The as-prepared material exhibits excellent cycling stability and outstanding rate performance. Charge/discharge capacities of 266 mA h g−1 at 0.100 A g−1 and 200 mA h g−1 at 1.000 A g−1 are reached for Li2TiO3–coke. A cycling life-time test shows that Li2TiO3–coke gives a specific capacity of 264 mA h g−1 at 0.300 A g−1 and a capacity retention of 92% after 1000 cycles of charge/discharge.Anode material Li 2TiO3–coke was prepared and tested for lithium-ion batteries. The as-prepared material exhibits excellent cycling stability and outstanding rate performance.Anode material Li However, an anode material with high rate capability and high energy density is still not available. Presently, graphite is widely employed as an anode material in lithium ion batteries.3,4 However, graphite is not high rate capable.5 The spinel lithium titanate (Li4Ti5O12) is a high rate capable anode material, which has an almost zero-strain.6 The reduced volume change in lithium ion intercalation/deintercalation guarantees its high reversibility7,8 and long cycling life-time. The high lithium insertion potential (about 1.55 V vs. Li+/Li) could effectively avoid lithium plating over the anode in the charge process even at low temperature or high current density.9,10 Hence, regarding safety issues, Li4Ti5O12 represents a much better option than graphite. However, except the advantages of the spinel Li4Ti5O12, the high lithium ion insertion potential and the relatively low theoretical capacity (175 mA h g−1) of Li4Ti5O12 lead to a lower cell energy density, which limits the applications of Li4Ti5O12 in areas that need the batteries having higher energy density. Hence, searching for high performance anode material that has high specific capacity, high rate capability, long cycling life-time, and high safety is still the goal of researchers in battery investigation.Except for high energy density, long cycling life-time, and high safety, the application of rechargeable batteries in electric vehicles also requires that the batteries have high power density. In the manufacture of high-power density batteries, high rate capable cathode and anode materials are required.2TiO3 has a layer structure and a three-dimensional lithium ion diffusion network.11 In recent years, Li2TiO3 is often added into cathode materials, such as LiNix−y1−CoxMnyO2  and LiCoO2, to improve their rate capability and stability.12,13 Although, Li2TiO3 is a good lithium ion conductor, it is almost an electronic insulator. In order to making use of Li2TiO3 as an anode material, one must improve the electronic conductivity of Li2TiO3. Investigators usually add carbon materials14–18 into metal oxide electrode materials or directly prepare the metal oxide electrode materials in metal foam19,20 to improve their electronic conductivity. Since the electronic conductivity of Li2TiO3 is very poor, we choose to support relatively less amount of Li2TiO3 over relatively larger amount of coke to reach good electronic conductivity. Because petroleum coke has medium electronic conductivity and pores that lithium ions could easily diffuse in and out. The coke itself is a potential high rate anode material. In the present work, by making use of the good lithium ion conductivity of Li2TiO3 and the electronic conductivity of coke, we intend to use lithium hydroxide, TiO2, and petroleum coke to prepare a practically useful high rate anode material Li2TiO3–coke (LTOC) for lithium ion batteries.Monoclinic Li2TiO3 was prepared by the following method. TiO2 (5.6724 g), LiOH·H2O (5.9602 g), and H2O (50.0 ml) were mixed and milled at 180 rpm for 6 h to obtain a slurry. The slurry was transferred into a beaker. The milling container and beads were washed three times with DI water (10 ml for each time) and the washing out mixture was combined with the previous slurry to obtain a diluted slurry. Adding DI water to the diluted slurry to a total volume of 100 ml for spray dry. After spray dry, a powder was obtained, and then the powder was calcined at 850 °C for 4 h in air to obtain Li2TiO3.Li−1 in argon, then heated at 1200 °C for 6 h, and then cooled down to room temperature in argon to obtain the preheated coke, which was used as anode material and precursor for Li2TiO3–coke (LTOC) preparation.The oil coke was placed into a tube furnace and heated from room temperature to 1200 °C at a heating rate of 5 °C min2 (1.7067 g), LiOH·H2O (1.7933 g), surfactant (([n-C16H31(CH3)3N]CO3CH3) writing as QA, 0.9457 g), and distilled water (50.0 ml) were added into a ball mill container and milled at 180 rpm for 6 h to obtain a slurry. The slurry was transferred into a beaker. The mill container and beads were washed three times with DI water (10 ml for each time) and the washing out mixture was combined with the previous slurry to obtain a diluted slurry. Adding DI water to the diluted slurry to a total volume of 100 ml for spray dry. After spray dry, a powder was obtained, and then the powder was calcined at 850 °C for 4 h in argon to obtain LTOC.The LTOC was prepared through the following method. The preheated coke (6.5000 g), TiON-methyl-2-pyrrolidone), polyvinylidene fluoride (PVDF) binder (dissolved in N-methyl-2-pyrrolidone) were mixed according to the mass ratio of 85 : 10 : 1.5 : 3.5 to prepare a paste. The paste was coated on a copper foil (the thickness of the anode coating layer is 30 μm) with an applicator (frame-type), and then dried in a vacuum oven at 120 °C for 2 h to obtain the anode plate. The dried anode plate was cut into cycles with a diameter of 14 mm. The anode cycles were farther dried in a tubular furnace (filled with argon) at 140 °C for 5 hours, and then transferred into an argon filled glove box (Mikrouna) in argon atmospheres. The anode cycles together with the separator (PP), the lithium metal cycles (as the counter electrode), and the electrolyte solution  were used to assemble coin-type cells.The as-prepared anode material, conductive carbon black, carbon nanotubes . The X-ray Photoelectron Spectroscopy (XPS) measurements of samples were conducted on a XPS spectroscopy meter . The specific surface areas of the materials were measured by BET method .The crystal structure of the samples was analyzed by X-ray diffraction spectroscopy . The surface morphology and microstructure of the samples were characterized by SEM  and TEM . Laser Raman measurements were conducted on a laser Raman spectroscopy meter with an excitation wavelength of 532 nm  at a scan rate of 0.1 mV s−1 within 0.01–2.0 V. The EIS measurement of cells was also carried out on the electrochemical workstation . All tests were carried out at room temperature.The discharge/charge tests of the cells were carried out on a NEWARE CT-3008-5 V-10 mA system. The voltage window is 0.01–2.0 V. In the rate performance test, the cells were tested at current densities 0.033, 0.100, 0.200, 0.300, 0.400, 0.500, 0.600, 0.700, 0.800, 0.900, 1.000, 2.000, 3.000, 4.000, 5.000, and 0.100 A g−1 and the G band at 1580 cm−1). The ratio of the D band intensity (ID) to the G band intensity (IG) is 0.91 (ID/IG = 0.91), which indicates that the preheated oil coke is not graphitized  with lattice parameters a = 5.069 Å, b = 8.799 Å, and c = 9.759 Å. Hence, the pristine Li2TiO3 is monoclinic Li2TiO3. In LTOC, except from the broad peak between 20° and 30° (assigned to amorphous coke), there are peaks that exactly match that of monoclinic Li2TiO3. Hence, monoclinic Li2TiO3 is formed in LTOC.The XRD analysis shows that the coke has only a broad peak between 20° and 30° . The bro2TiO3, coke, and LTOC are given in 2TiO3 shows that the average size of Li2TiO3 crystals is about 1 μm. Petroleum coke has a particle size distribution from nanometers to tens of micrometers. In LTOC  and (111) planes, respectively (the inset of image (h) gives the selected area electron diffraction pattern of a Li2TiO3 crystal). The results are consistent with that observed in the SEM characterization.The high resolution TEM images  reveal t4+ ions reduced to Ti3+ ions at high calcination temperature (850 °C). However, the major part of titanium ions is still T4+ ions . The CV curves of coke  of Li2TiO3 in the oxidation and reduction processes are 5.66 × 10−8 and 1.249 × 10−8 cm2 s−1, respectively .26The lithium ion diffusion coefficients  values (about 2.0 Ω). The Rs of LTOC (29.0 Ω) is smaller than that of coke (34.3 Ω), but close to that of Li2TiO3 (27.7 Ω). The results indicate that the Li+ diffusion rate in LTOC should be higher than in coke, but lower than in Li2TiO3. Among Li2TiO3, coke, and LTOC, the big differences in resistance are found in the charge transfer resistance. Coke has the smallest charge transfer resistance among Li2TiO3, coke, and LTOC (Table S2Rct). The Li2TiO3 is almost an insulator to electrons. Although the addition of Li2TiO3 into coke reduces the electronic conductivity of coke (Rct of LTOC), but improves the Li+ conductivity of the material. The Re (2.01 Ω) and Rs (29.0 Ω) of LTOC are much smaller than the corresponding Re (10.6 Ω) and Rs (79.6 Ω) of literature reported spinel Li4Ti5O12.29 Table S2Rct. The vs. lithium metal anode. The coke cell has smooth discharge/charge curves. The discharge/charge curves of LTOC and Li2TiO3 cells have a very narrow discharge voltage plateau (10–20 mA h g−1) at about 1.5 V and a narrow charge voltage plateau at about 1.6 V, which is contributed by Li2TiO3,25 while the rest parts of the curves are smooth. This type of smooth discharge/charge curves offers people options for selecting any suitable final charge voltage in full cells to avoid lithium plating on anodes. It could be found that, over LTOC, the most part of the