5x Sypro orange Dye (Invitrogen). Thermal unfolding was measured as an increase in fluorescence signal in an 7500 Real Time PCR System (Applied Biosystems) after increasing temperature by 1 °C per min from 20 °C to 95 °C. Fluorescence data was normalized to peak values and melting curves were evaluated as described previously . Gels were stained using Coomassie Brilliant Blue-G (panreac applichem). Molecular weights were assigned using the PageRuler prestained protein ladder as a reference . Mycocypins proved to be resistant to unfolding in SDS-loading buffer. Therefore, increased concentrations of SDS (up to 15%) or urea (3 M) were added, if specified.Legumain was incubated with Mcp1a or Clt2 in a 1:1 molar ratio in a buffer composed of 20 mM citric acid pH 4 and 50 mM NaCl for 30 min at 37 °C. Subsequently, samples were analyzed by SDS-PAGE and mass spectrometry, utilizing an ESI-Orbitrap setup. Similarly, Kgp was incubated with Mcp1a-N74K or Mcp1a-I72A-N74K in a 1:10 molar ratio in Kgp assay buffer at 37 °C for 3 h and analyzed by SDS-PAGE and mass spectrometry. For mass spectrometric analysis, samples were desalted with C18 ZipTips (Merck Millipore), eluted from the tips with 50% acetonitrile in 0.1% formic acid and directly infused into the mass spectrometer  at a flow rate of 1 μl/min. Capillary voltage at the nanospray head was 2 kV. Raw data were processed with Protein Deconvolution 2.0 (Thermo Fisher Scientific). Masses were assigned to the protein sequence with the Protein/Peptide Editor module of BioLynx .C. nebularis clitocypin 2 (Q9P4A2), M. procera macrocypin 1a (B9V973), macrocypin 1b (B9V975), macrocypin 1c (B9V976), Mcp2a (B9V977), macrocypin 2b (B9V978), macrocypin 3a (B9V979), macrocypin 3b (B9V980), macrocypin 3c (B9V981), macrocypin 4a (B9V982), macrocypin 4b (B9V983), and macrocypin 5a (B9V984). A sequence alignment was prepared using Clustal W2 . Initial crystallization screening was performed in a sitting-drop vapor diffusion setup. 0.2 μl concentrated enzyme–inhibitor complex were mixed with 0.2 μl JBScreen Classic (Jena Bioscience) screen solution and equilibrated against 60 μl reservoir solution in 96 well INTELLI-PLATEs at 4 °C. After approximately 1 week, crystals appeared in a condition composed of 1.6 M ammonium sulfate and 500 mM LiCl, pH 5.2. For cryo-protection, a cryo-solution composed of the reservoir solution supplemented with 30% glycerol was added stepwise to the drops containing crystals before flash freezing in liquid nitrogen. A native x-ray diffraction data set was collected at beamline ID30A-3 (ESRF Grenoble) at a wavelength of 0.9677 Å and 0.1 ° oscillation range to a resolution of 2.2 Å.Similarly, the legumain–Clt2 complex was formed by incubation of legumain with Clt2 in a 1:1.1 molar ratio in a buffer composed of 20 mM citric acid pH 5.5, 50 mM NaCl, and 0.5 mM MMTS. After concentrating the complex to approximately 20 mg/ml final protein concentration, initial crystallization screening experiments were set up using the Hampton index screen (Hampton). After approximately 2 weeks, at 4 °C, we obtained an initial hit in a condition composed of 0.1 M sodium citrate pH 4.5 and 20% PEG 4000. Crystals were optimized by fine-screening around the originally identified condition and flash frozen in liquid nitrogen after incubation in a cryo-solution composed of the original condition supplemented with 30% ethylene glycol. An x-ray diffraction data set was collected at the ESRF in Grenoble at beamline ID30A-3 at a wavelength of 0.9677 Å and 0.2° oscillation range to a resolution of 1.8 Å.https://www.ebi.ac.uk/pdbe/pisa/).X-ray diffraction data was processed utilizing XDS . An initP. gingivalis Kgp, human cathepsin V, and A. thaliana legumain isoforms β and γ were prepared by superposing structures of human legumain in complex with Mcp1a or Clt2 onto structures of Kgp (pdb 4rbm), the Clt2-cathepsin V complex (pdb 3h6s), AtLEGβ (pdb 6ysa), and AtLEGγ (5obt) using TopMatch (Models of mycocypins in complex with TopMatch . Pymol wTopMatch .8AE4 (legumain in complex with clitocypin 2) and 8AE5 (legumain in complex with macrocypin 1a).The structures presented in this article have been deposited in the Protein Data Bank (PDB) with the accession codes This article contains The authors declare that they have no conflict of interest with the contents of this article.


The optimizedsprayed separator consisted of 50 nm Al2O3 particles,1 wt % poly(acrylic acid), and 5 wt % styrene–butadiene rubber,deposited from an 80:20 vol % suspension of water and isopropanol.Separators between 5 and 22 μm thick had consistent andsimilar porosity of ∼58%, excellent wettability, thermal stabilityto at least 180 °C, adequate electrochemical stabilityand high effective ionic conductivity of ∼1 mS cm–1 at room temperature in an EC/DMC electrolyte, roughly double thatof a conventional polypropylene separator. A sequentially depositedthree-layer LiFePO4/Al2O3/Li4Ti5O12 full cell, the first of its kind,showed similar rate performance to an identical cell with a conventionalpolypropylene separator, with a capacity of ∼50 mAh g–1 at 30 C. However, after cycling at 2 C for 400 cycles, Al2O3 separator full cells retained 96.3% capacity, significantlymore than conventional full cells with a capacity of 79.2% remaining.A porous, spray-deposited Al The separatoris a membrane designed to stop electronic contact between the anodeand cathode while providing pathways for lithium ion transport, andthis is conventionally achieved by using an porous electronic insulator,in which the pores become filled with the Li-ion conducting electrolyte.5 Commercial PE and PPseparators are manufactured by using an extrusion process, eitherdry  or wet.6 The dry process relieson forming pores between semicrystalline lamellar and amorphous regionsin a polymer film by stretching, while the wet process relies on stretchingbefore or after the removal of additives .6 For the dry process, the shape of the poresis characteristic to the technique, that is, slit-shaped pores, whilethe distribution and size of pores can be controlled by processingconditions.3 Polyolefin separators havebeen favored due to low cost, a mature manufacturing process, goodelectrochemical stability, acceptable mechanical strength, and anintrinsic “thermal shutdown” capability.7 However, with the desire for higher performance and safetyin LIBs, improvements to their relatively poor thermal stability (shrinkageat 90 °C) and still often poor wettability are a pressingrequirement.9Most commercialLIB separators consist of microporous semicrystallinepolyolefins, commonly polyethylene (PE), polypropylene (PP), or combinationsof both .4 Surface coating, grafting,and blending of conventional polyolefin separators to improve wetting,thermal, mechanical, and chemical characteristics have been effectivefor many polymers,13 ceramics/inorganics,17 and combinations thereof.21 However, the improvements imparted by these modificationsare often coupled with a complicated manufacturing process, increasedseparator thickness, a decrease in porosity, and/or debonding of