How kinesin motor proteins pick up and transport their cargo from one defined location to another is poorly understood. Serena et al. investigate how MKLP2 recognizes its cargo, the chromosome passenger complex, and promotes its transport away from chromosomes in anaphase. The Aurora B chromosomal passenger complex (CPC) is a conserved regulator of mitosis. Its functions require localization first to the chromosome arms and then centromeres in mitosis and subsequently the central spindle in anaphase. Here, we analyze the requirements for core CPC subunits, survivin and INCENP, and the mitotic kinesin-like protein 2 (MKLP2) in targeting to these distinct localizations. Centromere recruitment of the CPC requires interaction of survivin with histone H3 phosphorylated at threonine 3, and we provide a complete structure of this assembly. Furthermore, we show that the INCENP RRKKRR-motif is required for both centromeric localization of the CPC in metaphase and MKLP2-dependent transport in anaphase. MKLP2 and DNA bind competitively to this motif, and INCENP T59 phosphorylation acts as a switch preventing MKLP2 binding in metaphase. In anaphase, CPC binding promotes the microtubule-dependent ATPase activity of MKLP2. These results explain how centromere targeting of the CPC in mitosis is coupled to its movement to the central spindle in anaphase. Aurora B is a crucial mitotic kinase that regulates chromosome condensation in prophase, microtubule attachment to kinetochores during prometaphase and metaphase, and anaphase spindle microtubule dynamics and cytokinesis . These fA fundamental insight into the molecular mechanism underpinning the complex spatial and temporal pattern of CPC localization came from the identification of a ternary subcomplex formed by survivin, borealin, and the N-terminal region of INCENP . Further1-76, and the minimal module comprising the first 58 amino acids of INCENP [INCENP1-58]) has been proposed to be the minimal assembly required for targeting to centromeric chromatin in mitosis, CPC targeting is further enhanced by additional signals in both INCENP and borealin. The INCENP single α-helical domain is involved in chromatin and microtubule binding . In addition, the structure of dimeric survivin bound to an H3T3-phosphorylated peptide has been solved . Both complexes crystallized in the P65 space group and diffracted to 1.81 and 2.55 Å, respectively. We were able to identify the first five residues of the bound peptide (A1–Q5) for CPI58 and the first seven residues (A1–A7) for CPI80. However, we were unable to see INCENP from 45 onward, suggesting it might be flexible, as reasonably expected around the T59 CDK1 phosphorylation site. In both CPI58 and CPI80 complexes, H3pT3 was bound to survivin that, as previously described, is stabilized by a structural Zn2+ ion, coordinated by C57, C60, H77, and C84 , confirming a role for K62 in phosphate recognition  of ∼1 µM (0.8 ± 0.3 µM for CPI58 and 1.1 ± 0.5 µM for CPI80) and to the H3T3 nonphosphopeptide with a Kd of 4–6 µM  with purified components. Addition of CPI80 to assays resulted in a mobility shift in the 2.9-kb linear duplex DNA used . This shty shift . These fty shift . We usedty shift . CPI80 sectively . Both CPectively , and theR mutant  failed tlite DNA .Together, these data support the conclusion that the interaction of the CPC with chromatin is mediated by both selective binding of histone H3 by survivin and interaction of the INCENP RRKKRR-motif with DNA. There is a slight ∼1.25-fold but reproducible preference for α-satellite DNA that suggests there may be some sequence specificity to the interaction, but this requires further investigation. We then investigated the relationship between the mechanisms of chromatin binding from prophase to metaphase and CPC release from chromatin and subsequent localization to the central spindle in anaphase.T59E still coprecipitated phosphorylated histone H3 in metaphase  was used to give an unbiased estimation of molecular mass and stoichiometry. Recombinant CPI80 forms a 1:1:1 trimer of survivin, borealin100.3 kD , and dim100.3 kD . By contSurvivin E65A-H80A was able to form SEC-stable complexes  and 20 mM MPS1 inhibitor AZ3146 (Tocris Bioscience). Thymidine  and doxycycline  were dissolved in water. Commercially available polyclonal antibodies (pAbs) or mAbs were used for tubulin , cyclin B1 , GFP , Histone H3 , Histone H3pT3 , Aurora B , Survivin , Borealin , Histone H3pS10 , and PRC1 pT481 . Human CREST serum was obtained from Antibodies Inc. (15-234-0001). Sheep antibodies produced against full-length Aurora B, MKLP2 . Mammalian expression constructs for MKLP2, INCENP, and survivin were made in pcDNA5/FRT/TO (Invitrogen) modified to encode EGFP tag . Mutagenesis to introduce specific point mutations was performed using the QuickChange method (Agilent Technologies), whereas MKLP2 deletion mutants were obtained by overlapping PCR starting from the WT sequence. siRNA duplexes targeting the 3′ UTR were used to target endogenous MKLP2 (5′-CCACCUAUGUAAUCUCAUG-3′), INCENP (5′-GGCUUGGCCAGGUGUAUAU-3′), and survivin (5′-GCAGGUUCCUUAUCUGUCA-3′). As a negative control, the luciferase GL2 duplex was used. Bacterial expression constructs for INCENP and MKLP2 were made in the pFAT2-His6-GST plasmid, with a human rhinovirus (HRV) 3C protease cleavage site inserted between GST and the insert. The pETDuet-1 expression vector (Addgene) was used to coexpress survivin and borealin: the former was cloned into the multiple cloningsite I with an N-terminal His tag, the latter was cloned into the multiple cloning site II. Recombinant baculovirus carrying His6-tagged full-length MKLP2 cloned into the pFastBac vector was produced using the Baculovirus Expression System (Invitrogen).All DNA primers were obtained from Invitrogen. Coding sequences of MKLP2, INCENP, borealin, and survivin were amplified from human testis cDNA (Becton Dickinson) using the 2. For plasmid transfection and siRNA transfection, Mirus LT1 (Mirus Bio LLC) and Oligofectamine (Invitrogen), respectively, were used. In both cases, transfection mix was prepared in Opti-MEM. HeLa Flp-In T-REx cell lines with single integrated copies of full-length WT GFP-INCENP point mutants T59E, T59A, RRKRR, and truncation mutants amino acids 1–58, 1–80, 1–80 RRKKRR to hexa-alanine were created using the T-REx doxycycline-inducible Flp-In system and selection with hygromycin . Sf9 insect cells were grown in insect cell growth medium  at 27°C and atmospheric CO2.HeLa and HeLa Flp-In T-REx cells were cultured in DMEM with 1% (vol/vol) GlutaMAX (Life Technologies) containing 10% (vol/vol) bovine calf serum at 37°C and 5% CO4Cl in PBS and a 5-min cell permeabilization with 0.2% (vol/vol) Triton X-100 in PBS. Antibody dilutions were performed in PBS: affinity-purified antibodies were used at 1 µg/ml, whereas commercial antibodies were used according to manufacturers’ instructions. Hoechst 33258 was added to the secondary antibody staining solution at 0.3 µg/ml. Coverslips were mounted in Mowiol 4-88 mounting medium (EMD Millipore). Samples were imaged using a 60×/1.35 NA oil immersion objective on a BX61 Olympus microscope equipped with filter sets for DAPI, EGFP/Alexa Fluor 488, Alexa Fluor 555, and Alexa Fluor 647 (Chroma Technology Corp.) and a Photometrics Prime camera. Image stacks with spacing of 0.4 µm through the cell volume were taken and then converted in RGB TIFF files using MetaMorph 7.8 imaging software (Molecular Devices). Images were then cropped in Adobe Photoshop and placed into Adobe Illustrator to produce the figures.Cells were seeded in six-well plates on no. 1.5 thickness glass coverslips . In siRNA rescue assays, either doxycycline induction or plasmid transfection was performed 6 h before silencing. For synchronization, cells were treated for 18 h with 2 mM thymidine and washed three times in PBS and twice with growth medium. Cells were then fixed with 3% (wt/vol) paraformaldehyde in PBS followed by quenching with 50 mM NH3VO4, protease inhibitor cocktail [Sigma-Aldrich], and phosphatase inhibitor cocktail [Sigma-Aldrich]). The cell lysate was supplemented with 2 mM MgCl2 before addition of 40 U of Benzonase (Sigma-Aldrich) and then incubated for 30 min on ice, 30 min at room temperature, and 30 min on ice. Proteins were then clarified by centrifugation, and protein complexes were isolated from 3 mg of cell lysate using 3 µg of sheep anti-GFP or anti-MKLP2 antibodies and 20 µl of protein G Sepharose. Isolated complexes were washed once with lysis buffer and three times with wash buffer . Samples were then resuspended in 3× Laemmli buffer for Western blotting.HeLa cells were arrested with 0.1 µM nocodazole for 16 h, and mitotic cells were collected by shake-off and washed three times with PBS and twice with growth medium warmed to 37°C. Metaphase cells were collected after incubation in DMEM at 37°C for 25 min. Anaphase cells were collected at different time points after the addition of AZ3146. At each time point, cells were washed with ice-cold PBS. Cell pellets were suspended in mitotic lysis buffer . Cell lysates were then supplemented with 2 mM MgCl2 before addition