discharge/charge occurs below 1.5 V. Hence, by comparing with the spinel Li4Ti5O12 (major discharge/charge voltage plateau at about 1.55 V), the batteries having LTOC anode should give much higher full cell voltage and energy density than that having a Li4Ti5O12 anode . Coke has higher specific capacity  than Li2TiO3, but bad stability. Coke only retains 68.5% of it's initial specific capacity after 1000 discharge/charge cycles. The LTOC cell performances the best. The LTOC has the highest specific capacity of 264 mA h g−1. After 1000 of discharge/charge cycles, LTOC still retains 92.4% of it's initial specific capacity.2TiO3 at different current densities. The LTOC has the best rate capability. It starts with a specific capacity of 266 mA h g−1 at 0.100 A g−1 and ends with 200 mA h g−1 at 1.000 A g−1. High specific capacities of 160, 142, 126, and 118 mA h g−1 are obtained at even high current densities 2.000, 3.000, 4.000, and 5.000 A g−1, respectively (ESI Section 84Ti5O12 (about 160 mA h g−1).30−1 (at 0.100 A g−1), respectively . On the other hand, the introduction of Li2TiO3 into coke raises the D+Li of coke, and therefore, improves the rate capability of the material. Hence, the high specific capacity, the low discharge/charge voltage of LTOC vs. Li+/Li, the high rate capability, and the simple preparation method of LTOC make it a practical anode material to make high rate and high energy density batteries.In conclusion, the investigation show that the presence of coke carbon could inhibit the crystal growth of LiThere are no conflicts to declare.RA-009-C9RA02611H-s001


CCP20 loop, a known anti-CFH AAbs epitope. We next analyzed the dynamics of the C-terminal region of CFH and showed that the epitopes recognized by anti-CFH AAbs and VHH4 were the most flexible regions in CCP18-20. Finally, we conducted mutation analyses to elucidate the mechanism of VHH4 recognition of CFH and revealed that VHH4 inserts the Trp1183CCP20 residue of CFH into the pocket formed by the complementary determining region 3 loop. These results suggested that anti-CFH AAbs may adopt a similar molecular mechanism to recognize the flexible loop of Leu1181-Leu1189CCP20, leading to aHUS pathogenesis.Atypical hemolytic uremic syndrome (aHUS) is a disease associated with dysregulation of the immune complement system, especially of the alternative pathway (AP). Complement factor H (CFH), consisting of 20 domains called complement control protein (CCP1-20), downregulates the AP as a cofactor for mediating C3 inactivation by complement factor I. However, anomalies related to CFH are known to cause excessive complement activation and cytotoxicity. In aHUS, mutations and the presence of anti-CFH autoantibodies (AAbs) have been reported as plausible causes of CFH dysfunction, and it is known that CFH-related aHUS carries a high probability of end-stage renal disease. Elucidating the detailed functions of CFH at the molecular level will help to understand aHUS pathogenesis. Herein, we used biophysical data to reveal that a heavy-chain antibody fragment, termed VHH4, recognized CFH with high affinity. Hemolytic assays also indicated that VHH4 disrupted the protective function of CFH on sheep erythrocytes. Furthermore, X-ray crystallography revealed that VHH4 recognized the Leu1181–Leu1189 Atypical hemolytic uremic syndrome (aHUS) is a disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury . aHUS isCFH (155 kDa) forms a linear, chain-like structure consisting of twenty domains called complement control protein (CCP) 1 to 20 (CCP1-20), each comprising ∼60 residues , 8. Two In aHUS derived from CFH abnormality, CFH mutations , 2 and dβ-sandwich-type fold including four consensus cysteine residues forming two disulfide bonds and having an overall prolate spheroidal shape with N- and C- terminal lying close to opposite poles of the long axis  and the antigen, we employed X-ray crystallography and other biophysical techniques. Based on our data, we discuss the possibility that AAbs in aHUS adopt similar mechanisms to recognize the CFH, leading to pathogenesis.E.coli and purified by immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC) (KD SPR = 1.71 ± 0.66 pM) and a rather slow dissociation rate constant  . The purhy (SEC) . CFH18-2constant . The intconstant . AlthougWe evaluated the ability of VHH4 to inhibit CFH by a hemolytic assay using nonsensitized sheep red blood cells (SRBCs) that are generally employed to assess the cell-protective function of CFH. The C-terminal region of CFH (CCP19–20) binds to the abundant SA moieties present on the surface of SRBCs and protects erythrocytes from complement-mediated lysis. Thus, the dysfunction of the C-terminal region of CFH causes hemolysis of SRBCs .B), implying that VHH4 might have little influence on the CFH binding to C3b or CFI. Added to the fact that first, binding of CFH to both C3b and SA is required for CFH to be fully effective as a complement suppression cofactor to protect erythrocytes from hemolysis and, second, that mutants of CFH at the SA-binding region resulted in significantly weaker protecting activity than that offered by wildtype CFH . The crystal structure revealed that VHH4 recognized the loop Leu1181–Leu1189CCP20 belonging to the CCP20 domain using complementarity-determining region 3 (CDR3) (A). Importantly, this loop corresponds to a region previously reported to be the epitope of anti-CFH AAbs (4ONT) (B and C). Specifically, the binding of VHH4 pushed the loop of CFH toward the side of SA, thereby causing steric hindrance between the loop and SA (B and C). In addition, the conformation of the Leu1181–Leu1189CCP20 loop was altered by VHH4. The residue Trp1183CCP20 belonging to this loop, and necessary for the interaction with SA, is now surrounded by the VHH4 CDR3 loop (B and D). Collectively, these results suggested that VHH4 inhibits CFH binding to SA on the surface of the erythrocyte membrane  A. ImportCFH AAbs , 27. We s (4ONT) . The sups (4ONT) , B and Cp and SA , B and CDR3 loop , B and Dmembrane .Figure 4CCP20 loop, which is the epitope for VHH4 as well as for AAbs in aHUS, we conducted molecular dynamics (MD) simulations. We performed MD simulations using the crystal structure of CFH18-20 in the unbound form (PDB: 3SW0) . First, we calculated the RMSD and root mean square fluctuation (RMSF) of the Cα atoms in each domain . The RMSD and RMSF values revealed that CCP18 was the most rigid domain among these three domains (D and B–D). Intriguingly, large RMSF values were observed, especially in loops Leu1181–Leu1189CCP20 and Arg1210-Thr1217CCP20 . It is thus suggested that the Leu1181–Leu1189CCP20 loop, which is one of the functional regions, of CFH was also the most flexible regions in the C-terminal region of CFH. The pathogenesis of autoantibody-mediated aHUS is likely to be caused by the binding of AAbs to a functional site of CFH such as this loop, thus inhibiting the CFH binding to the SA moiety. Also, looking into the steps necessary for the generation of AAbs and given that antibodies have the tendency to recognize a rigid portion of antigens (To characterize the L1181–L1189B: 3SW0)  , remarkably long among VHHs  were predicted to contribute to the interaction. A hydrogen bond was formed between Trp1183CCP20 and Thr108VHH4 , a residue deeply inserted into the CDR3 loop of VHH4, and subjected it to SPR analyses. The mutant W1183ACCP20 showed significantly lower affinity to VHH4 than CFH18-20 WT. The estimated affinity was lower than 10−6 M  due to the slower association and much faster dissociation phases than VHH4 WT . Considering that Arg1182CCP20 engaged in an intramolecular interaction with Glu1198CCP20 in the apo form, the heat generated via the direct Arg1182CCP20–Asp121VHH4 interaction upon the VHH binding to CFH is likely to be composed by loss of Arg1182CCP20–Glu1198CCP20 interaction. The result showing that no change of binding enthalpy was observed by D121AVHH4 mutation also supports that finding.D121AFig. 8D,  and 2, iKD Y118A/KD WT = 1.28 × 106) with faster dissociation in the mutant Y118AVHH4  and large loss of enthalpy change  compared with WT  by ITC   . Conside(CCP1-8) , 42, theCCP20 side chain into its CDR3 loop resulting in significant conformational changes of this residue with respect to that in the absence of antigen. Given that Trp1183CCP20 was suggested to be important for CFH binding to SA based on structural analyses in a previous report , the same region known to be an epitope of AAbs in aHUS, and characterized the interaction between the antibody and antigen in detail. Our results revealed how the antibody specifically recognizes such a flexible loop. Although further analyses using AAbs from patients will be required to elucidate the molecular mechanism, our results provide important insights to understand how autoantibodies recognize CFH and cause aHUS.2.All CFH constructs were expressed using the same method. The DNA sequence encoding human CFH CCP18-20 with a myc tag and His-tag at the C-terminus for SPR and ITC measurements and with only His-tag at the C-terminus for crystallization were cloned into pcDNA 3.4 vector . Recombinant CFH proteins were expressed using Expi293 cells  following the manufacturer’s methods. The cells were cultured for 7 days after transfection at 37 °C and 8% COPurification methods were identical for all constructs. The supernatant was collected and filtered followed by dialysis against a solution composed of 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 5 mM imidazole. IMAC was conducted using Ni-NTA Agarose . The proteins were eluted with 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 200 mM