modifyingmaterials during cell assembly and cycling.23 Lee et al. provide an overview of the variousmodification techniques.1Researchers have focusedon two methods to produce separators withhigher performance: (a) modification of existing polyolefins and (b)identification and application of alternative materials and manufacturingtechniques.25 poly(ether ether ketone) (PEEK),26 polytetrafluoroethylene(PTFE),27 and poly(phenylene sulfide) (PPS)28 offer higher mechanical strength, good chemicaland electrochemical resistance, and superior thermal resistance. Liuet al. showed that an electrospun polyimide/poly(vinylidene fluoride-co-hexafluoropropylene) (PI/PVDF-HFP) core/sheathseparator could provide higher tensile strength (53 MPa), improveddimensional stability at 150 °C, and improved electrochemicalperformance over a Celgard PP separator.29 However, manufacture of separators based on higher performance polymerscan be challenging due to their poor solubility in common solvents.Liu et al. recently showed that the thermally induced phase separation(TIPS) technique could overcome this challenge by preparing a thin(∼30 μm) PPS separator with high porosity (∼70%),excellent mechanical strength (∼120 MPa tensile strength and∼7.5 GPa Young’s modulus), high thermal stability(>200 °C), and improved electrochemical performanceinLFP half-cells over conventional PE.28Becauseof the issues and compromises associated with polyolefinmodification, there have been efforts to identify alternative polymericseparators. High performance polymers, such as polyimide (PI),30 Common drawbacks include, in particular, the difficultyin creating a mechanically stable, thin, free-standing membrane andthe often comparatively high material density (increasing the inactivemass in the cell).32 Takemura et al. presented thefirst example of a free-standing ceramic-based separator,33 where Al2O3 particlesof 0.01 and 0.3 μm were combined with PVDF in varyingratios. The thermal shrinkage of the resulting separator was negligibleup to 150 °C and decreased with reducing particle sizeand a higher powder-to-binder ratio. Cells based on an optimized oxideseparator had similar capacity retention to cells using Celgard separatorsover 500 cycles.33 Zhang et al. produceda free-standing 37 μm Al2O3 separatorfrom 100 to 300 nm particles, 6 wt % styrene–butadiene rubber(SBR), and 1 wt % poly(ethylene glycol) (PEG)34 and an aqueous process. On its own, SBR binder was unable to stabilizea free-standing film, while a combination of 1 wt % PEG and 6 wt %SBR produced a mechanically stable membrane. A subsequent heat treatmentat 55 °C to remove the PEG resulted in dimensional stabilityat 130 °C for 40 min, an ∼0° contact anglewith the electrolyte, and improved rate performance in LiNi1/3Co1/3Mn1/3O2/graphite full cellswhen compared with conventional PE.34Ceramic and inorganic separators have also received attention dueto their high wettability, excellent thermal stability, and comparativelyhigh stiffness (to resist deformation).35 Friesen et al. described how commercial LIB cells can make use ofa 2–10 μm thick coating of 500 nm Al2O3 particles directly deposited onto LIB anodes in combinationwith a polymer separator, as an alternative to coating the polymerseparator directly.36 Jung et al. directlycoated 1–5 μm Li7La3Zr2O12 (LLZO) particles with 10–20 wt % PVDF-HFP directlyonto graphite-based electrodes.37 A free-standingmembrane of the same type had a thickness of 30 μm, aporosity of ∼40%, and no dimensional change after 1 h at 150 °C.Although the separator displayed a relatively poor electrolyte uptakeof ∼20%, the rate and cycle performance were improved overcells using PP separators. Significantly, the LLZO separator suppressedthermal runaway in a pouch cell during nail penetration tests, whilethe PP separator cell surged to ∼280 °C. The thermaland dimensional stability of ceramic particles stopped direct contactbetween the anode and cathode, suggesting that in some cases and incomparison to conventional separators ceramic particle separatorsmay improve cell safety.37 Other examplesof direct deposition have shown similar improvements to thermal performancebut generally produced membranes between 40 and 200 μm thick,which increases the inactive cell volume and mass considerably.40Most ceramic/inorganic particle separators are formed by usinga slurry deposition method, so direct deposition onto the electrodeis an obvious route to remove processing steps in the manufactureof LIBs. For example, Lagadec et al. stated, “With time, weexpect separators to be viewed as a continuation of the electrodesthemselves (perhaps even directly coated on them) with both structuraland chemical functionality in mind.”41 The separator consisted ofPVDF, poly(methyl methacrylate) (PMMA), and fumed SiO2 ata ratio by weight of 27:9:4, deposited from a mixture of acetone and N,N-dimethylformamide (DMF), while therest of the components were deposited from N-methyl-2-pyrrolidone(NMP). A separator thickness of 200 μm was said to benecessary to prevent internal shorting due to solvent penetrationduring deposition of the second electrode onto the separator. Despitethe relatively thick separator, the cells showed good performancein an Li4Ti5O12–LiCoO2 (LTO–LCO) full cell for 65 cycles and could powerLEDs after being printed onto the side of a mug.41Taking direct deposition further, Singh et al. showed thata layer-by-layerspray deposition technique enabled the fabrication of a functioningfull cell including the current collectors, anode, cathode, and theseparator.4 (LFP) positive electrodes to assesstheir ability to enable stable cycling of positive electrode-basedhalf-cells. The separator that yielded the best performance was thencharacterized further and compared with the performance of a commercialCelgard separator in LTO-based negative electrode half-cells for oxideseparators of various areal densities. Spray-deposited full cellswith a thin oxide separator layer were deposited via a continuoussequential procedure, which at the time of writing is the first reportof this type, as similar prior work has instead relied on direct depositionof much thicker separators.41 We show thatthis arrangement provides improved wetting, thermal, rate, and degradationcharacteristics compared with a conventional Celgard 2500 polypropyleneseparator. Full experimental details are given in The work described here advances previous examplesof direct depositionof separators by providing a simple, potentially “greener”spray deposition route, paired with spray-deposited cathodes and anodes,for the sequential layer-by-layer processing of full cell assemblies.While inorganic layers have been used in combination with polymericseparators to improve thermal runaway and safety characteristics,an inorganic separator formed as an integral part of the manufactureof the full cell assembly has not been reported previously. The principalobjective was to assess the promise of thin (5–22 μm)spray-deposited oxide layers in a simple electrode–separator–electrodeintegrated manufacturing sequence, as shown in 22.14  was