of 40 U of Benzonase and incubated for 30 min on ice, for 30 min at room temperature, and for 30 min on ice. Proteins were then clarified by centrifugation, and 1 mg of cell lysate was analyzed by SEC using a Superose 6 10/300 column . All chromatography steps were performed at 4°C using a Prime Plus System . Eluted fractions were then analyzed by Western blot.GFP-MKLP2 (WT or Δ636-652)–transfected HeLa cells were arrested with 0.1 µM nocodazole for 16 h, and mitotic cells were collected by shake-off. Cell pellets were suspended in mitotic lysis buffer  were cultured in Luria–Bertani media in the presence of 100 µg/ml ampicillin and 34 µg/ml chloramphenicol. DNA transformation was performed by heat shock, and protein expression was induced overnight at 18°C after the addition of 0.1 mM IPTG. Cells were harvested by centrifugation at 4°C and lysed in wash buffer , supplemented with 0.5 mg/ml lysozyme, 5 U/ml DNase (Roche), and 1 tablet of Complete Ultra Mini EDTA-free Protease Inhibitor Cocktail (Roche) per liter of culture. For CPC purification, INCENP and survivin–borealin pellets were lysed together. After sonication and centrifugation, the supernatant was incubated for 2 h with Glutathione Sepharose 4B  and then loaded into a column. After washing with wash buffer, protein complexes were eluted with 20 mM reduced glutathione. Tags were cleaved away from protein complex during overnight dialysis at 4°C against wash buffer, in the presence of the GST-tagged HRV 3C protease . The CPC was then purified by SEC in wash buffer supplemented with 10% glycerol using a HiLoad 16/600 Superdex 75-pg column  followed by GSTrap 4B columns . If necessary, the complex was further purified by anion exchange using a Mono Q 5/50 GL column , with a gradient of NaCl. MKLP2 purification was similarly performed, but using a HiLoad 16/600 Superdex 200-pg column . MKLP2:CPC complexes were obtained after incubation of CPC variants and MKLP2557-668 in a 1:1 molar ratio for 30 min in ice in binding buffer , followed by SEC using a HiLoad 16/600 Superdex 200-pg column. MKLP2- and CPC-binding assays were performed on an analytical Superdex 200 increase 10/300 column . All chromatography steps were performed at 4°C using an EttanLC System .BL21-CodonPlus (DE3)-RIL NT equivalent to previous work , CPI80 , and MKLP2596-668 were dialyzed against 50 mM MES, pH 6.8, and 2 mM DTT and concentrated up to 10 mg/ml. For peptide binding, H3pT3 (ARTPKQTARKSTG) was incubated with the CPC in a 2:1 molar ratio. CPINT crystals were obtained in 50 mM MES, pH 6.0, 5% (wt/vol) polyethylene glycol (PEG) 3350. CPI58:H3pT3 crystals were obtained in 0.2 M lithium sulfate, 0.1 M MES, pH 6.5, and 20% (wt/vol) PEG 400. CPI80:H3pT3 crystals were obtained in 0.15 M ammonium sulfate, 0.1 M Hepes-NaOH, pH 7.0, and 20% (wt/vol) PEG 400. MKLP2596-668 crystals were obtained in 0.1 M sodium citrate, pH 5.6, 20% (vol/vol) 2-propanol, and 20% (wt/vol) PEG 400. When needed, 20% (vol/vol) PEG 400 or 2-methyl-2,4-pentanediol, prepared using mother liquor, was used as cryo-protectant. CPINT diffracted to 3.5-Å resolution at the P14 beamline operated by EMBL Hamburg at the PETRA III synchrotron . CPI58:H3pT3 and MKlp2596-668 crystals diffracted to 1.81 and 1.43 Å, respectively, at the I24 beamline at Diamond Light Source synchrotron . CPI80:H3pT3 crystals diffracted to 2.55-Å resolution at the ID23-1 beamline at the European Synchrotron Radiation Facility  synchrotron.CPI2QFA (5LEF (https://pymol.org/2/).CPC data were processed and integrated using DIALS , and cry2QFA  as a mod2QFA  and STAR2QFA  and PDB FA , H2A (119–129: KKTESHHKAKG), and H2ApT121 (119–129: KKTPESHHKAKG). Lyophilized peptides were dissolved against ITC buffer . Protein samples were dialyzed overnight at 4°C against the same ITC buffer. All the binding experiments were performed on a MicroCal PEAQ-ITC instrument  at 25°C. Protein concentration was 50 µM, whereas peptide concentration was 400 µM. First titration was performed using 0.5 µl of peptide, followed by 19 2-µl injections applied 180 s apart. Data were fitted using MicroCal PEAQ-ITC software with a 1:1 binding model.All peptides were synthesized by Peptide Protein Research Ltd: H3 (1–12: ARTKQTARKSTG), H3pT3 .Assays were performed with BamHI-linearized pBluescript plasmid (0.15 pmol), 3.6 µM the different CPC complexes, and control proteins in 20 µl of 100 mM NaCl, 20 mM Tris-HCl, pH 7.4, and 1 mM DTT for 2 h on ice. Reactions were analyzed on a 0.8% (wt/vol) agarose gel in the presence of 0.8 µg/ml ethidium bromide. For CPI80 titration, the concentration range indicated in Kd calculation were performed using the NanoTemper analysis software.MST experiments were performed on a Monolith NT.115 instrument using a blue filter (NanoTemper Technologies) and Alexa 488–labeled DNA for non–α-satellite DNA  and a centromeric human α-satellite DNA consensus  glycerol. Elution was monitored via online static light-scattering , differential refractive index  and UV  detectors. Data were analyzed using the ASTRA software package (Wyatt Technology).6-tagged MKLP2 used to infect 4 × 107 insect cells in 20 × 15-cm dishes with a multiplicity of infection of 10. To infect cells, the medium was removed, and the virus was added in 3 ml of insect growth medium per dish. Dishes were gently rocked for 1 h; every 10 min the dishes were rotated by 90°. After 1 h, 17 ml of insect cell growth medium was added, and the cells were left for 60 h before harvesting by centrifugation at 200 g. To obtain M-phase, CDK-phosphorylated MKLP2, the cells were treated for 3 h with 100 nM okadaic acid before harvesting. Cell pellets were washed once in ice-cold PBS and then lysed in 10 ml of IMAC20 , and protease inhibitor cocktail (Sigma-Aldrich) for 20 min on ice. A cell lysate was prepared by centrifugation at 100,000 g in a TLA100.3 rotor (Beckman Coulter) and then loaded onto a 1-ml HisTrap FF column  at 0.5 ml/min. The column was then washed with 30 ml of IMAC20 and eluted with a 20-ml linear gradient from 20 to 200 mM imidazole in IMAC20, collecting 1-ml fractions. Peak fractions judged by SDS-PAGE and absorbance at 280 nm were concentrated using Ultracel-10K centrifugal filters (EMD Millipore) according to the manufacturer’s instructions to a final volume of ∼1 ml. Samples were then buffer exchanged using 5 ml of Zeba Desalt Spin columns (Perbio) into 20 mM Tris-HCl, pH 8.0, 300 mM NaCl, and 1 mM DTT. Protein samples were snap-frozen in 15-µl aliquots and stored at –80°C for further use.Recombinant baculoviruses encoding His2, 0.5 mM EGTA, and 60% [vol/vol] glycerol) and 0.5 µl 100 mM GTP at 30°C for 20 min. To stabilize microtubules, 8 µl of 2 mM paclitaxel in 750 µl of tubulin buffer  was added to the microtubule polymerization reaction. This 3.75 µM tubulin stock mix was stored at room temperature until further use. A microtubule premix was created by mixing 1 ml of reaction buffer , 10 µl of 2 mM paclitaxel, 235 µl of Taxol-stabilized microtubules, 240 µl of 1 mM 2-amino-6-mercapto-7-methylpurine riboside, and 12 µl of 0.1 U/µl purine nucleoside phosphorylase. Reactions were set up in 96-well plates by mixing the proteins of interest in a total volume of 7.5 µl. Reactions were set up in 96-well plates by mixing the protein of interest in 150 µl of microtubule reaction mix. To start the assay, 10 µl of 10 mM ATP was added to each well. This was then rapidly transferred to a 37°C plate reader  set to read absorbance at 360 nm. Readings were acquired every 30 s for 1 h. An inorganic phosphate standard curve was created in the same assay buffer and used to convert absorbance to nanomoles of hydrolyzed ATP.A commercial enzyme-linked inorganic phosphate assay  was used to measure MKLP2 ATPase activity. Taxol-stabilized microtubules were produced by incubating 45 µl (7.4 mg/ml) of porcine tubulin, 4.5 µl of cushion buffer  or at the central spindle , divided by the fluorescence measured into the cytoplasm (fcytoplasmic). H3pS10 relative intensity (fpS10/fGFP-INCENP) was calculated normalizing the fluorescence value for H3pS10 intensity (fpS10) to the respective INCENP fluorescence value detected on centromeres (fGFP-INCENP) of the same cell. All fluorescence values were corrected by subtracting background intensity.INCENP localization either on centromeres or the central spindle was calculated as a ratio between the fluorescence measured, respectively, on chromatin .Statistical analysis for immunofluorescence data was performed using the unpaired MST data were analyzed by two-way ANOVA, considering dilution factor and DNA sequence (centromeric α-satellite or non–α-satellite) as independent variables, using StataMP 14.0 (Stata Corp.). A significance level of P < 0.05 was accepted as statistically significant.NT showing crystal contacts involving the INCENP N terminus and further structural details of phospho-histone binding to CPI58 and CPI80 are presented in Review HistoryClick here for additional data file.Table S1lists data collection and refinement statistics for structures.Click here for additional data file.Table S2shows original data traces for the MST experiments.Click here for additional data file.