imidazole. The proteins were dialyzed against a solution composed of PBS (pH 7.4). The final purification was performed by SEC using HiLoad 16/60/20 Superdex 75 pg column  at 4 °C equilibrated in PBS, pH 7.4.CD spectra of CFH18-20 were collected in a JASCO J-820 instrument using a 1 mM path-length quartz cell. Measurements were carried out at 25 °C in PBS, pH 7.4. Sample concentration was 5 μM. The background  was subtracted, and the spectra were analyzed using spectra manager software (JASCO Co).E. coli XL-1 Blue followed by VCS M13 helper phage infection, and phage production was induced in the presence of 1 mM of isopropyl-1-thio-β-D-galactopyronoside (IPTG). Phage was precipitated from the bacterial supernatant with PEG/NaCl and resuspended in 1% bovine serum albumin/PBS. The VHH antibodies were selected by three rounds of biopanning using microtiter wells, and selected antibody sequences were analyzed.Library construction from the peripheral blood B cells obtained from immunized alpaca and antibody selection was conducted as described in a previous study . BrieflyE. coli (Merck Darmstadt). IPTG (0.5 mM) was added to the growing cells when the A600 value reached 0.9 ∼1.1 to induce expression of proteins, and overnight culture was harvested. The E. coli pellet was resuspended in 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 5 mM imidazole, and then the cells were disrupted by sonication. IMAC was conducted using Ni-NTA Agarose . The proteins were eluted with 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 200 mM imidazole. The proteins were dialyzed against a solution composed of PBS (pH 7.4). The final purification was performed by SEC using HiLoad 26/60/20 Superdex 75 pg column  at 4 °C equilibrated in PBS, pH 7.4.All VHH constructs were expressed using the same method. The DNA sequence encoding VHH with His-tag at the C-terminus was cloned into a pRA2 vector . VHHs weA600 value reached 0.6. After 16 h at 28 °C, culture was harvested. The E. coli pellet was resuspended in 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 5 mM imidazole, and then the cells were disrupted by sonication. IMAC was conducted using Ni-NTA Agarose . The proteins were eluted with 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 200 mM imidazole. Ulp1 protease was added to cut the His-SUMO tag and dialyzed against a solution composed of 20 mM Tris-HCl, pH 8.0, 500 mM NaCl, and 5 mM imidazole. 2 nd IMAC was conducted to remove the His-SUMO tag and Ulp1 protease and flow through was dialyzed against a solution composed of PBS (pH 7.4) to collect C3d with no Histag. The final purification was performed by SEC using HiLoad 26/60/20 Superdex 75 pg column  at 4 °C equilibrated in PBS, pH 7.4.The DNA sequence encoding C3d was cloned into pET SUMO vector. C3d were expressed using BL21 (DE3) expression system (Merck Darmstadt). To induce expression of proteins, 0.5 mM IPTG was added to the growing cells when the The DNA of each mutant was prepared by site-directed mutagenesis PCR using the KOD-Plus Mutagenesis Kit . The protocol was slightly modified, as we used KOD One PCR Master Mix  instead of polymerase KOD-Plus for the inverse PCR step. Expression and purification methods of mutants were the same as that of wildtype proteins.CCP20 , Y118AVHH4, D121AVHH4 , and L104AVHH4 . The association time was 120 s, and the dissociation time was 1800 s. The assays were carried out in PBS, pH 7.4, and Tween 20 (0.005%) at 25 °C. The data were collected by single-cycle kinetics. The data were analyzed with the BIAevaluation software .The kinetic parameters of CFH–VHH4 interactions were determined using a Biacore 8K instrument . CFH18-20 was immobilized on a CM5 Biacore sensor chip  at around 300 resonance units using the amine-coupling method according to the manufacturer’s methods. After immobilization, VHH4 was injected into the sensor chip at a flow rate of 30 μL/min. The range of each concentration VHH4 was 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM for all the constructs except for the case of W1183AThe interaction analyses between C3d and the complex of CFH18-20 and VHH4 were performed using a Biacore T200 instrument . C3d was immobilized on a CM5 Biacore sensor chip at 400 response units using the amine-coupling method according to the manufacturer’s methods. After immobilization, the complex of 5 μM CFH18-20 and VHH4 was injected into the sensor chip at a flow rate of 30 μL/min for 120 s.CCP20 (15 μM), Y118AVHH4 (15 μM), and D121AVHH4 (10 μM), and the VHH4 concentration was 50 μM except for the case of W1183ACCP20 (150 μM), Y118AVHH4 (150 μM), and D121AVHH4 (100 μM). The thermodynamic parameters were calculated by the fitting of titration curves using ORIGIN 7.0 software .The thermodynamic parameters of interactions between CFH18-20 and VHH4 were evaluated using an iTC200 microcalorimeter . Samples were dialyzed against PBS, pH 7.4. CFH18-20 was injected into the cell, and VHH4 was injected into the syringe. The CFH18-20 concentration was 5 μM except for in the case of W1183A2, and 10 mM EGTA, pH7.2–7.4) to make 100-μl solution, and the mixture was incubated with 100 μl of SRBCs  at 37 °C for 30 min. As a blank sample, each plasma was also diluted with AP-CFTD buffer containing 50 mM EDTA and tested in the same way. The reaction with SRBCs was quenched by the addition of 1 ml of VBS-EDTA buffer . After the centrifugation, the absorbance of the supernatant was measured at 414 nm using a plate reader . The hemolytic assay was performed in duplicate, and the mean value was shown in the graph. The detailed information of O72 is described in the method of “Hemolytic assay was performed as previously described . SRBCs wFluid-phase cofactor activity of CFH was measured by using a C3b proteolysis assay . To assePurified CFH18-20 with His-tag and VHH4 was dialyzed against 20 mM Tris-HCl, pH 8.0, and 200 mM NaCl, respectively. After dialysis, the proteins were mixed to achieve a molar ratio of CFH to VHH4 of 1:1.2 and concentrated to a volume of 5 to 6 ml for SEC purification using Amicon Ultra-15 10K . SEC purification was performed using HiLoad 16/60/20 Superdex 75pg column , in an AKTA system (Cytiva) at 4 °C. After purification, the protein was concentrated to 6 mg/ml (145 μM). Crystallization was conducted using an Oryx8 instrument (Douglas Instruments) using commercial screening kits, PEG/ION 1 . The crystals were obtained by mixing the complex with a crystallization solution composed of 200 mM sodium phosphate monobasic monohydrate and 20% w/v polyethylene glycol 3350 at 20 °C. Suitable crystals were harvested, incubated in the same solution supplemented with 20% glycerol, and transferred to liquid nitrogen for storage until data collection.3SW0) . Diffraction images were processed with the program MOSFLM and merged and scaled with the program SCALA or AIMLESS  of CCP4 3SW0)  and a fu3SW0)  with PHA3SW0) . The mod3SW0)  and buil3SW0) . Validat3SW0) . Data co2PQR  for 1 ns. Further simulations were performed with the NPT ensemble at 298 K for 1000 ns. The time step was set to 2 fs through the MD simulations. A cutoff distance of 12 Å was used for Coulomb and van der Waals interactions. Long-range electrostatic interactions were evaluated using the particle mesh Ewald method . Bonds i7WKI. All other data are available from the authors upon request. Please send request to Makoto Nakakido, nakakido@g.ecc.u-tokyo.ac.jp.The coordinates and structure factors for the structure of CFH–VHH complex have been deposited in the PDB under entry code This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Inflammasomes are known to contribute to brain damage after acute ischemic stroke (AIS). TAK1 is predominantly expressed in microglial cells and can regulate the NLRP3 inflammasome, but its impact on other inflammasomes including NLRC4 and AIM2 after AIS remains elusive. EPO has been shown to reduce NLRP3 protein levels in different disease models. Whether EPO-mediated neuroprotection after AIS is conveyed via an EPO/TAK1/inflammasome axis in microglia remains to be clarified. Subjecting mice deficient for TAK1 in microglia/macrophages (Mi/MΦ) to AIS revealed a significant reduction in infarct sizes and neurological impairments compared to the corresponding controls. Post-ischemic increased activation of TAK1, NLRP3, NLRC4, and AIM2 inflammasomes including their associated downstream cascades were markedly reduced upon deletion of Mi/MΦ TAK1. EPO administration improved clinical outcomes and dampened stroke-induced activation of TAK1 and inflammasome cascades, which was not evident after the deletion of Mi/MΦ TAK1. Pharmacological inhibition of NLRP3 in microglial BV-2 cells did not influence post-OGD IL-1β levels, but increased NLRC4 and AIM2 protein levels, suggesting compensatory activities among inflammasomes. Overall, we provide evidence that Mi/MΦ TAK1 regulates the expression and activation of the NLRP3, NLRC4, AIM2 inflammasomes. Furthermore, EPO mitigated stroke-induced activation of TAK1 and inflammasomes, indicating that EPO conveyed neuroprotection might be mediated via an EPO/TAK1/inflammasome axis.The online version contains supplementary material available at 10.1007/s12975-021-00948-8. Acute ischemic stroke (AIS) remains a leading cause of death and long-term disability worldwide and is associated with a high socioeconomic burden –3. The cPost-stroke neuroinflammation is one of the major contributors to secondary brain damage –13. The We have recently demonstrated that the deletion of microglial and macrophage (Mi/MΦ) TAK1 significantly reduced infarct sizes, neurological impairments, as well as the levels of