used as cathode active material and Li4Ti5O12  as theanode active material. Carbon black  was used as a conductive additive, and sodium carboxymethylcellulosesalt  was used as the dispersant and binder. LFPand LTO suspensions had component ratios of 85:5:10 and 90:5:5 ofactive material:CMC:carbon black by mass, respectively. LFP and LTOslurries were mixed according to the following method: (1) carbonblack and CMC solution were mixed by ball-milling for 15 min at 350rpm by using 10 mm diameter ZrO2 balls, (2) active materialand water were added until a slurry concentration of 40 wt % solidwas reached and then mixed for a further 15 min at 350 rpm, (3) aportion of the slurry was extracted and diluted with water to a solidconcentration of 1 g/dL, (4) the suspension was agitated via magneticstirrer, and (5) 1 min before spray deposition, the solution was dilutedwith IPA to a solid concentration of 0.8 g/dL and solvent ratio of80:20 vol % water:IPA.LiFePO2.22O3 , poly(acrylic acid) , and styrene–butadienerubber  as the oxide particles, dispersant, andbinder, respectively. Suspensions with a oxide:dispersant:binder ratioof 94:1:5 were prepared as follows: (1) Al2O3, predissolved dispersant, and deionized water were added into aball mill and milled for 10 min at 350 rpm with 10 mm diameter ZrO2 balls; (2) SBR solution was added, and the mixture ball-milledfor a further 10 min at 350 rpm; (3) a slurry equal to 0.4 g of thesolid component was diluted with water to a total volume of 80 mLand mixed for 1 min via magnetic stirring; (4) the diluted solutionwas sonicated for 5 min; and (5) the suspension was magnetically stirredand diluted with 20 mL of IPA to a final volume of 100 mL and a solventratio of 80:20 vol % water:IPA, 1 min before the spray depositionprocess.Spray-deposited separators consisted of <50 nm Al2.3Particle size and zeta-potential measurements of the suspended oxideparticles were characterized by using dynamic and electrophoreticlight scattering . For zeta-potentialmeasurements, suspensions were prepared as above until step 4, atwhich point they were diluted with an aqueous solution of NaCl toreach an oxide particle concentration of 0.1 g/dL and 10 mM NaCl.For size measurement, the suspensions were diluted with deionizedwater only to 0.1 g/dL. Diluted suspensions were stirred by magneticstirrer for 30 min before measurement.2.447 and shown schematically in –1 and atomizedin an industrial spray nozzle using compressed air at 0.3 bar. Duringatomization, the nozzle followed a preprogrammed zigzag (snake raster)pattern in the x–y planeover the spacers at a constant distance of 15 cm. As suspension dropletsdeposited on the hot spacers, the water and IPA components evaporatedalmost instantly to avoid resuspension of previously deposited material.Electrode material was deposited until an areal density of 2 and 2.3mg cm–2 was achieved for LTO and LFP, respectively.All the data reported here are for uncalendered electrodes and layers,and the potential benefits or otherwise of calendering at variousstages are discussed later.Electrode and separator layers were fabricated by using a spray-printingtechnique developed in our group, which has been described in detailpreviouslyFor separator characterization,the separator was deposited from suspension directly onto spacersand Al foil. For half-cells, the separator was spray-deposited ontoa preformed LTO electrode layer from suspension to coat the anodewith a thickness of 5–22 μm. Full cells were formed byusing the same process as that for half-cells up until the separatorwas deposited. Following this step, 3D-printed polymer masks wereplaced around the electrode–separator-coated spacers (to preventconnection of the second electrode with the cell casing around thecell perimeter), and the counter (LFP-based) electrode was then deposited.Full cell anode and cathode capacity was ensured to be a 1:1 ratioby measuring the spacers after deposition of each electrode and separatorlayer.2.5The mass and thickness of spacers and electrode/separator layerswere measured by using an analytical balance with a readability of0.1 mg  and a micrometer with anaccuracy of ±1 μm and a resolution of 1 μm. Electrode surface morphology was characterizedby scanning electrode microscopy . Cross sectionswere prepared by using a plasma focused ion beam system , and cross-sectional analysis was performed by usingthe built-in SEM and energy-dispersive X-ray spectroscopy (EDX) detectorsand associated software.2 were evacuated and held under vacuumfor 10 h in a surface area analyzer . The sample was then immersed in liquid nitrogen and dosed aliquotsof nitrogen for 37 steps of increasing pressure and then 30 stepsof decreasing pressure. After each aliquot, pressure was equalizedand the measurement performed. Manufacturer supplied software wasused to model the resulting data to provide an estimation of surfacearea and pore width distribution.The porosity was characterized by usingnitrogen gas surface adsorption. Oxide separators deposited onto foilof ∼25 cm–1 heating rate. For each sample, the TGA dishwas coated with a bed of inert oxide powder to protect the Pt cruciblefrom contamination. The Celgard 2500 separator was cut to the sizeof the dish, spray-deposited separators were scraped off the substrate,and powder samples were tested as-supplied. The mass loading of samples,dependent on the sample volume, was an average of ∼4 mg.Thermal properties of separatorsand their constituent componentswere characterized by thermogravimetric analysis  in nitrogen gas from 25 to 900 °C at a 10 °Cmin2.6Cells were assembledaccording to the type of separator, the cell type, and the characterizationtechniques required, including separator-only cells, half-cells, andfull cells for both conventional Celgard and spray-deposited separators.Separator-only cells  were assembled forlinear sweep voltammetry and impedance testing. Unless stated otherwise,components were supplied by MTI Corp. USA.2O3 separators, and combinations thereof weredried in a vacuum for 12 h at 150 °C in a vacuum oven(MTI Corp.) within a glovebox with an Ar atmosphere of <0.1 ppmof H2O and <0.1 ppm of O2. Half-cells, usingCelgard 2500 separators as an example, were assembled with the followingcomponents placed one after another in the center of a stainless steelCR2032 coin cell cup: (1) a stainless steel spacer coated with workingelectrode , (2) 75 μLof LiPF6 EC/DMC = 50/50 (v/v) electrolyte (Sigma-Aldrich),(3) a Celgard 2500 separator , (4) 75 μL of LiPF6 EC/DMC = 50/50(v/v) electrolyte, (5) Li foil ,(6) a stainless steel spacer , (7) a stainless steel wave spring (0.3 mm height),and (8) a stainless steel CR2032 coin cell cap. For Celgard full cells,the Li foil, i.e., component 5, was replaced with a spacer coatedwith a spray-deposited counter electrode. For linear sweep voltammetry,a spacer replaced the spray-deposited electrode, i.e., component 1.For impedance testing, both electrodes, i.e., components 1 and 5,were replaced with spacers.Spray-deposited electrodes,AlFor spray-deposited separator half-cells,150 μL ofelectrolyte was