We found that the DesAbs induce a change in structure of the Zn2+-stabilized Aβ40 oligomers, generating a simultaneous increase in their size and solvent-exposed hydrophobicity. We then observed that these increments in both the size and hydrophobicity of the oligomers neutralize each other in terms of their effects on cytotoxicity, as predicted by a recently proposed general structure–toxicity relationship, and observed experimentally. These results illustrate the use of the DesAbs as research tools to investigate the biophysical and cytotoxicity properties of Aβ oligomers.Alzheimer’s disease is associated with the aggregation of the amyloid-β peptide (Aβ), resulting in the deposition of amyloid plaques in brain tissue. Recent scrutiny of the mechanisms by which Aβ aggregates induce neuronal dysfunction has highlighted the importance of the Aβ oligomers of this protein fragment. Because of the transient and heterogeneous nature of these oligomers, however, it has been challenging to investigate the detailed mechanisms by which these species exert cytotoxicity. To address this problem, we demonstrate here the use of rationally designed single-domain antibodies (DesAbs) to characterize the structure–toxicity relationship of Aβ oligomers. For this purpose, we use Zn The development and progression of Alzheimer’s disease (AD) is characterized by the misfolding and aggregation of the amyloid-β peptide (Aβ), which eventually results in the formation of dense senile plaques rich in fibrillar Aβ aggregates ,2,3. IncIntense focus on inhibiting Aβ deposition has resulted in the characterization of numerous molecules, including small molecules, antibodies and molecular chaperones, which are able to prevent Aβ aggregation, and therefore, reduce the rate of Aβ oligomer formation, although no disease-modifying compound has yet been approved for clinical use ,11. Thes18-24) is a potent inhibitor of the microscopic step of primary nucleation of the Aβ42 amyloid fibril formation process. Moreover, its administration to a C. elegans model of AD was shown to reduce the toxicity implicit to Aβ42 aggregation when administered before or during the development of a pathological phenotype [40 (DesAb34-40) on stabilized cytotoxic oligomeric species of Aβ40 after their formation, which were generated from both primary and secondary nucleation pathways in the endogenous Aβ aggregation reaction [Given the advantages of using antibodies as research tools ,23, in preaction .40 oligomers, we first determined the effects of DesAb18-24 and DesAb34-40 on the in vitro aggregation of Aβ40. Then, we analyzed the effects of these DesAbs on the size and hydrophobicity of Zn2+-stabilized Aβ40 oligomers formed under near physiological conditions. We found that the DesAbs induced the formation of larger and more hydrophobic Aβ40 oligomers. As a control, we then showed that the natural product trodusquemine, which protects cell membranes from the cytotoxic effects of protein misfolded oligomers at substoichiometric concentrations [40 did not significantly change the degree of cytotoxicity of the oligomers towards SH-SY5Y neuroblastoma cells, indicating that the simultaneous increase in oligomer size and hydrophobicity produces antagonistic effects on oligomer toxicity.To investigate the relationship between the structure and the toxicity of Aβtrations , inducestrations ,34,35,36trations , we obse18-24 and DesAb34-40, which target the regions 18–24 and 34–40 of Aβ40, respectively. These antibodies were generated by grafting a complementary peptide into the third complementary determining region (CDR3) of a single-domain antibody scaffold, as described previously [In this work, we investigate two computationally-designed, single-domain antibodies, called DesAbeviously . 40 aggregation. Samples containing monomeric Aβ40 at a concentration of 10 µM  were prepared in the absence and presence of 20-, 10-, and 5-fold substoichiometric concentrations of the designed antibodies and incubated under quiescent conditions at 37 °C. The fibrilization process was monitored by means of thioflavin T fluorescence (ThT) in the presence of 20 µM ThT. We observed that DesAb18-24 strongly inhibits Aβ40 aggregation, causing a significant delay in the half-time of aggregation (t1/2) (42 [18-24 (2 µM), Aβ40 did not aggregate beyond t1/2 in this experiment on the time course of 25 h. Further experiments on DesAb34-40 revealed that this designed antibody was also able to interfere with the reaction, as observed by an overall delay in the aggregation kinetics (1/2 (40 aggregation. We first investigated the effect of these two DesAbs on Aβn (t1/2) a, which kinetics b. Intereics . In agreement with the turbidity measurements, we found that the incubation of the stabilized oligomers in the presence of increasing concentrations of DesAbs resulted in augmented light scattering e,f. As apendence . Taken tructures , and we 34-40 complex by phase-controlled atomic force microscopy (AFM) [40 oligomers were incubated as previously described at a concentration of 10 µM  in the absence and presence of 2 µM DesAb34-40 , and diluted in buffer by a factor of 20 before conventional, manual deposition atop an atomically flat mica surface. In the absence of DesAb34-40, oligomers were 1–5 nm in height  ,46. Stabn height , in agreeriments .42 oligomers at superstoichiometric concentrations, whereas the effects were not significant at substoichiometric concentrations [The natural product trodusquemine has been characterized for its ability to displace protein misfolded oligomers from the surfaces of cell membranes, resulting in a marked decrease in oligomer-induced cytotoxicity towards neuroblastoma cells. Its protective effects were observed at physiological concentrations of this aminosterol, corresponding to equimolar or substoichiometric concentrations of the molecule relative to oligomers, where greater than equimolar concentrations of trodusquemine demonstrated its intrinsic toxicity to cells . Furthertrations . 40 oligomers similarly to our DesAbs, and thus to obtain a control case for this study, we incubated stabilized Aβ40 oligomers at a concentration of 5 µM  in 20 mM Tris, 100 mM NaCl, pH 7.4, in the absence and presence of increasing concentrations of trodusquemine . After incubation, ANS was added to a final concentration of 15 µM . The incubation of Aβ40 oligomers with trodusquemine increased the maximum fluorescence intensity and caused a further blue-shift of its wavelength, with a well-defined dose dependence  and subsequently diluted by a factor of five in buffer prior to spraying atop an atomically flat mica surface  were formed and incubated in the absence and presence of increasing concentrations of DesAb18-24 and DesAb34-40  for 1 h at 37 °C, and subsequently added to the cell culture medium of SH-SY5Y cells for 24 h at 37 °C. These concentrations were selected to match the biophysical experiments. The cells were also treated with the corresponding concentrations of antibodies in the absence of oligomers, which was not observed to induce deleterious changes in cellular viability. Aβ40 oligomers were found to decrease the viability of the cells by 23.4 ± 3.6%, and this toxicity was not changed significantly upon the addition of either DesAb when co-incubated with oligomers at these concentrations  and at the same molar ratios studied above, i.e., 0.5 µM, 1 µM and 2 µM antibodies . Again, dictions . 40 oligomers incubated with trodusquemine at the superstoichiometric concentrations that are necessary to observe a significant clustering effect of the oligomers [It was not possible to carry out the MTT assays for Aβligomers . Indeed,ligomers . Such an2+-stabilized Aβ40 oligomers, which we adopted as models of the Aβ oligomers that may form in synaptic regions where Zn2+ tends to be abundant [Aβ oligomers are highly cytotoxic species associated with the aggregation reaction of Aβ, which is central to the onset and development of AD ,5,6,7. Eabundant . We usedabundant  or theraabundant . 42, HypF-N, and islet amyloid polypeptide through a clustering-based mechanism [18-24 and DesAb34-40, and trodusquemine at superstoichiometric concentrations, Aβ40 oligomer size was significantly increased upon interaction with the antibodies and the aminosterol.An increase in size alone, in the absence of other physicochemical changes, has been well documented to suppress oligomer cytotoxicity. Indeed, high concentrations of molecular chaperones, including clusterin and aβ-crystallin, have been shown to reduce the cellular toxicity of oligomers comprised of Aβechanism ,33,35. Techanism ,35. In t18-24 and DesAb34-40 and trodusquemine at superstoichiometric concentrations, however, Aβ40 oligomer hydrophobicity was significantly increased upon interaction with the antibodies and molecules. In addition to size, hydrophobicity is a key determinant of oligomer cytotoxicity ,36. A deThe DesAbs modified the structure of the oligomers in a way that is expected to increase their cytotoxicity by increasing their hydrophobicity. At the same time, the DesAbs could also be expected to decrease the toxicity of the oligomers by increasing their size. Thus, by using the DesAbs as molecular tools, we leveraged a well-described size–hydrophobicity formula  to predi18-24 and DesAb34-40 to small molecules. Trodusquemine and other molecules within the aminosterol family possess the unique ability to bind to cell membranes and prevent the deleterious association of oligomers via a displacement-based mechanism ,56,57. GBased on these results, we anticipate that it may become possible in future systematic studies to obtain more insights into the structure-toxicity relationship of oligomers with new DesAbs targeting disease-related proteins. Utilization of in vitro assays capable of predicting the toxicity of oligomers by probing their structural and physicochemical properties could facilitate enhanced throughput preclinical drug discovery studies by limiting in part the need of more expensive and time-consuming cell culture experiments.Trodusquemine was synthesized as a hydrochloride salt at a purity greater than 97%, stored as a lyophilized powder ,59 and sE. coli Rosetta-gami 2 , cells were grown, harvested and lysed, and the antibodies (circa 18 kDa) were purified using a Ni2+-NTA Superflow column  [18-24 is MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPKLEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIGWVRRAPGKGEEWVASIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAAGSVFVGTEAEEEAAAWGQGTLVTVSSGT and that of DesAb34-40 is MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDPKLEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIGWVRRAPGKGEEWVASIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAAGSIRETALLYEEEAAAWGQGTLVTVSSGT. Antibodies were computationally designed as previously described ,37. Compes circa  kDa werees circa  kDa were40, was expressed in the Escherichia coli BL21 Gold (DE3) strain  and purified as described previously [280 = 1400 L mol−1 cm−1 [The recombinant Aβ(M1-40) peptide (sequence MDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV), denoted as Aβeviously . Samplesl−1 cm−1 . 