TNF-α, IL-1β, and IL-6 in the periinfarct zone after transient middle cerebral artery occlusion (tMCAo) . AlthougThe hematopoietic growth factor erythropoietin (EPO) seems to have a regulatory impact on the aforementioned downstream effectors of TAK1. Although EPO has been approved for the treatment of renal anemia and has been successfully tested in multiple preclinical and clinical trials addressing different neurological diseases –42, the Cx3cr1creER-Tak1fl/fl mice and subjected these and the creER-negative Tak1fl/fl control mice to 30 min of tMCAo followed by 6 and 72 h of reperfusion. EPO was administered directly, 24 and 48 h after tMCAo. Oxygen-glucose-deprivation (OGD) was performed in murine microglial BV-2 cells to assess the impact of TAK1- and NLRP3-inhibition on the NLRC4 and AIM2 inflammasomes. In total, our data indicate that EPO may convey neuroprotection via an EPO/TAK1/inflammasome axis.To address these questions, we induced a tamoxifen-dependent conditional deletion of Mi/MΦ TAK1 utilizing All experimental procedures were approved by the Animal Care Committee of the RWTH Aachen University and the District Government of North Rhine Westphalia in Recklinghausen, Germany (LANUV ID 84-20.04.2015.A292). All procedures were conducted in accordance with the ARRIVE guidelines. This study was not pre-registered.Cx3cr1CreER mice (B6J.B6N(Cg)-Cx3cr1tm1.1(cre)Jung/J; Stock No: 025524, Jackson Laboratories) were generated on a C57BL/6 background [Map3k7 (Tak1fl/fl) were crossed with Cx3cr1CreER mice (Jackson Laboratories). Littermates carrying the respective loxP-flanked alleles but the lacking expression of Cre recombinase (further referred to as “Tak1fl/fl”) served as control. To induce the Cre recombinase, 6 to 8-week-old mice were injected twice subcutaneously with 4 mg tamoxifen  dissolved in 200 μL corn oil  in a period of 48 h. The mice were bred by the Institute for Laboratory Animal Science and Experimental Surgery, Faculty of Medicine, RWTH Aachen University.Mice were housed and handled according to the guidelines of the Federation for European Laboratory Animal Science Association (FELASA) in a pathogen-free, temperature-controlled (20–24 °C) facility with a 12/12-h light/dark cycle and access to pelleted food and water ad libitum. ckground . Mice ca2 / 67% N2O) and was maintained with 1% isoflurane. The regional cerebral blood flow (rCBF) was monitored with a laser Doppler probe  above the left MCA territory  or sham surgery was performed for 30 min followed by 6 or 72 h of reperfusion as previously described . In brieTAK1fl/fl, 32 Cx3cr1creER-Tak1fl/fl) to either 30 min of tMCAo or sham surgery followed by a reperfusion period of 6 or 72 h  after 5 to 10 min reperfusion was absent. Furthermore, animals with brain hemorrhage, seizures, extensive weight loss (>20% of baseline), missing infarction in TTC-staining (described below) and those that did not develop sufficient neurological deficits (mNSS <5) were excluded. Mice that died during the post-surgical observation period (72 h) were excluded from all analyses, except the mortality rate between genotypes and treatment.Recombinant human EPO (rhEPO)  was diluted in 0.9% NaCl. EPO in a dosage of 5000 U/kg body weight or 0.9% NaCl (vehicle control) was injected intra-peritoneally directly after tMCAo, 24 and 48 h after reperfusion as previously described . In the To evaluate the general status and focal neurological impairments after 30 min of tMCAo or sham surgery, a modified neurologic severity score (mNSS) was utilized , 48. ThiInfarct volumes were measured using the 2,3,5-triphenyltetrazolium chloride (TTC) staining method as previously described , 37, 45.2. The cells were cultured in Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum  and 0.5% penicillin–streptomycin . Cells were sub-cultured at a level of approx. 80% confluence. Trypsin/EDTA  treatment was used to detach cells for splitting.The murine microglial BV-2 cell line was originally generated by Blasi and colleagues  and has 2). We monitored the oxygen levels , temperature (set at 37 °C), as well as the pressure  as previously described , 37. AftViability of the cells was assessed by cell counting . In addition, secreted lactate dehydrogenase (LDH) levels were measured in the cell supernatant using the CytoTox 96 Non-Radioactive Cytotoxicity Assay  according to the manufacturer’s protocol. A lysis-control after application of Lysis solution (Triton X-100) was used as a positive internal control.2O  served as no template control (NTC) and primer efficiencies were calculated using the Pfaffl method [Hprt) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) were measured at cycle threshold  and relative quantification was calculated by the ΔΔCt method using the qbase+ software . A list of used primers is given in Table Gene expression analyses were performed with brain tissue biopsies from the peri-infarct areas (Bregma 0 ± 1 mm) using a stereomicroscopic approach. After dissolving and homogenizing the tissue in PeqGold , the total RNA was extracted using peqGold RNA TriFast as previously described . Compleml method . The tarProtein analysis was performed by western blot as previously described . In brieAfter euthanasia, the brains were fixed with 4% paraformaldehyde (PFA) before the paraffin-embedding process. Staining was performed on 5-μm sections from Bregma ±0 regions. First, the sections were deparaffinated and incubated for 10 min with citrate buffer (pH 6.0) in a microwave. The slices were blocked for 30 min followed by incubation with the first primary antibody at 4 °C overnight. Then the first secondary antibody was incubated for 1 h at RT. Afterward, the second primary antibody was incubated for 3 h at RT followed by the second secondary antibody for 1 h at RT and DAPI . We counted double-positive cells in twelve selected fields and expressed the percentage of double-positive cells/100 cells in the (peri-) infarct zone. A list of the used antibodies is given in Table After removal of the medium, cells were fixed with 3.7% Formaldehyde in PBS for 30 min at RT and washed three times with PBS. Then, cells were permeabilized by incubation with 0.2% Triton X-100 in PBS for 10 min at RT, followed by blocking with IFF buffer for 1 h. The primary antibody diluted in PBS was applied and incubated overnight at 4 °C. The following day the secondary antibody diluted in PBS was applied for 1 h at RT. In addition, cell nuclei were stained with 4′,6-Diamidin-2-phenylindol (DAPI) . Immunocytochemical staining was performed using antibodies against Ionized calcium-binding adaptor molecule 1 (IBA1) to detect microglia. Visualization was performed with a Leica fluorescence microscope . A list of antibodies used in this study is given in Table Caspase-1 (CASP1) activity was determined in the cells and the supernatant using the Caspase-Glo® 1 Inflammasome Assay  according to the manufacturer’s instructions. The luminescence was measured using the multiplate reader .IL-1β enzyme-linked immunosorbent assay (ELISA) was performed according to manufacturers’ protocols and as described previously   infarct sizes were twice as large compared to Cx3cr1creER-Tak1fl/fl mice (58.4 mm3 ± 2.93 mm3) [a = 0.05), expected attrition of less than 5%, and a power of 80%. Power analysis was carried out with G*Power. The number of animals, technical, and experimental repeats are indicated in the corresponding figure legends including the statistical tests employed.The investigators/surgeons were blinded to genotype and experimental groups during experiments and analysis. Animals were identified by a technical assistant not involved in the analyses by earmarks that assigned numbers to each, which were announced to the investigator only after finishing experiments and analysis. Randomization was carried out using sorting by random numbers . Data analysis and visualization were performed using GraphPad Prism . Residuals were analyzed for normal distribution using the Shapiro-Wilk and D’Agostino-Pearson omnibus normality test. Variance homogeneity was tested using the Bartlett test or the Spearman’s rank correlation test for heteroscedasticity. For the identification of outliers, a ROUT test was utilized. In case of significances in normality and/or variance homogeneity, values were BOX-COX-transformed after calculation of the optimal lambda and used for statistical analysis. Intergroup differences were tested by ANOVA two-way or three-way followed by Tukey post hoc test (multiple groups). Data are given as arithmetic means ± SD. .93 mm3) . Here, wCx3cr1CreERTakfl/fl) mice and respective control mice (Takfl/fl) to study the effect of TAK1 and Erythropoietin (EPO) on inflammasome contribution to AIS. The primary endpoint of the study was the evaluation of infarct sizes in TTC-stained brain sections of both genotypes after 6 or 72 h of reperfusion  . We also detected a comparable reduction in infarct sizes after EPO administration. Although EPO application evoked a tendency of reduction of infarct sizes in both genotypes after 6 h of reperfusion, an almost 50% reduction of infarct sizes was observed after 72 h of reperfusion in the control animals . EPO in Cx3cr1creER-Tak1fl/fl mice led to a further reduction of infarct volumes by 63% , suggesting a synergistic effect of EPO and deletion of Mi/MΦ TAK1  Fig. . In contAK1 Fig. .Fig. 1MTak1fl/fl mice were also associated with higher post-ischemic neurological impairments and weight loss. While the sham group  recovered rapidly from surgery and exhibited hardly any deficits during the entire observation period, tMCAo in both genotypes caused considerable neurological deficits, especially