dispensed on top of an electrode–separator-coatedspacer and assembled from step 5. For three-layer spray-depositedfull cells, a spacer coated with a working electrode, separator layer,and counter electrode was placed in the coin cell cup, 150 μLof electrolyte was dispensed on top, and assembly continued from step6. For linear sweep voltammetry, a spacer coated with a separatorlayer was placed in the coin cell cup, 150 μL of electrolytewas dispensed on top, and assembly continued from step 5. For impedancetesting, a spacer coated with a separator layer was placed in thecoin cell cup, 150 μL of electrolyte was dispensed intothe cell, a spacer was placed on top, and assembly was continued fromstep 6.The assembled cells were crimped by using a coin cellcrimper  and cleaned with ethanol after removalfrom the glovebox. Cells were allowed to rest for at least 20 h beforeinitial cycling was performed.2.7Electrochemical characterization was performed by using electrochemicalimpedance spectroscopy ,linear sweep voltammetry , andgalvanostatic cycling .d is the separator thickness, R0 is the effective resistance of the electrolyte, and A is the separator area.The separator-only cell EIS response was measured between 200 kHzand 1 Hz at open circuit voltage, 20 h after construction.Fitting of separator EIS data was performed according to an R-CPEequivalent circuit, where R is a resistor in series with a constantphase element (CPE). The effective resistance of the electrolyte throughthe separator was estimated from the intersection of a best fit ofthe Nyquist plot with the real impedance axis. The effective ionicconductivity of the separator was calculated from the effective resistanceand physical measurements of the separator according to+ at a rate of 1 mV s–1.Linear sweep voltammetry measurementswere collected on a potentiostat/galvanostat/ZRAsystem . After resting for 20h, spacer/separator/Li cells were subjected to a linear voltage sweepbetween 2 and 6 V vs Li/LiLTO–Li coin cells were galvanostaticallycharged and dischargedfor three cycles at each rate between 0.1 and 1 C and cycled fivetimes at each rate between 2 and 30 C before one cycle at 0.1 C. Long-termcycling was performed at 2 C for 400 cycles after rate cycling. Fullcells of LTO–LFP were galvanostatically charged and dischargedbetween 1 and 2.5 V, with two extra cycles at 0.1 C after ratecycling and seven cycles at 2 C to reach a steady state before long-termcycling. Capacity loss per cycle during long-term cycling was calculatedby using linear regression for both half-cells and full cells.33.1To achieve a functional spray-deposited oxide separator, i.e.,controllable and reproducible thickness, acceptable roughness, balanceddensity/porosity, and short resistant, an initial optimization wasperformed for the separator suspension, substrate choice, and depositionparameters.2O3 particles used in the sprayedseparators in water as a function of several common aqueous thickenersand pH modifiers. With no dispersant, the Al2O3 particles had a zeta-potential of 15.9 mV and an intensity-weightedmean hydrodynamic diameter (Z-average mean) of 421nm at pH 7. On decreasing the pH to 4.3, the zeta-potential increasedto 37.6 mV, suggesting greater suspension stability, and the Z-average mean reduced to 204 nm. Although altering thepH induced a more stable dispersion, acidic and alkaline conditionscan corrode Al foil commonly used as one of the current collectors,and large pH modifications were not seen as viable for direct depositionof LIB separators. Carboxymethyl cellulose (CMC) is often favoredin the literature as an aqueous binder due to its relatively low environmentalimpact. Adding 1 wt % CMC reduced the particle Z-averagemean to 220 nm. However, a zeta-potential magnitude of only −19.8mV suggested only limited stabilization. Alternatively, poly(acrylicacid) (PAA) improved the zeta-potential magnitude to −27.0mV and provided an acceptable Z-average mean of 225 nmafter 30 min. The magnitude of polydispersity in terms of additiveswas in the order none > pH > CMC > PAA, i.e., a narrowingof the particlediameter distribution and the degree of agglomeration from left toright. Therefore, on the basis of the similarity of the Z-average mean and the polydispersity index (PDI), but the improved(reduced) number mean diameter and (increased magnitude) zeta-potentialover CMC, PAA was selected for further experimentation.2O3 tended to flake off in small circular areas,likely caused by tensile capillary forces during drying. 2O3 particles maintained adhesion to the electrode layer andsubstrate.Styrene–butadiene rubber(SBR) was selected as a model binder due to its known compatibilityand widespread use in aqueous manufacture of LIB anodes. SBR emulsionis insoluble in water upon drying, which reduced the risk of suspensionof previously deposited layers in subsequent droplets. Suspensionof previously deposited material could have caused short circuitsby generating pathways of conducting material through the separator layer during deposition. Stainless steel circular spacersfrom 2032 coin cells were selected as substrates to facilitate simple,punch-less assembly into standard cells. In assembling coin half-cellswith an electrode and separator deposited on thin Al foil, an Li chipof diameter smaller than the separator and with exact alignment mustbe used to avoid short circuits. Alternatively, the use of the spacerraised the contact point of the Li foil into the middle of the celland retained the protective conformal coating of the separator layer,removing the chance of contact between the Li foil and the cell cupor working electrode, as shown in 49A separator with a uniform structureand low roughness was desired . Separators with excessively heterogeneous structures willresult in a heterogeneous current density distribution, leading tolocalized heating and degradation of the electrode and separator,which could lead to physical contact and short circuits between theanode and cathode.2O3 separator deposited from water alone (W) onto an LFP electrode,while 2O3 deposited from water with20 vol % IPA (IPA), which had a qualitatively smoother surface. Someagglomerates persisted, most with a diameter of 1–3 μm,up to a maximum of ∼4 μm.2O3 (W)and Al2O3 (IPA) are shown in 2O3 separator deposited from water alonehad a lower areal density and greater thickness, which were likelydue to the increased roughness. Droplets of water commonly depositsuspended particles in heterogeneous patterns due to a weak Marangoniflow during evaporation.50 A mixture oflow and high boiling point liquids can suppress the effect, whichcould explain the improvement gained by including 20 vol % IPA inthe suspension. Overall, the physical properties of Al2O3 deposited from water with 20 vol % IPA were favoredover that of Al2O3 deposited from water alone.Surface roughness measurements of Al2O3 deposited from water alone could not charge due to shortcircuits between the anode and cathode caused by significant variationsin separator thickness. In contrast, the preferred Al2O3 separator deposited from water with 20 vol % IPA mitigatedany short circuits and was chosen for subsequent in-depth comparisonwith a commercial polymer separator.Three LFP half-cells with sprayed separators fromeach solventmixture were assembled. The LFP half-cells with Al3.22O3 separator physical properties, thermalstability, wettability, impedance, and electrochemical performancein LTO-based (anode) half-cells were investigated. A Celgard 2500PP monolayer separator was used for comparative purposes.Given their encouragingproof-of-concept demonstration when sprayed onto a cathode, spray-depositedAl2O3 particles, PAA, SBR in a 94:1:5solid ratio, and 80:20 vol % water:IPA solvent were spray-depositedonto spacers precoated with an ∼14 μm LTO-basedelectrode. 