40 at a concentration of 10 µM in the absence or presence of 0.5 µM, 1 µM, and 2 µM DesAbs. All samples for the kinetic measurements were supplemented with 0.2 mM ethylenediaminetetraacetic acid (EDTA) [For kinetic experiments, samples were prepared with monomeric Aβd (EDTA) . ThT was40 was solubilized overnight in 300 µL hexafluoroisopropanol (HFIP) at 4 °C. Solvent was evaporated with a gentle flow of nitrogen gas, and the protein was resuspended in DMSO to 2.2 mM. Two sonication steps of 10 min were preformed, after which the protein was diluted at a concentration of 100 µM in 20 mM sodium phosphate buffer, 200 μM ZnCl2, pH 6.9 for 20 h at 20 °C. Samples were centrifuged  to a pellet and the supernatant was removed [First, 1 mg of lyophilized Aβ removed . Oligome40 oligomers  were incubated  in the absence and presence of increasing concentrations of DesAbs  at. Samples containing trodusquemine were incubated  at an oligomer concentration of 5 µM  in the absence or presence of the small molecule at concentrations ranging from 5 µM to 50 µM. Subsequently, ANS was added to a final concentration of 30 µM or 15 µM, respectively . Emission spectra were recorded using a plate reader (BMG Labtech) with excitation at 380 nm. Duplicate samples are representative of three independent experiments that gave consistent results. All spectra were background subtracted to the buffer.First, 10 µM AβSamples from the ANS preparation were analyzed using a plate reader (BMG Labtech) with spectral scanning. Duplicate samples are representative of three independent experiments that gave consistent results. All spectra were background subtracted to the buffer.40 oligomers  were incubated for 2 h in the absence and presence of increasing concentrations of DesAbs  at 20 °C. SLS measurements were performed using the Malvern Zetasizer Nano S instrument  with fixed parameters , equipped with a Peltier temperature controller (25 °C). A low volume (70 µL) disposable cuvette was used . Samples containing trodusquemine were incubated  at an oligomer concentration of 5 µM  in the absence or presence of the small molecule at concentrations ranging from 5 µM to 50 µM.First, 10 µM Aβ40 oligomers were prepared at a concentration of 10 µM  and incubated in the absence or presence of 0.5, 1.0 and 2 µM DesAbs, as described above. For sedimentation measurements, 50 µL aliquots of samples were centrifuged  for 10 min at 13,000× g and 20 °C. Supernatant was removed and protein was thoroughly resuspended in 20 µL buffer prior to spotting the nitrocellulose membrane (2 µL per spot) . Membranes were blocked with skim milk (Sigma-Aldrich) for 1 h, after which time the primary antibody 6E10  was incubated at a 1:1000 dilution overnight at 4 °C. The Alexa488-conjugated secondary antibody  was subsequently incubated for 1 h at a 1:5000 dilution at 20 °C. Fluorescence was detected using a Typhoon Trio Imager . Membranes were prepared in duplicate, and representative images are shown. ImageJ was used for quantifications.Aβ40 oligomers in PBS overnight at 4 °C. Blots were then probed with the mouse monoclonal anti-amyloid β antibody (clone 6E10)  overnight at 4 °C, and then with goat anti-mouse IgG (H+L) secondary antibody and AF488 conjugate  for 2 h at 25 °C. In order to assess the binding of the DesAbs, dot blots were prepared as follows: 4 μM solutions of DesAb were spotted (3.5 μL) onto a 0.2 μm pore size nitrocellulose membrane corresponding to total deposited antibody of 50, 25, 12.5 and 6.3 µg DesAb. The blots were blocked in PBS, 5% BSA overnight at 4 °C. Subsequently, they were incubated in solutions containing 5 μM Aβ40 oligomers  were incubated in the absence and presence of 2 µM DesAb34-40, as previously described, and diluted by a factor of 20 before deposition atop an atomically flat mica surface. AFM samples preparation was carried out at room temperature by deposition of a 10 µL drop of solution deposited for 2 min to a freshly-cleaved mica surface. Salt was washed with water, and samples were dried by the gentle flow of nitrogen and stored in a sealed container until imaging using a Park NX10 AFM  with scan rates < 0.4 Hz and PPP-NCHR cantilevers with an 8 nm nominal radius . To visualize the trodusquemine-oligomer complex, Aβ40 oligomers  were incubated in the absence and presence of 25 µM trodusquemine for 1 h as previously described. These samples were diluted in buffer by a factor of five and subsequently sprayed at 100 µL/h for 1 min. at room temperature using a recently described microfluidic device [−1, MikroMasch, Wetzlar, Germany). For all samples, standardized scanning conditions were established and maintained while measuring the samples, such that a phase change on the order of ≈Δ20° upon the interaction of the tip with the sample was not exceeded; changes corresponding to dissipated energy during the sample–tip interactions in excess of this threshold can lead to artifacts in the height quantification caused by deformations induced in the sample by the tip [Aβc device  atop an  the tip ,45,61. T the tip ,61.3 (1:1) and supplemented with 10% FBS, 1 mM glutamine and 1.0% antibiotics. Cell cultures were maintained in a 5% CO2 humidified atmosphere at 37 °C, and grown until they reached 80% confluence for a maximum of 20 passages [Human SH-SY5Y neuroblastoma cells  were cultured in DMEM, F-12 HAM with 25 mM HEPES and NaHCOpassages ,63. Cell40 oligomers  were incubated in the absence and presence of 20:1 to 5:1 molar ratios of the designed antibodies in cell culture medium for 1 h at 37 °C under shaking conditions, and then added to the cell culture medium of SH-SY5Y cells seeded in 96-well plates for 24 h. The 3--2,5-diphenyltetrazolium bromide (MTT) assay was performed as previously described [Aβescribed ,64.t-test. p < 0.05 was accepted as statistically significant. Data are available from the authors upon request.Statistical analyses were performed in GraphPad Prism 8 . All error bars denote standard error of the mean (SEM). One-way ANOVA was used for multiple comparisons as a function of increasing concentration of DesAbs relative to oligomers incubated in buffer or cell culture medium (with Bonferroni’s multiple comparisons test). Comparisons between untreated cells and oligomer-exposed cells were performed using an unpaired, two-tailed Student’s


Plasmodium falciparum. It remains a major global health problem, and there is no highly effective vaccine. A parasite protein called RH5 is centrally involved in the invasion of host red blood cells, making it—and the other parasite proteins it interacts with—promising vaccine targets. We recently identified a protein called P113 that binds RH5, suggesting that it anchors RH5 to the parasite surface. In this paper, we use structural biology to locate and characterize the RH5 binding region on P113. These findings will be important to guide the development of new antimalarial vaccines to ultimately prevent this disease, which affects some of the poorest people on the planet.Malaria is a deadly infectious disease primarily caused by the parasite  Plasmodium falciparum RH5 is a secreted parasite ligand that is essential for erythrocyte invasion through direct interaction with the host erythrocyte receptor basigin. RH5 forms a tripartite complex with two other secreted parasite proteins, CyRPA and RIPR, and is tethered to the surface of the parasite through membrane-anchored P113. Antibodies against RH5, CyRPA, and RIPR can inhibit parasite invasion, suggesting that vaccines containing these three components have the potential to prevent blood-stage malaria. To further explore the role of the P113-RH5 interaction, we selected monoclonal antibodies against P113 that were either inhibitory or noninhibitory for RH5 binding. Using a Fab fragment as a crystallization chaperone, we determined the crystal structure of the RH5 binding region of P113 and showed that it is composed of two domains with structural similarities to rhamnose-binding lectins. We identified the RH5 binding site on P113 by using a combination of hydrogen-deuterium exchange mass spectrometry and site-directed mutagenesis. We found that a monoclonal antibody to P113 that bound to this interface and inhibited the RH5-P113 interaction did not inhibit parasite blood-stage growth. These findings provide further structural information on the protein interactions of RH5 and will be helpful in guiding the development of blood-stage malaria vaccines that target RH5. Plasmodium, and in 2018, it was responsible for an estimated 228 million clinical cases  –15 and R–subgenus , and thein vitro . It was P. falciparum P113. Antibodies were first tested by enzyme-linked immunosorbent assay (ELISA) to identify those which bind to the N-terminal cysteine-rich region of P113 which contains the RH5 binding site   assay, we found that neither anti-P113 monoclonal antibody was able to inhibit parasite growth in blood culture, even at concentrations that far exceeded the concentrations needed to inhibit the interaction in vitro and at which a monoclonal antibody targeting RH5 (R5.016)   and B. U 320 nM) , we asse 320 nM) . Because(R5.016)  showed >(R5.016) .2 (638 Å2 from VH and 212 Å2 from VL), with the interaction involving 15 hydrogen bonds, together with surface charge complementarity.To better understand the function of the N-terminal region