in the initial phase of the reperfusion (up to 6 h post-stroke)  compared to the control animals (severe injury 10–15 points). Both Tak1fl/fl (p < 0.05) and Cx3cr1creER-Tak1fl/fl mice (p < 0.01) gained clinical benefits from EPO administration over the entire observation period of 72 h after stroke. The EPO-dependent beneficial clinical outcomes were also reflected in the weight measurement since both genotypes displayed significantly lower weight loss compared to the vehicle group (p < 0.001)  Fig. . The del01) Fig. .These data suggest that both EPO and the deletion of Mi/MΦ TAK1 reduce infarct sizes and improve clinical outcomes. However, EPO-mediated protective effects do not appear to depend fully on Mi/MΦ TAK1, but the combination of EPO and Mi/MΦ TAK1 deletion seems to synergistically provide greater cytoprotection.Tak1 and the inflammasomes Nlrp3, Nlrc4, and Aim2 after stroke compared to sham surgery  . A similar pattern was evident in the expression of Nlrp3 and Nlrc4. Again, EPO reduced the expression of both inflammasomes in both genotypes  and Nlrc4 (p < 0.01) than the Cx3cr1creER-Tak1fl/fl mice 72 h after tMCAo. In contrast, mRNA levels of Aim2 and of the adaptor protein Asc revealed no genotype-specific differences  as well as after 72 h (p < 0.05) of reperfusion. In addition, EPO significantly reduced mRNA levels of Casp1 in both genotypes and at both reperfusion times (p < 0.05)  Fig. . Howeverpes Fig. . Tak1fl/ces Fig. . The tra05) Fig. . Similar05) Fig. .Fig. 2Ppro-Il-1β. Furthermore, EPO appears to reduce the priming signal of inflammasomes.This data shows that cerebral ischemia among other priming stimuli, such as ligands for toll-like receptors (TLRs), NLRs , or cytokine receptors, is able to upregulate the transcription of inflammasomes and p = <0.0001). A similar pattern was observed in the protein levels of pTAK1 . However, after 72 h of reperfusion, the stroke group still presented with higher levels of TAK1 and pTAK1 compared to the sham group, though the difference was not as prominent as at the earlier reperfusion timepoint (6 h) .To further assess the influence of cerebral ischemia on the activation of TAK1 and the inflammasomes NLRP3, NLRC4, and AIM2, we first compared the protein expression of TAK1 and its phosphorylation (pTAK1) in mice after tMCAo or sham surgery Fig. . The posAK1 Fig.  (p = <0. h) Fig. . EPO admp < 0.05) at both reperfusion timepoints . Cerebral ischemia also resulted in a significant increase of protein levels of the adaptor protein ASC . Again, EPO was able to significantly reduce the protein levels of the aforementioned proteins in both reperfusion phases.Next, we investigated the influence of stroke and EPO-administration on the activation of the inflammasomes NLRP3, NLRC4 and AIM2, and their downstreaming cascade Fig. . We founnts Fig. . Howevernts Fig. . EPO redASC Fig. , activatASC Fig. , and theASC Fig.   . In the latter, a significant reduction of protein levels of NLRP3, NLRC4, and AIM2 (p < 0.05) was observed compared to controls at both reperfusion periods  in both genotypes and at both times of reperfusion.To investigate a possible association between TAK1 and the observed post-ischemic upregulation of the NLRP3, NLRC4, and AIM2 inflammasomes, we utilized biopsies from the peri-infarct zones of 05) Fig. . This di05) Fig. . EPO redods Fig. . Howeverods Fig. . Here, wIn conclusion, our data suggest that Mi/MΦ appear to be a major source of TAK1 and pTAK1 expression after stroke. Mi/MΦ TAK1 seems to play an eminent role in the regulation and activation of NLRP3, NLRC4, and AIM2 inflammasomes. Furthermore, the regulatory effect of EPO on inflammasomes appears to depend in part on Mi/MΦ TAK1.2 < 1%) followed by different reperfusion timepoints revealed an increase in cell death, which was abrogated by 5Z-7-Oxozeaenol, but not upon MCC950 treatment    as well as the adapter protein ASC after OGD (03) Fig. . Similar01) Fig. .p < 0.02). MCC950 administration mitigated the OGD-induced upregulation of NLRP3, but increased protein levels of NLRC4, AIM2, and ASC (p < 0.001). CASP1 activity and IL-1β levels showed no significant difference compared to the control condition  using the search terms “NLRP3,” “NLRC4,” and “AIM2” in combination with “stroke,” “tMCAo,” and “cerebral ischemia” revealed 15–25 times the number of studies for NLRP3 263) than for NLRC4 (10) and AIM2 (15). TAK1 is reported to regulate the activation of the NLRP3 inflammasome , 66, 67 3 than fomulation . In lineHexb as a stable microglial core gene [Hexb as a microglial-specific promotor to avoid co-tracing of other monocytic cells.Using C-X3-C motif chemokine receptor 1 (Cx3cr1), which is also expressed e.g. by macrophages, as a promoter for the conditional knockout of TAK1, raises the question to what extent the protective effects are indeed mediated only by microglial cells. However, it has previously been shown that this probability might be rather small due to differences in the turnover rates. In fact, while microglial cells lack TAK1 at 4 weeks after tamoxifen administration, other macrophage-like cells  exhibit normal TAK1 levels again after this period , 20, 22.ore gene . Future creER-Tak1fl/fl mice. Furthermore, we demonstrated that EPO dampened stroke-induced activation of TAK1 and the inflammasomes along with their downstream cascade. However, the reduction of the protein levels of the inflammasomes was not evident after the deletion of Mi/MΦ TAK1, indicating that EPO-dependent regulation of the inflammasomes might be conveyed through TAK1. It is debatable whether the protein levels of inflammasomes in the Cx3cr1creER TAK1fl/fl mice are reduced by the knockout to the point that further reduction by EPO is no longer apparent or significant. However, since EPO administration downregulated the downstreams , it might be possible that EPO via TAK1 only has a regulatory effect on inflammasome sensors, not on the downstreams.A large number of studies have already shown that the pleiotropic and anti-inflammatory cytokine EPO has a regulatory impact on the downstream effectors of TAK1  as well as on IL-1β, IL-18, and the inflammasome NLRP3 , 71, 72.There is little literature on the regulation of TAK1 gene expression by EPO in the brain, however, NF-κB may play a crucial role here. Takahashi and colleagues identified a potential NF-κB -binding sequence in the promoter region of TAK1 in a colon cancer cell model . Upon biEPO has been clinically approved for the treatment of renal anemia for many years and has shown solid safety and efficacy after stroke in several preclinical studies , 75, 76,In conclusion, we demonstrated that a deletion of Mi/MΦ TAK1 as well as the administration of EPO reduced infarct sizes and neurological impairments after tMCAo in mice. In addition, we address major knowledge gaps related to EPO-dependent regulation of the NLRP3, NLRC4, and AIM2 inflammasomes after acute ischemic stroke. We provide evidence that microglia/CAMs and in particular their TAK1 are main contributors to post-ischemic neuroinflammation. Furthermore, we demonstrated that TAK1 regulates the expression and activation of the NLRP3, NLRC4, and AIM2 inflammasomes . Moreover, functional inhibition of NLRP3 is compensated by an upregulation of NLRC4 and AIM2 after ischemia. In addition, we demonstrated that EPO mitigated stroke-induced activation of TAK1 and the inflammasomes along with their downstream cascade, which was not evident after Mi/MΦ TAK1 deletion. In conclusion, our data indicate that monocytic TAK1 is crucially involved in the regulation of the inflammasomes in the brain after stroke and that EPO conveyed neuroprotection after stroke might be mediated via an EPO/TAK1/inflammasome axis.Online Resource 1Intraoperative laser doppler flowmetry revealed no differences between genotypes. (A) Schematic illustration of the treatment protocol for both genotypes, 4HT injections and EPO as well as NaCl treatment. (B) Scheme of protocol summarizing the number of total animals (64 mice). Exclusion criteria are described in material and methods section. (C) Changes in ipsilateral Laser Doppler flowmetry by intraluminal during MCAO procedure were monitored. The baseline blood flow was considered as 100% for all mice. Changes in LDF after occlusion of CCA, MCA and reperfusion are demonstrated. Abbreviations: bp: base pairs; CCAo: Common Carotid Artery occlusion; MCAo: Middle Cerebral Artery occlusion; LDF: Laser Doppler flowmetry. Online Resource 2. Immunohistochemical analysis of the NLRP3, NLRC4 and AIM2 inflammasomes in microglial cells, astrocytes and neurons after acute ischemic stroke. Immunofluorescence of brain slices after 30 min of tMCAo and 72 h of reperfusion in Tak1fl/fl and Cx3cr1creER –Tak1fl/fl treated with NaCl or EPO. (A + E) Representative images of double staining of a inflammasome such as NLRP3, NLRC4 or AIM2 (green) and a cell marker like NeuN, GFAP or Iba1 (red); nuclei were counterstained with DAPI (blue). (B-D + F-H) Double positives cells per 100 cells in the periinfarct zone are given in percentage. Data are presented as mean ± SD. Differences were tested by Mann-Whitney (n = 4). A list of used Antibodies is given in Table Online Resource 3. In vitro stroke model, cell viability and IL-1β protein levels in murine microglial cells. (A) Schematic illustration of cell cultivation and treatment protocol of BV-2 cells for viability assessment. (B) Environmental conditions during hypoxia in the chamber and the supernatant. (C + D) LDH release in supernatant in % of lysis control during normoxia and after OGD with different concentrations of 5Z-7-Oxozeaenol and MCC950. (E) LDH release in the supernatant in % of lysis control during normoxia and after OGD at different reperfusion timepoints (n = 4). (F) Viability in % of total cell count during normoxia and after OGD at different reperfusion timepoints. (G) LDH release in the supernatant in % of lysis control under CTR, 5Z-7-Oxozeaenol and MCC950 during normoxia, 0 and 6 h after OGD (n = 4). (H) Concentration of IL-1β was detected directly after OGD and after 6 h reperfusion by ELISA given in pg/ml supernatant. All bars represent means ± SD, individual data points are shown. For statistical analysis (C/D) a Kruskal-Wallis test followed by Dunn’s multiple comparisons test was performed. For statistical analysis (F) a 2way ANOVA followed by Tukey’s multiple comparisons test was performed. Comparison of OGD for each treatment group #p < 0.05 compared to the corresponding normoxia, *p < 0.05 for intergroup comparison. Online Resource 4. HIF-1α, TAK1 and pTAK1 levels after OGD in murine microglial cells. (A) Representative immunoblotting images of HIF-1α, TAK1, pTAK1 and ACTIN after 90 min of OGD and normoxia. (B-D) Protein-levels of HIF-1α, TAK1 and pTAK1 in cell lysate were detected by immunoblotting (n = 4). ACTIN served as loading control. Quantification of protein-levels by densiometric analysis. For statistical analysis a 2way ANOVA followed by Tukey’s multiple comparisons test was performed. Bars represent means ± SD. *p < 0.05. (PDF 3087 kb)