2O3 electrode/separator composites, where the approximately 5, 11, and22 μm thick Al2O3 separator layersare termed Al2O3 (5 μm), Al2O3 (11 μm), and Al2O3 (22 μm) where needed, respectively. 2O3 separators with Celgard 2500. The relatively high densityof Al2O3 (3.95 g cm–3) resultedin a higher mass per unit volume (PP density assumed at 0.9 g cm–3). To obtain the same areal density, or better, thanCelgard 2500, the Al2O3 separator should be∼7 μm thick, i.e., approximately a third to aquarter of the Celgard thickness, assuming similar porosity. The Al2O3 separator porosity was estimated as 58 ±3, 59 ± 5, and 60 ± 10% for 22, 11, and 5 μm thickness,respectively, slightly higher than that of Celgard 2500.52Suspensions comprising of50 nm Al2O3 separators. The Al2O3 separatorcoated conformally onto the underlying sprayed LTO-based layer. From 2O3 (5 μm) had an estimated deviation fromthe mean line of ±1 μm, while Al2O3 (22 μm) was slightly rougher at ∼±2 μm.Al2O3 (5 μm) had a few large poresof diameters of 200–300 nm, while Al2O3 (22 μm) had pores with diameters up to ∼400 nm.2O3 separator layers on Al foil. A separator layer fabricated from 300 nmAl2O3 particles was manufactured to provideadditional comparison. A separator consisting of 300 nm Al2O3 was unable to hold charge when tested in LFPhalf-cells, suggesting that particle size (or pore diameter) was likelya critical factor when successfully fabricating spray-deposited separators.Nitrogen gas surface adsorption (BET) was usedto investigate thespecific surface area and mesoporosity of Al2O3 and 300 nm Al2O3 are shown in 2O3 displayed a hybrid shape between a classical type IIand type IVa behavior,53 comprising adsorption–desorptionhysteresis.The BET isotherms of the Celgard and separators fabricated by using50 nm Alp/p° tends to 1. A type IVa isotherm is typical of mesoporous solidsand is generated due to pore condensation, where the adsorbing gascondenses to a liquidlike state at a pressure below p°. However, a final saturation plateau, typical of a type IVa,was not observed, indicating a structure with both mesoporosity andmacroporosity.53According to IUPAC, type II behavior is typicalof nonporous ormacroporous solids, as multilayer formation of adsorbed nitrogen appearsto increase without limit as 54 The distinctive isothermof the separator using 50 nm Al2O3 likely representsa porous powder bed with little or no pore blocking, similar to isothermsof nonporous nanoparticles.57H1 hysteresis isusually associated with a narrow range of uniformpores. However, it has also been attributed to spherical/cylindricalpore structures with wide openings through which there is immediateaccess to the vapor phase.2O3 SSA = 9.4 m2 g–1 with no distinctivehysteresis loop, indicating little or no mesoporosity. For the separatorusing 50 nm Al2O3 SSA = 44.4 m2 g–1, while for Celgard SSA = 41.3 m2 g–1. The separator using 50 nm Al2O3 had an estimated density of 3.77 g cm–3, ∼4times greater than that of Celgard at 0.9 g cm–3, so the difference in the surface area per unit volume was muchhigher than The specific surface area(SSA) was calculated from the BET isothermsand is shown in 2O3 had little mesoporosity, represented by a low cumulative mesoporevolume of 0.01 cm3 g–1. The cumulativemesopore volume of the Celgard and the separator using 50 nm Al2O3 were 0.28 and 0.20 cm3 g–1, respectively. The Celgard separator showed a narrower pore widthdistribution (PWD) with an average pore width of 22 nm. The separatorusing 50 nm Al2O3 had an average pore widthof 17 nm and a broader PWD in the mesopore range, with minimumpore widths of <10 nm.Vsep is the volume of the separator, and Vsolid, Vmeso, and Vmacro are the volumes occupied by the solid,mesopore, and macropore components of the separator, respectively,as shown for the Celgard and Al2O3 separatorsin 2O3 and Celgard. Celgard had a significantly larger pore volumeper gram of material (Vpore) of 1.36 cm3 g–1 compared with that of Al2O3 at 0.34 cm3g–1, displaying an advantage of polymer-based separators. The ratioof mesopores to macropores was 0.59:0.41 for the separator using 50nm Al2O3, compared with 0.21:0.79 for the Celgardseparator. The proportion of mesopores in the separator using 50 nmAl2O3, assuming a monomodal pore distribution,suggested a fine-scale average pore width near that of the averageparticle diameter of ∼50 nm.To estimate the volume of each componentin the separators, a simplerelation was used:2O3 and 50 nm Al2O3 powders, and the spray-deposited separator using50 nm Al2O3 between 25 and 900 °C. 60 The first peak (2% mass loss at 50–130 °C) was simplyevaporation of absorbed water. Stage 1 of polymer decomposition (massloss of 11% at 160–280 °C with a peak at 240 °C)was attributed to reversible anhydride formation.61 Stage 2 (mass loss of 22% with a peak at 330 °C)was due to decarboxylation of the anhydride and release of CO2. Finally, a broad peak, related to chain scission of thepolymer, occurred above 350 °C, and led to a further massloss of 55%. A mass of 10% carbonaceous residue remained above 530 °C.Styrene–butadiene rubber decomposed in a single step between320 and 520 °C with a DTG peak at 460 °C and lefta residual mass of 4.5%. The small peak at 150 °C wasmost likely due to volatilization of processing additives.622O3 powder in 2O3 powderreduced in mass gradually to 95.3% at 900 °C. 2O3 powders lost mass below 100 °C, which was attributedto evaporation of physically adsorbed water. In terms of normalizedmass loss, both powders lost 35% of their total mass loss below 100 °Cand followed similar trends up to 900 °C. An equilibriumbetween aluminum oxide–hydroxides and Al2O3 occurs at ∼370 °C (below 200 bar), and this maybe evident in the DTG of 50 nm Al2O3 and thenormalized mass loss of 300 nm Al2O3.63 Therefore, the slight differences between theAl2O3 powders above 150 °C weremost likely due to slightly different hydroxide surface species, andoverall the mass loss of 50 nm Al2O3 powderwas as expected.The 300 nm Al2O3 separatorcomprised a94:1:5 composite of 50 nm Al2O3 particles, PAAand SBR, and displayed similar mass loss behavior to 50 nm Al2O3 powder until 300 °C, along withsome underlying degradation of the small quantity of the PAA consistentwith 2O3, the Al2O3 surface species, and/orone or more of the polymer components at lower temperature. At 900 °C,the 50 nm Al2O3 powder had 95.3% of originalmass remaining, while the separator using 50 nm Al2O3 retained 91.2%. This difference was slightly less than expectedfrom the 6 wt % of PAA and SBR combined in the oxide separator butlies within the likely error of the technique.The optimized Al65 A high contact angle (poor wetting)is usually expressed as a relatively high electrolyte resistance acrossthe separator and unfilled pores may result. 