of P113, we determined its crystal structure. A protein containing residues 1 to 197 was expressed in HEK293 cells and purified for crystallization. While this did not crystallize alone, we were able to use a Fab fragment of the noninhibitory P3.2 antibody as a crystallization chaperone. A complex of P113 residues 1 to 197 bound to the P3.2 Fab fragment formed crystals that diffracted to 1.95-Å resolution. The structure was determined by molecular replacement, using the structure of the Fab fragment of antibody 9AD4  as a seaThe structure of the N-terminal region of P113 reveals two closely interacting domains. Both are formed from a four-stranded antiparallel β-sheet which packs against an α-helix  and 3. L1–197 in complex with the Fab fragment of 10C8, with RH5, or with a peptide containing residues 9 to 27 of RH5 previously shown to contain the P113 binding site , was subcloned into the P113-bio vector  using a Gibson assembly cloning kit (NEB). Primers were designed to introduce a thrombin site between the P113 gene fragment and the coding sequence for Cd4-His6 on the P113-bio vector. The resulting plasmid DNA construct was used to transfect Expi293F human cells using the Expi293F transfection kit and Expi293F expression medium (Thermo Fisher). Cells were harvested by centrifugation at 1,000 × g for 30 min and resuspended in 1× phosphate-buffered saline (PBS) supplemented with 30 mM imidazole. Cell lysis was performed using a cell disruptor at 10 kpsi pressure, and the cell lysate was centrifuged at 50,000 × g for 30 min at 4°C. The soluble fraction was incubated for 1 h at 4°C using a Ni-NTA (nitrilotriacetic acid) resin (Qiagen). The resin was washed with 4 resin volumes (RV) of buffer A followed by elution with 3 RV of 1× PBS supplemented with 0.5 M imidazole. The eluted protein was desalted against 1× PBS buffer and digested with thrombin protease  in a ratio of 1 unit per μg at room temperature (RT). The reaction mixture was loaded into a gravity flow column preloaded with Ni-NTA beads to remove Cd4-His6, obtaining tagless P1131–197.To produce P113g for 3 min. PEG solution was aspirated, and cells were resuspended initially in ClonaCell-HY Medium B and thereafter in ClonaCell-HY Medium C (Stemcell Technologies) and incubated overnight . On the following day, cells were pelleted (10 min at 400 × g), resuspended in 10 ml ClonaCell-HY Medium C and 90 ml ClonaCell-HY medium D (semisolid HAT-selection media), and plated out into 100-mm petri plates. On day 13, colonies were picked manually and transferred to HT-selection media. Culture supernatants were screened for specificity by ELISA, and positive cultures were single cell sorted using a Beckman Coulter Legacy MoFlo MLS high-speed cell sorter (BD). Monoclonal cultures were expanded in Dulbecco modified Eagle medium (DMEM) (Sigma) containing l-glutamine, penicillin-streptomycin (Pen/Strep), and ultralow fetal calf serum (FCS), and antibodies were purified from the supernatant by gravity flow protein G columns. For monoclonal antibody production, hybridoma cells were pelleted at 1,000 × g and the supernatant containing antibody P3.2 was filtered with 0.22-μm filter units  before being loaded onto Protein G resin (Thermo Fisher) previously equilibrated with 20 mM HEPES, 150 mM NaCl, pH 7.4. The resin was subsequently washed with 3 resin volumes (RV) of the same buffer before MAb P3.2 (Thermo Fisher) was eluted using 3 RV of 0.2 M glycine, pH 2.5. The pH of the solution was immediately neutralized with 1 M Tris-HCl, pH 7.5, to a final concentration of 50 mM, and MAb P3.2 was desalted in 1× PBS. Papain digestion was performed overnight at room temperature (RT) using the Pierce Mouse IgG1 Fab and F(ab′)2 kit (Pierce), and Fc was separated from Fab fragments using protein A resin. Fab P3.2-containing aliquots were stored at −80°C in 1× PBS. Monoclonal antibody 10C8 was prepared in a similar way except that SJL strain mice were coimmunized with both full-length P113EE and P113-Y1-N653 proteins . All procedures on mice were performed in accordance with the terms of the UK Animals (Scientific Procedures) Act Project Licence and were approved by the University of Oxford Animal Welfare and Ethical Review Body. Mice were immunized intramuscularly (i.m.) with 20 μg of the entire ectodomain of P113  adjuvantproteins . Immunizproteins . HybridoProtein expression levels and monoclonal antibody binding were quantified by ELISA essentially as described previously . BrieflyAvidity-based extracellular interaction screening (AVEXIS) was performed essentially as described previously  as described previously . Biotiny1–197 was combined with Fab P3.2 in a 1:1.2 (wt/wt) ratio for 1 h at room temperature before the lysine methylation method as described previously (−1 using 30-kDa-cutoff concentrators (Millipore) before injection into an S75 16/600 column  previously equilibrated with 20 mM Tris-HCl, 150 mM NaCl, pH 7.4. Fractions containing the P1131–197:Fab P3.2 complex were pooled and concentrated to 10 mg ml−1 as appropriate for crystal formation based on use of a precrystallization kit (Hampton Research). Crystallization trials in 96-well plates were set up at 4°C with the sitting-drop method using commercial screens  with 100 nl reservoir plus 100-nl protein drops. Diamond-shaped crystals appeared in 7 to 10 days in 0.1 M Mg acetate, 0.1 morpholineethanesulfonic acid (MES) (pH 5.8), 22% (wt/vol) PEG 4000 and were supplemented with 20% (wt/vol) 2-methyl-2,4-pentanediol (MPD) before being flash cooled in liquid N2. Two data sets from a single crystal were collected at 100 K at beamline I03 of the Diamond Light Source. The crystal belonged to space group P41212 with unit cell dimensions a = 97 Å, b = 97 Å, c = 178 Å, and α = β = γ = 90°. The data sets were indexed and reduced to 1.95-Å resolution using autoPROC , after pruning the side chains using chainsaw  and man1–197 (163/197 amino acids) was initially built as a polyalanine model in PHENIX Autobuild, followed by multiple runs of restrained refinement in PHENIX (resolution range 50.58 to 1.95 Å). Reiterated model building was performed in COOT (6Z2L). Crystallography data and refinement statistics are reported in The majority of P113 in COOT , and str in COOT  before d−1, to allow the removal of salts, and were then separated using a C18 column with a linear gradient of 5 to 65% acetonitrile in water, both supplemented with 0.1% formic acid, for 9 min at flow rate of 40 μl min−1. The temperature at which liquid chromatography was performed was set at 0°C to reduce back-exchange. Peptide mapping of P113 was performed by using nondeuterated samples in triplicate, and only unique peptides present in all three data files were selected for deuterium uptake data analysis. Apo P113 protein digestion provided a list of 1,649 peptides. After applying selection filters, and after manual inspection, 59 peptides were selected for analysis. These peptides provided ∼95% sequence coverage with many overlapping peptides. For labeling experiments, apo P113, P113-10C8, or P113-RH5 was incubated for 20 s, 10 min, and 2 h in deuterated buffer. All HDX-MS experiments were performed in duplicate. Sequence coverage and deuterium uptake were analyzed by using ProteinLynx Global Server (Waters) and DynamX (Waters) programs, respectively. Leucine enkephalin at a continuous flow rate of 5 μl min−1 was sprayed as a lock mass for mass correction.HDX-MS was performed using a Waters HDX manager composed of a nano-Acquity ultrahigh-pressure liquid chromatograph (UPLC) coupled to a Synapt G2-Si (Waters) mass spectrometer. Samples were prepared by 11-fold dilutions from 7 μM apo P113 in deuterated or nondeuterated and P113-10C8 or P113-RH5 complex in deuterated 20 mM HEPES, 150 mM NaCl (pH 7.4) buffer. The pH of the sample was reduced to 2.3 by adding 50% (vol/vol) 150 mM HCl. The apo protein and complexes were digested in-line using a pepsin-immobilized column at 20°C. The peptides generated from pepsin digestion were trapped on a micro-peptide trap for 2 min at a flow rate of 200 μl minin vitro growth of P. falciparum 3D7 parasites was assessed using a standardized procedure. The MAbs were buffer exchanged against incomplete culture medium , concentrated with centrifugal filter devices , and sterilized by filtration through a 0.22-μm spin filter  before use in the assay. These samples were tested in a serial 3-fold dilution with a start concentration of 1 mg ml−1 IgG in triplicate in a one-cycle growth inhibition activity (GIA) assay using human O+ erythrocytes parasitized with mid-trophozoite stages of P. falciparum prepared by 5% sorbitol treatment on the previous day. Parasite growth after approximately 44 h of culture was determined by a biochemical assay specific for parasite lactate dehydrogenase . All other data are available from the authors on request.The coordinates and structure factors associated with this work are available at the Protein Data Bank (PDB:


The ongoing development of drug resistance in HIV continues to push for the need of alternative drug targets in inhibiting HIV. One such target is the Reverse transcriptase (RT) enzyme which is unique and critical in the viral life cycle—a rational target that is likely to have less off-target effects in humans. Serendipitously, we found two chemical scaffolds from the National Cancer Institute (NCI) Diversity Set V that inhibited HIV-1 RT catalytic activity. Computational structural analyses and subsequent experimental testing demonstrated that one of the two chemical scaffolds binds to a novel location in the HIV-1 RT p51 subunit, interacting with residue Y183, which has no known association with previously reported drug resistance. This finding supports the possibility of a novel druggable site on p51 for a new class of non-nucleoside RT inhibitors that may inhibit HIV-1 RT allosterically. Although inhibitory activity was shown experimentally to only be in the micromolar range, the scaffolds serve as a proof-of-concept of targeting the HIV RT p51 subunit, with the possibility of medical chemistry methods being applied to improve inhibitory activity towards more effective drugs. Human immunodeficiency virus (HIV) remains as one of the major global pandemics, with millions infected worldwide . CurrentWhile integrase inhibitors are recommended by the World Health Organization (WHO)  as a firBoth NRTI and NNRTI are used in ART, each inhibiting via different modus operandi. NRTIs are nucleotide analogues and bind to the nucleotide binding site to competitively block reverse transcription , leadingHIV-1 RT is a heterodimer of the p66 and p51 subunits. The larger p66 subunit contains five subdomains: fingers, palm, thumb, connection, and RNase H. Despite having an identical sequence to that of p66, excluding the RNase H subdomain, the p51 subunit forms a more compact structure . HoweverIn our search for alternative NNRTIs against HIV-1 RT, we serendipitously found two inhibitory chemical scaffolds that exhibited RT inhibition. While inhibiting only at micromolar range, one of the compounds elicited the possibility of non-canonical targeting at the RT p51 subunit. We therefore attempted to investigate the underlying mechanism for their interactions on the RT using computational docking and molecular dynamic (MD) simulations, finding a possible alternative HIV-1 RT inhibition mechanism via the RT p51 subunit.50) at 50 nM in our assay, and in agreement to previous reports [50 at 17.13 µM and 139.31 µM, respectively  Diversity Set V, we serendipitously identified two chemical compounds, NSC48443 and NSC127133  that inhibited HIV-1 RT activity using the reverse transcriptase—quantitative polymerase chain reaction (RT-qPCR) assay . In thes reports ,25, the ectively B.To identify the binding sites of compounds 1 and 2 on the HIV-1 RT heterodimer structure (including p66 and p51 subunits), blind docking experiments were performed using AutoDock Vina , followeCompound 1 occupied the site on p66 with more favorable predicted binding energies than the binding site on p51 , and witOn the other hand, compound 2 maintained stable binding to the p51 subunit in three out of six successful setups for the MD simulations—each setup had a unique initial compound 2 conformation . In the Results of our independent triplicates confirmed that the inhibition did not occur during the GAPDH qPCR step since PCR product bands were still detected when the compounds were added only right after the cDNA synthesis step A. In sepp < 0.05) of relative GAPDH expression levels were observed between those expressed in p66 only, and in the whole heterodimer RT , in agreement with previously established enzyme assays [50 at 17.13 µM and compound 2 with IC50 at 139.31 µM.In this study, we demonstrated that HIV RT p51 could be targeted for novel RTIs, showing the molecular interactions that can form with such inhibitors by experimentally screening 40 compounds of the NCI Diversity Set V, serendipitously finding two compounds that could inhibit the HIV-1 RT. Using RT-qPCR to estimate the inhibition efficiency with a comparison to known NNRTI nevirapine as the positive control  for compound 1 . StructuComputational analyses showed that both compounds interact with the RT p51 subunit (with less pronounced propensity for compound 1 at its second binding site in p51), distant from the polymerase site located on p66 A, suggesIt should be noted that compound 2 also interacted weakly  compared to the direct inhibition of compound 1 on p66 (IC50 17.13 µM).Previous studies ,31,32 shDespite inhibiting the RT function only at a micromolar range, the two compounds can serve as scaffold for further development, e.g., in fragment-based ligand design , given tOur structural conservation analysis showed the compound 2 binding region on HIV-1 p51 to be conserved across multiple RTs . Since vIn conclusion, we demonstrated the feasibility of inhibiting HIV RT via the p51 subunit on a previously identified druggable pocket, revealing possible RTI scaffolds, and the necessary structural engagements for further improvements. Despite inhibiting only at micromolar range, the scaffolds are a promising starting point for further development of new HIV-1 RT lead candidates and even potential broad-spectrum RTIs.RNA was extracted from EXPI293F  cells using TRIzol Reagent  according to the manufacturer’s recommendations. The RNA was treated with RNase-free DNase I  and stored at −80 °C.http://dtp.cancer.gov) and Nevirapine  were reconstituted in dimethyl sulfoxide (DMSO) and stored at 4 °C. The chemicals were initially screened at 300 µM, and potential hit compounds were then used at various concentrations: 1000 µM, 100 µM, 10 µM, and 1 µM.Forty NCI Diversity Set V chemical compounds from the National Cancer Institute (NCI) Developmental Therapeutic Program’s Open Compound Repository, National Institutes of Health (NIH) (2), 5.0 µL of DEPC-Treated Water, 1.0 µL of RT p66 (0.33 µg/µL), 2.0 µL of RT p66 (0.17 µg/µL), 4.0 µL of candidate inhibitors and 1.0 µg of DNase-treated RNA. The reaction mixes were prepared on ice, followed by incubation in ProFlex 3x 32-Well PCR System (Applied Biosystems) at 25 °C for 18 min, followed by 37 °C for 1 h, and RT termination at 85 °C for 5 min. Reaction mixtures containing DMSO only, without RT were used as the negative control.All of the library compounds were first warmed to 37 °C for 10 min for better solubility prior to addition into the RT reaction mix. To form HIV-1 RT, Sino Biological HIV-1 reverse transcriptase p51  and p66  subunits were both used to form the RT complex for cDNA synthesis. The 20 µL cDNA synthesis mix is as follows: 0.5 µL of random hexamers , 0.5 µL of Oligo d(T)s , 1.0 µL of RiboLock RNase Inhibitor , 1.0 µL of dNTP mixture , 4.0 µL of 5 TM. The GAPDH qPCR reactions were setup according to manufacturer recommendations, containing the reagents PowerUpTM SYBR® Green Master Mix (2X) , Human GAPDH Forward Primer (5′– ACAACTTTGGTATCGTGGAAGG –3′) and Human GAPDH Reverse Primer (5′– GCCATCACGCCACAGTTTC –3′). The final concentration of DMSO in the reaction mixture is adjusted to 0.25%. Cycling conditions were set at 1 cycle of 50 °C (2 min), 95 °C (2 min), 40 cycles of 95 °C (5 s), 60 °C (30 s). A final melt curve was performed at 95 °C (15 s), 60 °C (ramp rate at 0.3 °C/s), and finally 95 °C (15 s). Independent triplicates were performed with “No RT”, “No Primer”, and “No Template Controls” (NTC). GAPDH amplification was quantified using comparative threshold cycle (Ct) as the following ΔCt method:The quantification of gene expression was obtained from Applied Biosystems StepOnePlusAgarose gel electrophoresis analysis was carried out on the qPCR products using 2% agar gels. Bands were validated using GelApp . Densito50) of the compound is defined as the amount of compound required to block HIV-1 RT activity by 50%. The best-fit curves were plotted using the relative amount of the amplified GAPDH as calculated in the previous step (using ΔCt method). Curve fitting was applied using Hill equation [50 was obtained directly from the calculated best fit curve parameters using AAT BioQuest IC50 calculator [The half-maximal inhibitory concentration  followed by Dunnett’s multiple comparison tests were performed using R software (version 3.6.2)  to compaThe chemical structures of the two compounds (NSC48443 and NSC127133 from the NCI/DTP Diversity Set V) were retrieved from PubChem library, Compound Identifier (CID): 241,217 and 278,037 (particularly from the ligand code 6B3 in this entry) for compounds 1 and 2, respectively. Wild type HIV-1 RT structure (Protein Data Bank (PDB): 3T19) was processed as previously described .We performed rigid blind docking using the program AutoDock Vina  to find We simulated the whole HIV-1 RT p66-p51 with the selected compound conformations using GROMACS (version 2018) , with thHydrophobic contacts were calculated using a 4.0 Å cut-off between the ligand heavy atoms and protein carbon atoms. The number of water molecules around the ligand was determined using a cut-off of 3.5 Å between the ligand heavy atoms and water oxygen atoms. Hydrogen bonds were calculated between donor and acceptors of the ligand and the protein using a cut-off of 3.5 Å, with a maximum angle between donor-hydrogen atom and acceptor of 30°. To determine stable positions of the ligand in each binding site, we calculated distances between the center of mass of the ligand and of the binding site residues, which were defined via the LigPlot+ program .g_mmpbsa [in) were used to estimate the polarizability of the binding sites, e.g., εin = 2 (hydrophobic), εin = 8 (polar), or εin = 20 (highly charged). The external dielectric constant εout was set to 80 (for water). The calculations were performed using the last 50 ns of each MD simulation trajectory for all independent replicas.The molecular mechanics Poisson–Boltzmann Surface Area (MM-PBSA) method was applied to calculate the binding energies of the compounds and their corresponding binding sites on the HIV-1 RT subunits, using g_mmpbsa . Variousg_mmpbsa ,61,62  RefSeq Databases) was first performed using ClustalW . The res