The structure features and hydration degree of this acid were characterized by IR, UV, XRD and TG-DTA. Its proton conductivity was studied by electrochemical impedance spectroscopy (EIS). The EIS measurements demonstrated that H5SiMo11VO40·8H2O showed excellent proton conduction performance with proton conductivity reaching 5.70 × 10−3 S cm−1 at 26 °C and 70% relative humidity. So, it is a new solid high proton conductor. The conductivity enhances with the increase of temperature, and it exhibits Arrhenius behavior. The activation energy value for proton conduction is 21.4 kJ mol−1, suggesting that the proton transfer in this solid acid is dominated by Vehicle mechanism.A molybdovanadosilicic acid H −3 S cm−1 at room conditions.A novel Keggin-type proton conductor shows high proton conductivity, reaching 5.70 × 10 What attracts us most is that the structure and proton conductive property of HPAs vary with the component elements of heteropolyanion changes. According to our recent researches,17 vanadium have great impact on the thermal stability and proton conductivity of heteropoly acids. Hence, in this work, based on Keggin-type HPAs, a mono-vanadium-substituted molybdovanadosilicic acid H5SiMo11VO40·8H2O is synthesized, and its structure, hydration and proton conductive properties have also been investigated.Keggin-type HPAs, which are an important branch of HPAs, occupy an unique place at the forefront of the HPAs field because of their many advantages, including chemical stability and convenience of synthesis.2.2.1.2SiO3·9H2O was dissolved in 100 mL deionized water, and added 26.62 g Na2MoO4·2H2O to the above solution. Then this mixed solution was heated to boil for 30 min. After that, 50 mL aqueous solution which contained 1.22 g NaVO3 was added to the mixture. The mixture was continuously heated at 90 °C for 2 h with the assistance of stirring under the condition of pH = 2.5 (adjusted by 1 : 1 H2SO4 solution). Lastly, the cooled solution was extracted with 30 mL ether in sulfuric acid environment. The powder H5SiMo11VO40·8H2O was obtained after the concentrated etherate solution was dried in vacuum. The color of the product is orange-red and the morphology of this solid is as shown in 5SiMo11VO40·8H2O were collected by inductively coupled plasma mass spectrometer (ICP-MS). The content of water was figured out by thermogravimetry. Found: Si: 1.37%; Mo: 54.06%; V: 2.49% and H2O: 9.30%. Calculated for H5SiMo11VO40·8H2O: Si: 1.43%; Mo: 53.87%; V: 2.60% and H2O: 9.18%. The theoretical values are in good agreement with the experimental results.2.84 g of Na2.2.−1. UV spectrum was got using SHIMADZU UV-2550 UV-Vis spectrophotometer.Element content was measured on a THERMO ELECTRON PQ3 inductively coupled plasma mass spectrometer (ICP-MS). The Fourier-transform infrared spectroscopy (FTIR) spectrum was performed on a NICOLET NEXUS470 FT/IR spectrometer using KBr as pellets, and the resolution is 4 cmθ = 3–40° at the rate of 0.02° s−1. The crystal data was collected using graphitemono-chromatic Mo–K radiation (0.71073 Å) at 293 K, and the data sets were corrected by empirical absorption correction using spherical harmonics, implemented in SCALE3 ABSPACK scaling algorithm. The thermal stability of the sample was carried out using simultaneous thermogravimetry (TG) and differential thermal analysis (DTA) technique, measurements were performed using a Shimadzu thermal analyzer in a nitrogen stream from room temperature to 600 °C, with the scanning rate of 10 °C min−1.Powder X-ray diffraction (XRD) pattern was recorded on a BRUKER D8 ADVANCE X-ray diffractometer in the range of 2All chemicals were of analytical grade and used without further purification.2.3.4 to 0.01 Hz.At ambient condition, the crushed sample was pressed at 25 MPa into a compacted pellet. The diameter of the obtained tablet is 10.00 mm and the thickness of it is 1.02 mm. The proton conductivity was measured using a cell: copper | sample | copper, in which copper slices were attached to the two sides of the tablet as electrodes. Complex impedance measurements were carried out on a VMP2 multichannel potentiostat electrochemical impedance analyzer over a frequency range from 9.99 × 103.12O40]n−. The SiO4 tetrahedron is located in the center of the twelve MO6 octahedra, which can be split into four groups of three edge-shared octahedra, M3O13. Furthermore, the M3O13 units are connected together by shared corners to each other. So, there are four kinds of oxygen atoms in [SiM12O40]n−: Si–Oa, M–Ob–M, M–Oc–M and M–Od, where each separately represent inner oxygen, corner-sharing oxygen, edge-sharing oxygen and terminal oxygen. −1, the IR spectrum of H5SiMo11VO40·8H2O exhibits four characteristic peaks. They are 958 cm−1, 908 cm−1, 860 cm−1 and 781 cm−1, which are assigned to νas(M–Od), νas(Si–Oa), νas(M–Ob–M) and νas(M–Oc–M) vibration modes of heteropolyanion SiMo11VO405−. For comparison, these four peaks of H4SiMo12O40 are 957 cm−1, 904 cm−1, 855 cm−1, 770 cm−1.18 The similarity of vibration bands demonstrates that the heteropoly acid H5SiMo11VO40 has the Keggin structure as its parent acid H4SiMo12O40 does.19 Obviously, all of these peaks have slightly shifted, which caused by the more negative charge carrier and the shift symmetry of molecular structure of H4SiMo11VO40·8H2O. Furthermore, there are two strong absorption peaks at 3410 cm−1 and 1627 cm−1 ascribed to the stretching vibration of O–H bond and the bending vibration of H–O–H bond.IR is a useful method for investigating the structure information of heteropoly compounds. There are four kinds of oxygen atoms in ·5H2O. What's more, it is noticed that there are two obvious peaks in DTA curve. The endothermic peak observed at 90 °C is believed to be the processes of the dehydration, and exothermic peak of 383 °C is attributed to the irreversible decomposition of heteropoly acid to individual oxides . It demonstrates that the decomposition temperature of H5SiMo11VO40·8H2O is 383 °C. According to the reported literature,22,23 the decomposition temperature of H4SiMo12O40·2H2O is 365 °C, indicating vanadium-containing heteropolyanion has better thermal stability than its parent acid.The hydration degree and thermal stability of H5SiMo11VO40·8H2O, and inset is the equivalent circuit, where R1 is the bulk resistance, C1 represents a constant phase element of the double layers, R2 denotes the charge transfer resistance and W1 is the finite length Warburg element of solid diffusion. The proton conductivity of HPA is calculated using the following equation: σ = L/(RS) . By calculation, the proton conductivity of H5SiMo11VO40·8H2O is 5.70 × 10−3 S cm−1 at 26 °C and 70% relative humidity. It shows a higher conductivity than H4SiMo12O40·12H2O (2.03 × 10−4 S cm−1),24 which illustrated that the incorporation of V element can increase hugely the proton conductivity of silicomolybdic acid. So, this heteropoly compound is a high proton conductor. Additionally, CPE for double layers from the equivalent circuit is 3.59 × 10−3 F, indicating that this acid has the potential for application in energy storage device, such as supercapacitors.The proton conductivity is one of the most important properties of heteropoly acids. −2 S cm−1 at 60 °C and 70% relative humidity. The relationship between proton conductivity and temperature is consistent with Arrhenius equation: σ = σ0 expEa/κT).31 Hence, we can speculate that the proton migration of H5SiMo11VO40·8H2O occurs by a mixing mechanism and Vehicle mechanism is predominant.So far, there are two major recognized proton conduction mechanisms: Vehicle and Grotthuss mechanism.4.5SiMo11VO40·8H2O, which was characterized by IR, XRD of powder, TG-DTA. This acid shows a good proton conductivity of 5.70 × 10−3 S cm−1 at 26 °C and 70% relative humidity. The proton conductivity enhances with the increase of temperature, and the proton conduction mechanism is dominated by Vehicle mechanism due to the activation energy of 21.4 kJ mol−1. It is a novel solid high proton conductor, which may be applied as solid electrolyte in the fields of fuel cell and supercapacitors.In this work, we have reported the synthesis of a vanadium-substituted heteropoly acid HThere are no conflicts to declare.