2O3, with contact angles of 64.9 ± 5.5°, 35.8 ±1.2°, and ∼0°, respectively. A coating of 50 nm Al2O3 only 5 μm thick reduced the contactangle of the spacer from 36° to ∼0°.The performance of separators reliessignificantly on their wettability with the electrolyte.3.32O3 separators were fabricatedwith 50 nm Al2O3. The equivalent series resistance,determined by modeling the impedance response by using –2 for 5, 11, and 22 μmthick Al2O3 separators and a Celgard separator. 2O3 separators.The effective ionic conductivity of the Al2O3 separators was estimated as 0.98 ± 0.05 mS cm–1, roughly double that of Celgard 2500 of 0.50 mS cm–1. The higher ionic conductivity and lower equivalent series resistancewere attributed to the superior wettability of the Al2O3 separator.6 EC/DMC 50/50 v/v electrolyte, as shown in –2 upto 4 V and then a continual rise in current density after ∼4.5V. The small peak in current density at 4.2–4.3 V was due tothe oxidation of water .67 The current density of the Al2O3 separatorcell up to 4 V was ∼2.4 μA cm–2, slightlyhigher than that of the Celgard separator cell.The stability of the separatorsunder electrochemical conditions was studied by using linear sweepvoltammetry on spacer/separator/spacer coin cells in 1 M LiPF2O3 separator cell was relativelylarge at 11.6 μA cm–2 over 4.2–4.6V and was likely due to the difficulty in removing excess water fromthe hydrophilic PAA dispersant and high surface area Al2O3 particles. The current density of the Al2O3 separator cells reduced to 7.5 μA cm–2 at ∼5 V, close to 4.5 μA cm–2 of the Celgard separator cells. Above 5.1 V, the currentdensity for the Al2O3 separator was ∼2times that of Celgard. Overall, the Al2O3 separatordisplayed adequate voltage stability up to 5 V, but there may be roomfor further improvement with the use of more stable polymer bindersand improved drying procedures.The water oxidationpeak for the Al2O3 (5 μm),Al2O3 (22 μm), and Celgard wereinvestigated between 0.1 and 30 C, as shown in 2O3 separators produced consistent capacities witha maximum difference in capacity of 2% at 30 C. Between 0.1 and 10C, all types of separators facilitated an initial discharge capacityof 170 ± 2 mAh g–1, with the largest differencein capacity of only 2.8% between the Al2O3 separators(136.2 ± 1.0 mAh g–1) and the Celgard separator(132.5 ± 1.9 mAh g–1) at 10 C. However, athigher rates, Al2O3 (5 μm) outperformedCelgard by 8.1% with a capacity of 125.5 ± 1.0 mAh g–1 compared with 116.1 ± 2.3 mAh g–1 at 20 Cand by 27.8% with a capacity of 118.3 ± 1.2 mAh g–1 compared with 92.5 ± 6.4 mAh g–1 at 30 C.Similarly, Al2O3 (22 μm) was within±2 mAh g–1 of Al2O3 (5 μm)at all tested C rates. The Al2O3 separatorsevidenced a relatively low standard deviation between cells, whicharose due to their excellent wetting and fine-scale microstructuraluniformity.The performance of LTO/separator/Lihalf-cells containing Al–1 at 2C on the first cycle (assumed as 100% capacity). After 400 cycles,Al2O3 (5 μm), Al2O3 (22 μm), and Celgard retained capacities of97.5 ± 0.9, 96.0 ± 0.4, and 91.5 ± 1.4%, witha capacity loss per cycle of 0.0068, 0.0093, and 0.0210%, respectively.The 2–3 times greater capacity retention for cells using Al2O3 separators was again ascribed to their significantlylower impedance and improved wetting.Cycle stability was assessed at 2 C over 400 cycles,as shown in 2O3 separator provided consistent, highperformance enabled by the excellent conformal coating and even coverage.It is worth recallingthat the sprayed electrodes in the half-cellstested here were not calendared. Nonetheless, even the 5 μmthick Al3.445 and (2) conformal coating of presprayed substrates. Combining theseattributes could enable coating of LIBs onto a variety of substrates,including nonflat geometries.Previous work has shown two advantageous propertiesof spray deposition: (1) the ability to process multiple layers ina continuous operation, due to “on-the-fly” changingof deposition conditions and incremental drying of layers,2O3 separatorwas deposited onto electrode #1, (e) 3D printed masks were positionedover the LTO/Al2O3-coated spacers, and (f) electrode#2 was deposited onto masked LTO/Al2O3. 2O3-coated spacers, leadingto short circuits on cell assembly, as shown in the schematic diagramin 2O3 separator fullcell were examined by SEM imaging and EDX analysis, as shown in 2O3 separator, Fe (purple)the LFP and stainless steel spacer, and C (green) in both the LTO(anode) and LFP (cathode). In this case the LTO-based layer had slightlylarger variations in thickness of up to 4 μm. Nonetheless,the 10 μm Al2O3 separator layerconformally adopted the surface topography. The LTO/separator interfacehad excellent contact while the separator/LFP interface had some poresof 2–3 μm diameter.Cross sections of an Al2O3 separator full cells and conventional full cellsusing a polypropylene Celgard 2500 separator. The cells were fabricatedwith an LFP:LTO capacity ratio of 1:1 according to 0.1 C capacitiesmeasured previously, and all electrodes were manufactured by usingspray deposition. The Celgard full cells had a capacity of 137.2 ±0.4 mAh g–1 at 0.1 C, while those with a Al2O3 separator had a similar capacity of 136.3 ±0.8 mAh g–1. Between 5 and 30 C, the Celgard 2500separator enabled a greater capacity at the start of each set of fivecycles, but capacity reduced each cycle. On the other hand, the cellsusing a Al2O3 separator increased in capacityduring each set of cycles until capacities were almost equal. After30 C, Celgard and Al2O3 separator full cellsrecovered to capacities of 134.8 ± 0.5 and 136.2 ± 0.8 mAhg–1 at 0.1 C, respectively.2O3 separatorfull cells had initial capacities of 108.6 ± 1.5 and 109.0 ±1.3 mAh g–1, reducing to 86.0 ± 1.4 and 104.9± 1.9 mAh g–1 after 400 cycles. The averageCoulombic efficiency of each cell was 99.8%. After 400 cycles, Celgardand Al2O3 separator full cells had final capacitiesof 79.2 ± 1.3 and 96.3 ± 1.8% and a capacity loss per cycleof 0.0354 and 0.0106%, respectively.Cycle stability was examined over 400 cycles at2 C directly afterthe rate testing. Celgard and Al2O3 separator after 400 cycles, 0.0106%,was slightly larger than 5 and 22 μm thick Al2O3 separator half-cells with 0.0068 and 0.0093%, respectively.However, the capacity of the LFP was intrinsically less stable thanthe LTO under long-term cycling, so a poorer capacity retention inthe LFP/LTO full cells was expected.Relating the degradationof Celgard full cell capacity to thatof LTO/Celgard half-cells 10, the c2O3-based separatorsperformed at least as well as the commercial Celgard polymer separator,but with notable improvements in cell capacity retention in integratedfull