Dmc1 on wheat chromosome 5D has been identified as a candidate for the maintenance of normal chromosome synapsis and crossover at low and possibly high temperatures.The meiotic recombination gene Triticum aestivum L.) variety ‘Chinese Spring’. At low temperatures, asynapsis and chromosome univalence have been observed before in Chinese Spring lines lacking the long arm of chromosome 5D (5DL), which led to the proposal that 5DL carries a gene (Ltp1) that stabilises wheat chromosome pairing at low temperatures. In the current study, Chinese Spring wild type and 5DL interstitial deletion mutant plants were exposed to low temperature in a controlled environment room during a period from premeiotic interphase to early meiosis I. A 5DL deletion mutant was identified whose meiotic chromosomes exhibit extremely high levels of asynapsis and chromosome univalence at metaphase I after 7 days at 13 °C, suggesting that Ltp1 is deleted in this mutant. Immunolocalisation of the meiotic proteins ASY1 and ZYP1 on ltp1 mutants showed that low temperature results in a failure to complete synapsis at pachytene. KASP genotyping revealed that the ltp1 mutant has a 4-Mb deletion in 5DL. Of 41 genes within this deletion region, the strongest candidate for the stabilisation of chromosome pairing at low temperatures is the meiotic recombination gene Dmc1. The ltp1 mutants were subsequently treated at 30 °C for 24 h during meiosis and exhibited a reduced number of crossovers and increased univalence, though to a lesser extent than at 13 °C. We therefore renamed our ltp1 mutant ‘ttmei1’ (temperature-tolerant meiosis 1) to reflect this additional loss of high temperature tolerance.We initially assessed the effects of low temperature on meiotic chromosome synapsis and crossover formation in the hexaploid wheat (The online version of this article (10.1007/s00122-019-03508-9) contains supplementary material, which is available to authorized users. In plants, male reproductive development is highly sensitive to adverse environmental conditions including high and low temperatures , which assembles between the paired chromosomes  must form between each pair of homologs, to forge a physical connection between the chromosomes. These physical connections can be seen cytologically and are called chiasmata. COs enable genetic information to be exchanged between chromosomes and are also needed for accurate chromosome segregation and balanced gametes in the daughter cells. Once COs have fully formed (at pachytene), the SC is disassembled (at diplotene), at which point the homologs are only connected via their chiasmata. They remain connected until the chromosomes segregate at anaphase I.In bread wheat, which is an allopolyploid with three homeologous (related) diploid genomes (AABBDD), COs only form between homologs and not between homeologs, ensuring that the three genomes behave as diploids during meiosis. Normally, at metaphase I, only ring bivalents (with two chiasmata) and occasional rod bivalents (one chiasma) are present. At metaphase I, the 21 bivalents align on the equatorial plate before segregating at anaphase I.Correct pairing and segregation of homologs is vital for maintaining the stability and fertility of the genome. Errors in meiosis can lead to aneuploidy or infertility. High and low temperatures can induce a variety of meiotic aberrations in plants including changes in the frequency of chiasma formation (CO frequency) Elliott . ReductiChinese Spring is one of the more heat-sensitive wheat cultivars  by Hayter and Riley , but in plants that are tetrasomic for 5B (44 chromosomes) synapsis is normal  ® Breeders’ Array probe set  var. ‘Chinese Spring’ by gamma irradiation of 1000 seeds at the International Atomic Energy Agency, Vienna (500 seeds irradiated at 250 Gy and 500 at 300 Gy). M1 deletion lines were self-fertilised. M2 lines were genotyped using Kompetitive allele-specific PCR (KASP) to identify plants with interstitial deletions of 5DL. These deletion mutants were exposed to 13 °C for 7 days to identify plants with asynaptic chromosomes at metaphase I of meiosis. Two other Chinese Spring-derived genotypes were used as controls for the KASP genotyping and in the temperature treatment experiments, the standard euploid (wild type) form, (AABBDD) and the nullisomic 5D-tetrasomic 5B (N5DT5B) genotype, which lacks chromosome 5D. Chinese Spring lines with homozygous terminal deletions of chromosome 5DL were obtained from TR Endo, Kyoto University, Japan. These were 5DL-1, 5DL-2, 5DL-5, 5DL-9 and 5DL-13  at 20 °C (day) and 15 °C (night) with a 16-h photoperiod (lights on between 10:00 and 02:00) and 70% humidity. Wheat seedlings were grown to the 2–3 leaf stage, and approximately 5 cm of leaf material was harvested into 1.2-ml collection tubes containing 3-mm tungsten-carbide beads in a 96-well format on dry ice. DNA was extracted using a method based on the protocol available at https://wheat.pw.usda.gov/GG3/, were used to map the breakpoints of the five Chinese Spring 5DL terminal deletion lines  markers from the Wheat Breeders’ 820 K Axiom® array , using Python Data Matcher https://github.com/wingenl/python_data_matcher, which merges files with common features. The BioMart data mining tool from Ensembl Plants  using the following programme: hot start at 94 °C for 15 min, followed by ten touchdown cycles  and then 30 cycles of amplification . Fluorescent signals from PCR products were read in a PHERAstar microplate reader (BMG LABTECH Ltd.). If tight genotyping clusters were not obtained, an additional 5 cycles  were performed. Genotyping data were analysed using KlusterCaller software (LGC Genomics).Assays were set up as 5 μl reactions in a 384-well format and included 2.5 μl genomic DNA template (15–30 ng of DNA), 2.5 μl of KASP 2 × Master Mix (LGC Genomics), and 0.07 μl primer mix. Primer mix consisted of 12 μl of each tailed primer (100 μM), 30 μl common primer (100 μM) and 46 μl dHPlants were initially grown in a CER at 20 °C (day) and 15 °C (night) with a 16-h photoperiod (lights on between 10:00 and 02:00) and 70% humidity until Zadoks growth stage 39 . Anthers were sampled from the first (oldest) 5 tillers only. Using an M80 stereo microscope , anthers were dissected from the two largest florets in each spikelet. Only anthers at metaphase I were scored, so, to determine the meiotic stage, one anther from each floret was stained with acetocarmine and squashed under a cover slip to extrude the PMCs, which were then examined using a DM2000 light microscope (Leica Microsystems). The three anthers within any floret are synchronised in meiotic development, so when PMCs with metaphase I chromosomes were identified, the two remaining anthers from the same floret were fixed in 3:1 (v/v) 100% ethanol/acetic acid, for cytological analysis. Anthers were incubated for at least 24 h at 4 °C before being transferred to 70% ethanol. Fixed anthers were washed with 0.1% sodium dodecyl sulphate for 3–5 min and then hydrolysed with 1 M hydrochloric acid for 10 min at 60 °C. They were then Feulgen-stained with Schiff’s reagent and squashed in 45% acetic acid. This allowed the chromosomes to be spread more widely to facilitate scoring of crossover. Images of metaphase I chromosomes were captured using a DM2000 microscope equipped with a DFC450 camera and controlled by LAS v4.4 system software (Leica Microsystems). For each cell, images were captured in up to 8 different focal planes to aid scoring.For each plant, 20–30 PMCs were blind scored from digital images. For each cell, the different meiotic chromosome configurations were counted. These were unpaired univalents (0 chiasmata), rod bivalents (1 chiasma), ring bivalents (2 chiasmata), trivalents (2–3 chiasmata), tetravalents (3 chiasmata) and pentavalents (4 chiasmata). Chiasma frequency per PMC was calculated separately for single and double chiasmata . All treatments were analysed by the Kruskal–Wallis test . Means were separated using the Dunn’s test with a probability level of 0.05. Statistical analysis was carried out between temperatures and between genotypes. Bar charts were plotted using Microsoft Excel (2016).ttmei1 mutant plants were immunolabelled with antibodies against the meiotic proteins ASY1 and ZYP1. PMCs from wild-type Chinese Spring and the ttmei1 mutant were embedded in acrylamide pads to preserve their 3D structure, and immunolocalisation of ASY1 and ZYP1 was performed as described previously  were used as secondary antibodies.To determine when meiosis is disrupted in the low-temperature experiments, PMCs from three wild-type and three ttmei1 replicates. Polyacrylamide-embedded PMCs were optically sectioned using a DM5500B microscope (Leica Microsystems) equipped with a Hamamatsu ORCA-FLASH4.0 camera and controlled by Leica LAS-X software v2.0. Z-stacks were deconvolved using Leica LAS-X software. Images were processed using Fiji, which is an implementation of ImageJ, a public domain program available from http://rsb.info.nih.gov/ij/  revealed that there are three homeologs of the Dmc1 gene in hexaploid wheat: TraesCS5A02G133000 (TaDmc1-A1) on chromosome 5A, TraesCS5B02G131900 (TaDmc1-B1) on 5B and TraesCS5D02G141200 (TaDmc1-D1) on 5D. The nucleotide sequences of the DNA, the coding sequences (CDs) and the promoter regions (including 1500 nucleotides downstream of the start codon) of the three TaDmc1 homeologs were compared.Multiple sequence alignment was carried out using ClustalX2 software  and rice (Oryza sativa Japonica). Gene IDs from Ensembl Plants are, for barley: HORVU5Hr1G040730.3 (HvDMC1) and for rice: Os12g0143800 (OsDMC1A) and Os11g0146800 (OsDMC1B).Multiple DMC1 amino-acid sequence alignment was also carried out between hexaploid wheat (Chinese Spring), barley (Dmc1 sequences from hexaploid wheat (T. aestivum) were also compared with those of its diploid and tetraploid progenitors, T. urartu (the A-genome donor), Aegilops tauschii (the D-genome donor) and T. dicoccoides (the AABB tetraploid progenitor). Gene IDs from Ensembl Plants are for T. urartu (TuDMC1): TRIUR3_13472; for Ae. tauschii (AetDMC1-D): AET5Gv20357200; for the T. dicoccoides A copy (TdDMC1-A): TRIDC5AG022500 and for the T. dicoccoides B copy (TdDMC1-B): TRIDC5BG023380.The ttmei1 M3 mutant plants was carried out to assess whether the gamma irradiation of Chinese Spring seeds had resulted in a duplication or insertion of Dmc1-D1 in other parts of the genome. Two different sets of Dmc1-D1 chromosome-specific primers were used to genotype three wild type and nine ttmei1 mutant plants. Primer set 1 consisted of two allele-specific primers: 5′ GAAGGTCGGAGTCAACGGATTcctgtcatgaaaccctgactC 3′ (including HEX tail [wild type]) and 5′ GAAGGTGACCAAGTTCATGCTcctgtcatgaaaccctgactT 3′ (including FAM tail [mutant]), and a common reverse primer: 5′ CcatctgtgctatGcgatAgaA 3′. Primer set 2 consisted of allele-specific primers: 5′ GAAGGTCGGAGTCAACGGATTgtagcttagctcctaaacctCaC 3′ (with HEX tail [wild type]) and 5′ GAAGGTGACCAAGTTCATGCTgtagcttagctcctaaacctCaT 3′ (with FAM tail [mutant]), and common reverse primer: 5′ GcgatAgaAtcttctgaaGtttgtG 3′.KASP genotyping of Chinese Spring wild type and Ltp1 phenotype, we first needed to narrow down our region of interest on chromosome 5DL. To facilitate this, we exposed five Chinese Spring lines with homozygous terminal deletions of 5DL to 13 °C for 7 days. All five had normal chromosome pairing at metaphase I. The breakpoints of these deletion lines were mapped using nine 5D-specific microsatellite markers , so we can infer that the length of 5DL is around 376 Mb. The length of the chromosome distal to Xgdm138 is 174 Mb, so almost half of the