Hearing relies on the transduction of sound-evoked vibrations into electrical signals, occurring in the stereocilia bundle of inner ear hair cells. The G protein-coupled receptor (GPCR) ADGRV1 and the multi-PDZ protein PDZD7 play a critical role in the formation and function of stereocilia through their scaffolding and signaling properties. During hair cell development, the GPCR activity of ADGRV1 is specifically inhibited by PDZD7 through an unknown mechanism. Here, we describe the key interactions mediated by the two N-terminal PDZ domains of PDZD7 and the cytoplasmic domain of ADGRV1. Both PDZ domains can bind to the C-terminal PDZ binding motif (PBM) of ADGRV1 with the critical contribution of atypical C-terminal β extensions. The two PDZ domains form a supramodule in solution, stabilized upon PBM binding. Interestingly, we showed that the stability and binding properties of the PDZ tandem are affected by two deafness-causing mutations located in the binding grooves of PDZD7 PDZ domains. The protein complex is thus referred to as Usher2 complex. It encompasses two transmembrane proteins, usherin and ADGRV1, exposing very large N-terminal extracellular regions  thought to form fibrous links in between stereocilia. Usherin is a single-pass transmembrane protein of largely unknown function. It contains multiple laminin and fibronectin type domains that are likely promoting extracellular matrix adhesion and local scaffolding. ADGRV1 (Adhesion G-protein Receptor V1) is a seven-pass transmembrane protein of the adhesion G protein-coupled receptor (GPCR)  family B pathway . The ADG pathway . These t pathway . The for pathway . However pathway . Interes pathway . Here weE. coli BL21 DE3 in LB or M9 minimal medium supplemented with 15NH4Cl and/or 13C-glucose, induced with 0.2 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and performed overnight at 18°C. Lysis is performed in 550 mM NaCl, 2 mM β-mercaptoethanol, 250 mM EDTA and complemented with anti-proteases (Roche Diagnostics) (buffer and pH as indicated for the respective experiment) using a Constant Systems Ltd. cell disruptor at 1.3 kbar. After 1 h centrifugation at 19000G, tagged proteins are purified from the supernatant by affinity chromatography using a GSTrap (Cytiva) column followed by overnight (on-column) TEV cleavage. The cleaved proteins are eluted in 300 mM NaCl, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), 250 mM EDTA complemented with anti-proteases . A last step of purification is performed by size-exclusion chromatography (SEC) using a Sephacryl S-100 HP 16/60 (Cytiva).All PDZD7 constructs are based on the murine long isoform (Uniprot entry E9Q9W7-1); PDZ1 (81-167), PDZ1ext (81-192), PDZ2 (206-292), PDZ2ext (206-313), P1P2 (81-313), N-PDZ1ext (1-188). All were expressed in fusion with a N-terminal GST tag followed by a Tobacco Etch Virus (TEV) protease site. Protein production is performed in E. coli BL21 DE3 in LB, induced with 1 mM IPTG for 3 h at 30 °C. Lysis is done in Tris HCl pH 7.5 50 mM, NaCl 300 mM, 2 mM β-mercaptoethanol and complemented with anti-proteases using a Constant Systems Ltd. cell disruptor at 1.3 kbar. After 1 h centrifugation at 19000G, the protein is purified from inclusion bodies retrieved in the insoluble fraction. Solubilization is performed using 6M guanidine hydrochloride and the resulting fraction is loaded on a HIStrap (Cytiva) column. Guanidine hydrochloride is gradually washed using Tris HCl pH 7.5 50 mM, NaCl 150 mM, 0.5 mM TCEP complemented with anti-proteases. The His-tag is removed by overnight (on-column) TEV cleavage. The cleaved protein is subsequently eluted and a last step of purification is performed by SEC using a Sephacryl S-100 HP 16/60 (Cytiva).ADGRV1 cytoplasmic domain is based on the murine long isoform  and is expressed in fusion with a N-terminal Histidine tag followed by a TEV protease site. Protein production is performed in 1H{13C/15N} cryoprobe. The 1H, 15 N and 13C backbone resonances assignment of PDZ1ext was carried out using 3D HNCA, HNCACB, HNCO, HNCOCA experiments (Tris HCl buffer adjusted to pH 6). The 2D 1H-15N HSQC spectra used to compare PDZ1, PDZ1ext, PDZ2 and PDZ2ext were recorded in Tris HCl pH 7.5 (50 mM). The 2D 1H-15N HSQC spectra used to compare PDZ1ext, PDZ2ext and P1P2 were recorded in Phosphate Buffer pH 6.5 (50 mM). Titration experiments of PDZ1 (WT), PDZ1ext (WT) and PDZ1ext (G103R) against ADGRV1pbm peptide were performed in Tris HCl pH 7.5 (50 mM). Averaged Chemical Shifts Perturbations (CSP) were calculated as: Δδavg = ((Δδ(1H))2+(Δδ(15N)*0.159)2)½. For the determination of dissociation constants (Kd) of each peptide/PDZ interaction, averaged CSP δavg was plotted as a function of the molar ratio (peptide/PDZ) for several peaks and fitted using nonlinear regression and assuming a simple complex formation model. The average and standard deviation on minimum 10 significantly shifting peaks were calculated for each complex.All experiments were recorded at 25°C on a Bruker 800 MHz spectrometer equipped with a triple resonance -COOH; 13,7 mM) are added to the 50 μl from the sample 1 hour before recording. Initial data processing was performed using sxCuBE, FOXTROT and PRIMUS. The radii of gyration were evaluated using Guinier approximation. The Dmax was determined from the distance distribution function P(r) obtained with the program GNOM.X-ray scattering data were collected at the SWING beamline at Soleil Synchrotron . 50 μl of sample are injected on a Superdex-75 5/150 GL size exclusion column in-line with the SAXS measuring cell. PDZD7 samples are prepared in the same buffer . The experiment is performed at initial concentrations of 119 μM (3.1 mg/ml) for P1P2 WT and 108 μM (2.8 mg/ml) for P1P2 G103R. For complexes, 2 μl of pH-adjusted ADGRV1pbm peptide . For the complex, 10 μl of pH-adjusted ADGRV1pbm peptide  were added to 300 μl of P1P2 sample. Interference profiles were analyzed with SEDFIT.The metadynamics simulation ran for 100 ns with four replicas for each experiment. The triple-strand conformation predicted by AlphaFold was used as starting point for PDZ1ext, and a homology model  based onp∗p0+(Fx-Fp)∗0.5∗(R∗I0+(1-R)∗x + K-((R∗I0+(1-R)∗x + K)2-4∗R∗(I0-x)∗x)1/2), where p0 is the initial protein concentration, I0 the peptide stock concentration, R the p0/I0 ratio, x the volume of added peptide in µl, Fp the fluorescence of PDZ constructs, Fx the fluorescence of the complex, and K the Kd. The values of p0, I0 and R are kept constant.Fluorescence titrations were performed on a Jasco V-730 spectrometer using quartz cell 109.004F-QS  with an optical path of 10 mm and with thick side to reduce the inner filter effect. PDZ1ext and P1P2 were prepared in Phosphate Buffer pH 6.6 (50 mM) at 6 and 2 µM respectively, while PDZ2ext was prepared in Tris HCl pH 7.5 (50 mM) at 5 µM. Variations in the quantum yield of fluorescence of tryptophans from the sample were monitored upon progressive addition of buffered ADGRV1pbm peptide. After peptide addition, the sample is homogenized by in-spectrometer magnet agitation for 30 s. The sample is left to rest for an additional 60 s before measurement. Excitation is set at 295 nm and fluorescence was measured at the maximum emission wavelength (346-349 nm) of the unbound form with a 5 nm bandwidth for both. Titration curves were fitted using the following formula: y = Fmax + K-((c + cmax + K)2-4c∗cmax)1/2/(2cmax). U is the Fnorm signal of the target, B the Fnorm signal of the complex, c the ligand concentration, cmax the final concentration of the ligand in the assay and K the dissociation constant.MST titrations were performed on a Monolith (Nanotemper) spectrometer using Monolith NT. LabelFree Premium Capillaries. The PDZ2 sample was prepared at 140 μM in MES pH6 (50 mM) and mixed (1:1) with a serial dilution (½) of ADGRV1pbm peptide . MST traces were read out at 20 s where signal to noise ratio was maximum and titration curves were fitted using the following formula: y = U+(B-U)∗(c + cNanoDSF experiments were performed on a Prometheus (Nanotemper) spectrometer using Prometheus NT.48 Series nanoDSF Grade High Sensitivity Capillaries. All samples were prepared in MES pH6 (50 mM) at micromolar concentrations. For the complexes, ADGRV1pbm peptide was added at a final concentration of 756 μM, diluting the samples by 5%.HeLa cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS). PDZD7 constructs correspond to the full-length murine sequence in fusion with a N-terminal strep tag and a C-terminal GFP, wild-type or carrying G103R/G228R variants, cloned in a PMT3 vector. The ADGRV1 construct corresponds to the murine β subunit of the receptor (5884-6298) cloned in a pRK5 vector. Plasmids were transfected in separate HeLa cultures using 25 μg (PDZD7) and 10 μg (ADGRV1) of DNA per 10 cm dish using TurboFect™ Transfection Reagent (ThermoFisher Scientific) at 3 μl per μg of transfected DNA. Mixes were prepared in volumes of 1 ml/plate (in DMEM), vortexed and incubated up to 30 min at room temperature, cell media were renewed before transfection. After drop by drop addition of the transfection mix, cells were maintained at 37°C for 14 h for protein expression. Cells were dissociated using Gibco™ Trypsine-EDTA , subsequently inactivated by addition of DMEM containing 10% FCS. Cells were counted, pelleted and resuspended at similar concentrations in PBS Tween 0.01%, Dodécyl-β-D-maltoside (DDM) 0.1% containing anti-proteases (buffer). Benzonase (Sigma) was added before sonication of the cells . An identical volume of ADGRV1 β transfected cell lysate was mixed to either PDZD7 WT, G103R or G228R transfected cell lysates. PDZ proteins have different expression levels, meaning that different amounts of sample are needed to saturate the dynabeads with the PDZ proteins. Pull down experiments have been performed several times allowing us to estimate the yield of expression of each PDZ construct for a given amount of cells. Therefore, volumes of added PDZD7 lysate were adjusted to compensate for expression disparities between WT and mutants and maintain a similar bait concentration in each mix. Lysates from non-transfected cells were added to each mix to level total cell content to ensure the same lysate concentration in the input fraction. Dynabeads™ Protein G (Invitrogen) with coupled α-GFP rabbit antibody (Invitrogen A11122) was added to each mix , then incubated 1 h on wheel (4°C). The supernatant was removed (unbound fraction) and beads were washed twice with buffer containing 10% BSA and twice with buffer without BSA. A final 40 μl wash of buffer was performed to check for remaining non-specific binding (wash fraction). The bait and bound proteins were eluted in 40 μl Bolt™ LDS Sample Buffer 4X by heating  and vortexing (15 s). Samples were loaded twice onto duplicate 4% polyacrylamide gels along with PageRuler™Plus protein ladder (ThermoFisher Scientific) and transferred to PVDF membranes. Membranes were blocked using 5% milk TBST. Target proteins were detected using mouse α-GFP  and rabbit α-ADGRV1  antibodies (1/2000 in 5% milk TBST), as well as secondary α-mouse and α-rabbit IgG antibodies coupled to HRP (1/2000 in 5% milk TBST). One duplicated gel was used for each primary antibody. Signal was generated using Immobilon Forte Western