cells and high C-rate response in LTO half-cells. A significantreduction in separator thickness was evidenced for the integratedfull cell compared to previous literature (from 200 to 5−10 μm).41 Several optimizations have been suggested tocontribute to the effectiveness of the sprayed Al2O3 separators. The mixed carrier liquid improved uniformityof deposited particles by influencing droplet evaporation, the smallaverage separator pore size (50 nm) blocked penetration of conductingmaterial from the second electrode layer or Li metal, and the combinationof high substrate temperature and the addition of SBR emulsion stabilizedlayers of separator material upon deposition. The Al2O3 separator in the full cell assembly was thinner than theCelgard separator, which contributed to the difference in capacityretention partly. However, as shown in 2O3 separatorwith similar thickness to the Celgard separator displayed significantlybetter capacity retention in a half-cell configuration. Therefore,it is suggested that the principal reason for the slower degradationof the Al2O3 separator containing cells is theirintrinsic improved wetting of the electrolyte .Although this undermines some of the single operation benefits ofthe current arrangements, it is relatively simple and effective toimplement. For convenience, the spray deposition process is normallyoperated with aqueous based suspensions/solvents, although it canalso be successfully implemented in an inert, dry atmosphere by usingorganic solvents for more air/water sensitive active materials. Solidelectrolytes have also been successfully codeposited for hybrid solidstate cathodes and separators based on ion-conducting polymers andinorganic electrolytes.69 The ability to controlsurface topography to mitigate separator defects is also useful aslarger area electrodes and one-step cells are fabricated in scale-upinvestigations. In this ongoing work, calendering is also used toadd the top-layer or “upper” current collector withsufficient cycle life stability and low impedance, for example, bywarm rolling of an Al foil precoated with a thin layer of thermopolymeronto the three-layer assembly.70 However,it should be noted that calendering of multilayers  may develop a tendency for short circuitsto form across the separator should there be regions of unusuallyhigh surface roughness.The spray depositionroute can also be used to fabricate successfullyLIB anodes and cathodes from a variety of different active materials,including those with particles diameters of up to 20 μm.When using larger diameter particles for spray-deposited three-layerfull cells in a single operation, i.e., similar to the LFP/Al42O3-based separators enabledstable, high rate, and long-term cycling of LTO/Li and LFP/LTO cells.Initial optimization of dispersant, binder, and solvent for spray-depositedseparators assessed suspension stability and deposition properties.An optimized Al2O3-based separator contained1 wt % PAA and 5 wt % SBR and was deposited from an aqueous suspensioncontaining 20 vol % IPA. Al2O3 separator surfacemorphology was more uniform with a 20 vol % IPA solvent than withwater alone, attributed to a greater Marangoni flow during dropletdrying that more efficiently distributed the 50 nm particles intoa relatively uniform layer.Spray-deposited Al2O3 separatorsbetween 5 and 22 μmthick had consistent and similar porosity of ∼58%, excellentwettability (contact angle ∼0°), thermal stability toat least 180 °C, adequate electrochemical stability, andhigh ionic conductivity of 0.98 mS cm–1, doublethat of a commercial PP separator. Furthermore, LTO half-cells with5 and 22 μm Al2O3 separators showed improvedrate performance and excellent cycle stability (0.007–0.009%capacity fade per cycle) compared with Celgard 2500 (0.021% capacityfade per cycle) due to the improved compatibility of the electrolyteand separator.Al2O3 separator, the first of its kind, showedsimilar rate performance to a conventional Celgard 2500 full cellwith a capacity of ∼50 mAh g–1 at 30 C. Uponlong-term cycling at 2 C, the Al2O3 separatorfull cells displayed excellent capacity retention per cycle of 0.0106%over 400 cycles compared with 0.0354% for the full cells with a Celgardseparator. After 400 cycles, Al2O3 and Celgardseparator full cells retained ∼96 and ∼79% of theirinitial 2 C capacity due to the lower impedance of the Al2O3 separator in the full cell assembly.A full cell manufactured in a sequential sprayprocess with a 10 μmAlFurtheravenues to improve the oxide separator performance includeselecting particles with lower density, improving the separator dryingstep, and using a dispersant with better dispersive and thermal propertiesthan PAA. Nonetheless, this work showed that a single deposition techniquecan be used to process both electrodes and separators for Li-ion batteriesand that cells manufactured by using this assembly technique can havesimilar or greater performance than those assembled with a conventionalPP separator.


RAD51AP1 and RAD54L further sensitizes human cancer cell lines to treatment with olaparib, a Poly (adenosine 5′-diphosphate-ribose) polymerase inhibitor, to the DNA inter-strand crosslinking agent mitomycin C, and to hydroxyurea, which induces DNA replication stress. We also show that the RAD54L paralog RAD54B compensates for RAD54L deficiency, although, surprisingly, less extensively than RAD51AP1. These results, for the first time, delineate RAD51AP1- and RAD54L-dependent sub-pathways and will guide the development of inhibitors that target HR stimulators of strand invasion.Homologous recombination DNA repair (HR) is a complex DNA damage repair pathway and an attractive target of inhibition in anti-cancer therapy. To help guide the development of efficient HR inhibitors, it is critical to identify compensatory HR sub-pathways. In this study, we describe a novel synthetic interaction between RAD51AP1 and RAD54L, two structurally unrelated proteins that function downstream of the RAD51 recombinase in HR. We show that concomitant deletion of  Homologous recombination (HR) is an essential DNA damage repair pathway critical for genome stability and tumor suppression. HR is altered in many different tumor types and has become an attractive target for the development of new anti-cancer therapies . AccuratRAD51AP1 and RAD54L are two RAD51-associated proteins that co-operate with the RAD51 filament in the capture of the DNA donor molecule and in formation of the D-loop . RAD51APThe RAD51AP1 and RAD54L proteins are unrelated structurally, but both upregulate RAD51 activity by enhancing the ability of the RAD51 filament to engage with the homologous double-stranded (ds)DNA donor  and in strand invasion . In thesThe mild phenotype of RAD54L-deficient human cells has been attributed to the existence of RAD54B, a RAD54L paralog . Human RRAD54L in human cells is compensated for by the RAD51AP1 protein. We show that simultaneous deletion of the RAD54L and RAD51AP1 genes further sensitizes human cancer cell lines to treatment with the DNA inter-strand crosslinking agent mitomycin C (MMC), to prolonged