HRP substrate.The N-terminal PDZ domains of PDZD7 paralogous proteins harmonin and whirlin possess C-terminal extensions with conserved proline and tryptophan residues . These f15N and 13C labeled sample of PDZ1ext to characterize the folding state of the PDZD7 PDZ1 C-terminal extension. We successfully assigned 98%, 99% and 100% of the , CO and  backbone resonances of PDZ1ext, respectively. Secondary structure analysis derived from HN, N, CA, CB and CO chemical shifts predicts backbone torsion angles consistent with a triple β strand fold of the PDZ1 extension , but higher than expected for a flexible unfolded tail. Moreover, the peak intensities of the β strand extension in the 1H-15N HSQC spectrum at pH 6.5 are similar to the ones of the PDZ core (data not shown), but are significantly reduced at higher pH (7.4) due to increased HN/H2O exchange. Altogether, these results are consistent with the formation of transient C-ter β-strands.We produced a Furthermore, the mapping of Chemical Shift Perturbations (CSP) calculated for backbone resonances between PDZ1 and PDZ1ext constructs highlights an area encompassing β2 to β4 strands and the α1-β4 loop of the PDZ core . This reFinally, we used metadynamics  enhanced15N-labelled PDZ2 constructs were stable for a few hours only, preventing the acquisition of 3D heteronuclear NMR experiments and the assignment of PDZ2 and PDZ2ext for an analysis at the residue level. Nonetheless, we recorded 2D HSQC spectra on PDZ2 and PDZ2ext samples . The  samples  and we o samples . The sub samples . Again, As previously stated, folding of PDZ extensions downstream PDZ1 and PDZ2 domains of harmonin and whirlin is necessary for the supramodular organization of the proteins. In particular, the two N-terminal PDZ domains of whirlin adopt a compact conformation with a hairpin-mediated interface. In PDZD7, our results showed that PDZ1 and PDZ2 also possess C-terminal folded extensions. We collected Small Angle X-ray Scattering (SAXS) data and determined geometrical parameters of the PDZD7 PDZ tandem , with a derived radius of gyration (Rg) of 27.8 Å and interatomic maximum distance (Dmax) of 109.5 Å. These parameters are comparable to what we previously measured on the PDZ tandem supramodule in whirlin  . The estThe superimposition of HSQC spectra of PDZ1ext, PDZ2ext and P1P2 in -COOH; PBM underlined). We measured apparent Kd values of our constructs towards ADGRV1pbm using several biophysical methods ) (We monitored the interaction between PDZD7 constructs and a peptide (ADGRV1pbm) corresponding to the last 13 residues of ADGRV1, encompassing its PBM (sequence VELRRIPIADTHLs (MST)) . First, We also showed that PDZ2ext has a similar affinity (Kd = 3.1 µM) for ADGRV1pbm compared to the PDZ tandem , but 15-fold higher than PDZ1ext (Kd = 47 µM). Taking advantage of the good superimposition of the isolated PDZ spectra on the tandem spectrum, we followed the saturation of each PDZ in the tandem as a function of ADGRV1pbm concentrations (data not shown). We confirmed that both PDZ domains in the tandem bind to ADGRV1pbm and that PDZ2 is saturated at lower ligand concentration than PDZ1, in agreement with the affinities measured for the isolated domains. Finally, we assessed if the presence of ligand further stabilizes a compact conformation of the P1P2 supramodule using SAXS and Analytical UltraCentrifugation (AUC) . For theWe measured the thermostability of PDZD7 constructs using nanoDifferential Scanning Fluorimetry (nanoDSF). As previously observed during the production and biochemical/biophysical characterizations of the domains, PDZ1ext is drastically more stable than PDZ2ext, with melting temperatures of 61.6°C and 38.5°C, respectively. Interestingly, the binding of ADGRV1pbm strongly increases the thermal stability of the unstable PDZ2ext (Tm increased from 38.5°C to 47.2°C), while the binding has no significant effect on the more stable PDZ1ext (Tm increased from 61.6°C to 61.8 °C) . The melThe P1P2 tandem of PDZD7 is preceded by 80 residues with no predicted secondary or tertiary structure, but which could contribute to the interaction with ADGRV1. Similarly, the 152 residues of ADGRV1 cytoplasmic domain upstream its PBM exhibit several stretches of conserved positions and could participate in the interaction. We first aimed at identifying potential secondary structures in both PDZD7 N-terminal region and ADGRV1 cytoplasmic domain. To this end, we produced both an extended PDZ1ext construct, including the N-terminus of the protein  and the whole ADGRV1 cytoplasmic domain of 152 residues.On the overlay of PDZ1ext and N-PDZ1ext HSQC spectra, there is no significant chemical shift changes on PDZ1ext due to the presence of the N-terminal region (CSPs <0.04 ppm). The latter is characteristic of a disordered region, with about 70 peaks (80 expected) of poor dispersion of HN chemical shifts (8–8.5 ppm) . This suTwo deafness-causing mutations of the same amino acid substitution are located at the same position in the binding groove of the first (G103R) and second (G228R) PDZ domains in PDZD7 . We firsThe mutation in PDZ1 (G103R) induces a significant destabilization of both isolated PDZ domains  and PDZ tandem . The second mutation (G228R) markedly destabilizes the isolated PDZ2ext, with a slow transition of fluorescence ratio (330/350 nm) initiated below 15°C, indicating progressive unfolding of the protein at room temperature, with a Tm that can be roughly estimated in the 30°C range . As a coWe characterized the potential effect of the two substitutions on the interaction with ADGRV1pbm using a panel of techniques. First, we confirmed by NMR that PDZ1ext G103R is well-folded, with significant perturbations (CSPs >0.04 ppm) only detected for residues spatially close to the G103R mutation . In addiWe could not measure the interaction between PDZ2 G228R and ADGRV1pbm, due to a low yield and severe aggregation of the mutated domain during biophysical experiments, consistent with its low melting temperature. For the same reasons, we are unable to produce the PDZ tandem carrying the G228R substitution, preventing us from measuring its affinity for ADGRV1pbm.To conclude, the substitution G103R prevents the first domain of the tandem from binding ADGRV1, but does not affect the binding capacity of the second domain. On the other hand, it significantly alters the overall thermostability of the supermodule even in complex with ADGRV1pbm. The other substitution G228R in PDZ2 has an even greater effect by targeting the more unstable PDZ domain and the best ADGRV1 binder in the tandem.in vitro using purified mutated PDZ constructs.Finally, we used pull-down assays to test the interaction between full-length PDZD7 wild-type and mutants , and ADGRV1 β subunit, encompassing the seven transmembrane segments and cytoplasmic domain. Proteins are expressed separately in HeLa cells and lysates are mixed after sonication. Addition of G sepharose beads coupled to an anti-GFP antibody  allowed to bind PDZD7. Three experiments were performed in parallel for each mutant; first a pull-down of ADGRV1 without bait to rule out unspecific binding (negative control), then with WT PDZD7 (positive control) and finally with the considered mutant. For each experiment, the same samples corresponding to the input, unbound, wash and elution fractions are loaded in equal amounts into two twin gels (top and bottom parts). Each gel is used to detect the presence of either PDZD7-GFP . TherefoPDZD7 and ADGRV1 are the main candidate proteins for regulating the signaling associated with the Usher2 complex. PDZD7 is primarily a scaffolding protein, with several protein-protein interaction domains (three PDZ domains and one HHD) likely recruiting several partners at the ankle links anchoring sites . Interes1H-15N spectra of the PDZ2 similar to the ones observed in PDZ1, suggesting comparable contacts with the PDZ core. Lastly, complex formation with ADGRV1pbm also leads to a change in dynamics of the extension for both PDZ, resulting in a loss of signals by NMR. Altogether, these results support an accessible 2-3 β strands conformation of each PDZ extension in solution.Here we show that both N-terminal PDZ1 and PDZ2 domains of PDZD7 can interact with the C-terminal PDZ binding motif of ADGRV1. Our results highlight a critical contribution of atypical PDZ domain C-terminal extensions to the binding, tuning affinity towards partners. Indeed, the presence of these extensions results in a ∼20 to 100 fold increase in affinity for each PDZ towards ADGRV1pbm. In addition, the ADGRV1 motif preferentially interacts with PDZD7 PDZ2, with a ∼15 fold higher affinity (Kd = 3.1 µM) compared to PDZ1 (Kd = 47 µM). Therefore, ADGRV1pbm binds to PDZ2 in the higher range of affinity than what is commonly found for PDZ domain interactions (1-100 µM). As monitored by NMR on PDZ1, the C-terminal extension of the domain adopts an original three β strands conformation in solution, that is at least partially in contact with the core of the PDZ , consistent with AlphaFold’s prediction. Interestingly, addition of the ADGRV1pbm peptide induces a change in dynamics of the extension, potentially favoring the folded state, and a compaction onto the PDZ core. The increase of affinity induced by the extension could arise from a modulation of the dynamics of the PDZ core in favor of the bound state, or from additional contacts between the extension and the partner upstream of its PBM, as observed in the homologous protein harmonin in complex with the protein SANS   PBM such as the one of ADGRV1 (-THL-COOH). By contrast, PDZD7 PDZ1 unusually exhibits a methionine at position 1 of its α2 helix (only four amongst the 266 human PDZ domains), more suitable for type 2 (-Φ-X-Φ-COOH) PBM binding. Therefore, PDZD7 PDZ1 might recruit other partners in the vicinity of ADGRV1  and ADGRV1 whole cytoplasmic domain (152 intracellular residues). We confirmed that PDZD7 residues upstream of the tandem do not exhibit tertiary structures, are largely extended and flexible in solution and do not induce perturbations onto the PDZ1 core. Similarly, we have shown that the isolated cytoplasmic domain of ADGRV1 is mostly unfolded and flexible. In terms of interaction, we measured similar affinities for ADGRV1 PBM or whole cytoplasmic domain with the PDZD7 constructs we tested. In conclusion, the PBM of ADGRV1 and the PDZ tandem of PDZD7 are the only determinants of interaction between the two proteins in our experiments. Altogether, our results highlight the critical role of flanking regions in PDZ interactions and supramodular organization, which has not been systematically documented thus far. Interestingly, five out of the six PDZ located in the N-terminal regions of the three paralogous proteins harmonin, whirlin and PDZD7 have their binding capacity modulated by domain extensions. These sequences have been conserved during evolution and could play a unique role as modulator of affinity and specificity. More generally, PDZ domains are known to form hetero and homotypic supramodules. One of the most extensively studied supramodule is the three-domains PDZ3-SH3-GK from the protein PSD-95 . In addiThe regulation of GPCR trafficking by PDZ-containing proteins has been well documented . However