exposure to replication stalling by hydroxyurea (HU), and to Poly (adenosine 5′-diphosphate-ribose) polymerase inhibition (PARPi). We also show that the RAD54L paralog RAD54B can substitute for RAD54L activity, but, surprisingly, to a lesser degree than RAD51AP1. Based on these results, we conclude that the activities of RAD51AP1 and RAD54L can underpin two major, mechanistically distinct routes for the completion of HR in human cells.In this study, we show that loss of RAD51AP1 and RAD54L, we generated RAD54L/RAD51AP1 double knockout (DKO) HeLa cell lines and compared the phenotypes of these DKO cells to HeLa cells deleted for either RAD51AP1 or RAD54L  and detected no significant differences in population doubling times . In fracRAD51AP1 and RAD54L single KO cells are moderately sensitized to the cytotoxic effects of MMC , and combined loss of both RAD51AP1 and RAD54L synergistically sensitized HeLa cells to olaparib , and combined loss of both RAD51AP1 and RAD54L synergistically sensitized Hs578T cells to olaparib exposure , possibly related to the role of RAD54L in catalyzing fork regression  and RAD54L single KO cells . These results show that replication stress leads to genome instability most prominently in RAD54L/RAD51AP1 DKO cells, which also show the most pronounced defect in fork restart.Next, we tested the consequences of HU-induced replication stress to cells with impaired replication restart. To this end, we determined chromatid gaps and breaks, and complex chromosome aberrations  in HeLa, single KO and  with HU . ExposurRAD54L/RAD51AP1 DKO cells , and at 4 and 20 h after release from a 2-h treatment with 0.5 µM MMC. RAD54B was present in anti-eGFP complexes from both untreated and MMC-treated cells ; RAD54L/RAD54B DKO HeLa cells are less sensitive to fork degradation.As observed earlier , CldU tred cells . In the RAD54L/RAD51AP1 and RAD54L/RAD54B DKO cells. Surprisingly, treatment with olaparib decreased the survival of both RAD54L/RAD51AP1 and RAD54L/RAD54B DKO cells to similar extent , the compensatory activity of RAD51AP1 is greater than that of the RAD54L paralog RAD54B . After tRAD54L KO cells with RAD54B knockdown show increased sensitivity to MMC but RAD54B-depleted RAD54L/RAD51AP1 double KO cells do not, this may be further evidence of RAD54B functioning in conjuncture with RAD51AP1. Nonetheless, RAD54L/RAD51AP1 double KO cells are significantly more sensitive to MMC than RAD54L/RAD54B double KO cells, which argues that RAD51AP1 has additional function(s) aside from working with RAD54B in repairing MMC-induced DNA damage. This could be together with another and yet to be identified translocase, or within transcriptionally active, decondensed regions of the genome in which RAD51AP1 was shown to promote HR , evidence of physical interaction and functional cooperation between RAD54B and RAD51 , and of omote HR .RAD54L/RAD51AP1 and RAD54L/RAD54B DKO cells  and RAD54L KO HeLa cells, that we described previously  KO plasmids each containing one of two different sgRNAs -A and sgRNA (54)-B; sgRNA (54B)-A and sgRNA (54B)-B; RAD54L; sc-403794-NIC-2 for RAD54B) and used to transfect single KO cells as described . RAD54L and RAD54B genomic DNA sequences were amplified by PCR using primer pairs flanking the sgRNA target sites (E. coli. Plasmid DNA was prepared using ZR Plasmid Miniprep-Classic Kit (Zymo Research) and submitted for sequencing. For each KO cell line, 15–20 individually cloned amplicons were analyzed by Sanger sequencing  KO plasmids described above  each encoding Cas9 nuclease and one of three different RAD51AP1-specific gRNAs targeting exons 2, 3 or 5/6 , followed by transfer into pLentiCMV/TO DEST#2  were performed, as described . To asseWestern blot analyses were performed according to our standard protocols . The folCell cycle analysis and flow cytometry were performed as described , except 5 cells were seeded in 6-well tissue culture plates and incubated at 37°C for 24 h before exposure to 4 mM hydroxyurea  in regular growth medium for 5 h, as described  at room temperature for 10 min. Images were acquired using Zeiss Axio-Imager.Z2 microscope equipped with Zen Blue software (Carl Zeiss Microscopy) using a 63× oil objective. One hundred metaphases were assessed per sample.For the assessment of chromosomal aberrations, 2 × 10escribed . After Hescribed . Cells w5 cell/ml and processed for fiber spreading as described (https://imagej.net).DNA replication progression was assessed by the single-molecule DNA fiber assay and essentially as described previously . Brieflyescribed . In a moescribed . Images RAD51AP1 single or RAD54L/RAD51AP1 double KO cells were transfected with peGFP-C1 or peGFP-RAD51AP1 and Lipofectamine2000 (Invitrogen). Twenty-four hours after transfection, cells were subjected to a medium change, treated with 0.5 µM MMC for 2 h or 10 µM olaparib for 24 h. Cells were washed twice with warm PBS, fresh medium was added, and cells were incubated for the times indicated. Cells were lysed in chilled lysis buffer containing 50 mM Tris-HCl, pH 7.5, 300 mM NaCl, and 0.5% NP-40, supplemented with EDTA-free protease inhibitor cocktail (Roche) and HALT phosphatase inhibitors (Thermo Fisher Scientific). For 1.5 × 106 cells, 25 μl of GFP-Trap® dynabeads (ChromoTek) were used to trap the ectopic proteins. Protein lysates were diluted to 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, and 0.1 unit DNase I (Gold Biotechnology) per µg protein, and mixed with the equilibrated beads at 4°C for 1 h with gentle rotation. The GFP-Trap® dynabeads were washed three times with 500 µl binding buffer, bound protein complexes were eluted in 40 µl 2× LDS buffer (Thermo Fisher Scientific) and fractionated on 7% NuPAGE Tris-Acetate gels (Thermo Fisher Scientific) and for Western blot analysis.The peGFP-RAD51AP1 expression vector is based on peGFP-C1 (Clontech) and has been described previously . RAD51AP6-RAD51AP1-FLAG in E. coli and its purification were carried out as described previously (Expression of (His)eviously . RAD54B eviously .6-RAD51AP1-FLAG (100 nM) or no protein were incubated with the equilibrated resin at 4°C for 1 h. Unbound protein was removed by centrifugation at 3,000 rpm for 3 min RAD54B (100 nM) was added to the washed resin in 100 µl binding buffer and incubated at 4°C for 1 h with gentle agitation in the presence of DNase I (1 U/µg protein). Supernatant was removed and RAD54B (100 nM) was added in a final volume of 100 µl and further incubated for 1 h at 4°C. The resin was washed three times in 200 µl binding buffer each, and bound protein was eluted in binding buffer containing 150 ng/μl 3× FLAG peptide (Sigma). Eluted protein was fractionated by 10% SDS-PAGE, transferred onto a PVDF membrane and detected by Western blot analysis.FLAG pull-downs were performed essentially as described . Brieflyp ≤ 0.05 was considered significant.GraphPad Prism 9 software was used to perform statistical analyses on data obtained from two to five independent experiments, as indicated. To assess statistical significance two-way or one-way ANOVA tests were performed.