chromosome arm could be eliminated from our search to find the gene responsible for the Ltp1 phenotype.To locate the gene(s) responsible for the ers Fig. . Line 5D1 plants were self-fertilised (four ears per plant where possible), and 1–2 M2 seeds from each ear were genotyped. A total of 2444 Chinese Spring M2 plants were genotyped, initially using 20 KASP markers located in the proximal half of 5DL. This identified 16 plants with deletions in 5DL  germinated and 378 plants (76%) were fertile. Of the 500 seeds irradiated at 300 Gy, 417 plants (83%) germinated and 372 (74%) were fertile. MLtp1 locus, so it suggests that Ltp1 is amongst the genes that have been deleted in the 22-F5 mutant.All 16 deletion mutants were exposed to 13 °C for 7 days during premeiotic interphase to early meiosis I. Fifteen plants had normal bivalent formation at metaphase I, but one deletion mutant, 22-F5, exhibited almost complete crossover failure after low-temperature treatment .Given the effects of low temperature on the 22-F5 mutant, we assessed whether it also showed reduced tolerance to high temperature. After 24 h at 30 °C, the 22-F5 deletion mutant did indeed exhibit a reduced number of crossovers and increased univalence, but to a lesser extent than at 13 °C  was around 19 as opposed to 21 in the wild type, and the chromosomes were still able to align on the metaphase plate with the exception of the occasional one or two univalent chromosomes . Univalents appeared more condensed than other chromosomes and were unable to align on the metaphase plate  available from Ensembl Plants. Functional annotations of the genes were retrieved from the file ‘FunctionalAnnotation.rds’ in https://opendata.earlham.ac.uk/wheat/under_license/toronto/Ramirez-Gonzalez_etal_2018-06025-Transcriptome-Landscape/data/TablesForExploration/FunctionalAnnotation.rds  were identified in the 4-Mb deletion region. Eighteen of the 41 genes were expressed during meiosis , which has a known meiotic phenotype, playing a central role in homologous recombination. It is a high confidence gene, and its expression level is around ten times higher in meiotically active tissues compared with non-meiotic tissues . The other 17 genes in the region are much less likely than TaDmc1 to be candidates for the TTmei1 phenotype because none of them could be attributed to any known meiotic function, and although they are expressed during meiosis, expression is proportionally higher in other tissues.Of the 18 genes expressed during meiosis in the Dmc1-D1 in other parts of the genome, three Chinese Spring wild-type and nine ttmei1 M3 mutant plants were KASP-genotyped using two different sets of Dmc1-D1-specific primers. As expected, with both primer sets, none of the ttmei1 mutant plants showed any amplification when compared with the wild-type plants. This indicates very clearly that Dmc1-D1 is completely deleted from the ttmei1 deletion line and has not been duplicated or inserted elsewhere in the genome as a result of the gamma irradiation.To assess whether gamma irradiation of the Chinese Spring seeds had resulted in a duplication or insertion of Dmc1 gene in hexaploid wheat: TraesCS5A02G133000 (TaDmc1-A1) on chromosome 5A, TraesCS5B02G131900 (TaDmc1-B1) on 5B and TraesCS5D02G141200 (TaDmc1-D1) on 5D. There are differences in gene expression levels between the three Dmc1 homeologs in meiotic anthers: TaDmc1-A1 has the lowest gene expression levels and TaDmc1-D1 the highest .BLAST searches against the Chinese Spring IWGSC RefSeq v1.0 sequence assembly IWGSC  revealedTaDmc1 homeologs were compared. The CDs of the A, B and D homeologs are highly conserved  with sequence identity ranging between 98.1 and 98.7% (Suppl. Table 3a). The DNA sequences are less similar , with sequence identity ranging from 82.1% (between A and B homeologs) to 89.0% (between A and D homeologs) (Suppl. Table 3a). Comparing the promoter regions of each of the three TaDmc1 homeologs showed a large insertion mutation (163 nt in size) at position 430 downstream of the start codon of TaDmc1-B1 .The nucleotide sequences of the DNA, the coding sequences (CDs) and the promoter regions of the three TaDmc1 homeologs , which encodes a predicted protein of 344 amino acids  and rice (Oryza sativa Japonica). There is a high level of amino-acid sequence conservation between the wheat, barley and rice proteins  and its diploid and tetraploid progenitors, T. urartu (the A-genome donor), Ae. tauschii (the D-genome donor) and T. dicoccoides (the AABB tetraploid progenitor) . The Dmc1 sequences of T. aestivum are quite conserved when comparing each gene copy with its ancestral genome copy. For the DNA sequences, the highest percentage of homology is between the D copy of T. aestivum and the ancestral gene of Ae. tauschii (99.8%), whilst the lowest homology is between the B copy of T. aestivum and the B-genome copy in T. dicoccoides (92.2%) (Suppl. Table 3c), due to a large insertion of 294 bp in intron 7 . The percentages of homology in TaDmc1-A1 versus T. dicoccoides_A and TaDmc1-A1 versus T. urartu are 98.6% and 98.4%, respectively (Suppl. Table 3c).T. aestivum and those of its diploid and tetraploid progenitors . Unlike those of T. aestivum, most of the Dmc1 gene copies in the wheat ancestors have multiple transcripts due to alternative splicing variants (as predicted by Ensembl Plants). TdDmc1-A (T. dicoccoides A copy) has 3 transcripts; TdDmc1-B (T. dicoccoides B copy) has 9 transcripts; and AetDmc1-D (Ae. tauschii) has 16 transcripts. TuDmc1-A  has a single transcript, but of all the ancestral DMC1 proteins in our comparisons, the encoded protein sequence of TuDMC1 from T. urartu is the one that diverges most from that of T. aestivum (Suppl. Table 3d). Of the amino-acid differences between the A-, B- and D-genome copies of DMC1 in T. aestivum, the single amino-acid substitution in the D-genome copy of T. aestivum at position 114 .Comparisons were also made between the amino-acid sequences of DMC1 proteins from ttmei1 M3 plants were exposed to 30 °C for 24 h. A higher number of mutant plants were treated because of the wide variation in the data for this group. Chromosome stickiness and clumping were observed in many of the cells at this temperature  that is probably responsible for the Ltp1 phenotype. Subsequent exposure of this ltp1 deletion mutant to 24 h of high temperature (30 °C) during the same developmental period led to a reduced number of crossovers and increased univalence, though to a lesser extent than at 13 °C. As this deletion clearly has an effect on chromosome pairing at 30 °C as well as at 13 °C, the mutant line was renamed ttmei1 to reflect both its reduced high temperature tolerance and its loss of Ltp1.Chinese Spring wild-type plants and mutant plants with interstitial deletions within chromosome 5DL were exposed to low temperature (13 °C) for 7 days during a period lasting from premeiotic interphase to early meiosis I. Microscopic examination of PMCs from these plants enabled us to identify a 5DL deletion mutant whose meiotic chromosomes exhibit extremely high levels of asynapsis and chromosome univalence. Within the deleted region of this mutant, we identified a candidate gene on 5DL , suggesting that the ttmei1 deletion is not necessarily linked to the occurrence of multivalents. The presence of high numbers of univalents and more rod bivalents than normal in ttmei1 mutants under low temperatures suggests a major problem with CO formation. Consistent with this, the ttmei1 line exhibits significant abnormalities of synapsis at low temperatures.Exposure of the 5DL mutant plants to 13 °C for 7 days identified a deletion mutant, ttmei1 mutant carries a 4-Mb deletion of 5DL. Within this deletion interval, there are 41 genes, 18 of which are expressed during meiosis. Amongst these genes, the strongest candidate for the low-temperature pairing phenotype is the meiotic recombination gene TaDmc1-D1. None of the other genes are known to have any meiotic function. Moreover, TaDmc1 is expressed most highly during early prophase I (Suppl. Table 2), which, in wheat, is when synapsis is initiated at the ‘telomere bouquet’ stage , there are two Dmc1 homologs, OsDMC1A and OsDMC1B, that probably arose through chromosome duplication  carries a single Dmc1 homolog, HvDMC1, and mutations in this gene lead to abnormal synapsis, multiple univalents and chromosome mis-segregation . In Chinese Spring, deletion of the region containing TaDmc1-D1 results in asynapsis at 13 °C, and it seems highly likely that this gene promotes low temperature tolerance. However, different dosages of the TaDmc1 alleles can affect the stability of synapsis at low temperatures. When chromosome 5A is present as a single dose in N5DT5B plants, it cannot compensate for the lack of 5D and is unable to stabilise synapsis at low temperatures, but when present as a double dose in N5DT5A plants it can compensate for the 5D deficiency and chromosome pairing at 12 °C is normal . The fact that TaDMC1-A1 can compensate for TaDMC1-D1 suggests that this amino-acid difference is not so important for low temperature tolerance.In hexaploid wheat, there are three copies of ins Fig. b, a subsTaDmc1 are highly conserved with their ancestral homeologs, which suggests that these copies are functional. The B-genome copy is less well conserved due to a large insertion of 294 bp in intron 7 . Dmc1-B1 has intermediate gene expression in Chinese Spring, higher than Dmc1-A1, but lower than Dmc1-D1, but it cannot compensate for the loss of the D copy at low temperatures even when present as a double dose. The inability of Dmc1-B1 to stabilise low-temperature pairing may arise from three amino-acid differences in TaDMC-B1 compared to the A- and D-genome copies, or from the 163-bp insertion in the promoter region of TaDmc1-B1. Alternatively, the 294-bp insertion in intron 7 of TaDmc1-B1 could be affecting gene expression, because although intronic sequences do not encode protein products, they can still have an effect on gene expression and gene functionality , synapsis at 12 °C is normal despite the absence of chromosome 5D  and 5BS (Ltp3) that also have a stabilising effect on homologous pairing and crossover at low temperatures  populations or by CRISPR/Cas9 genome editing. TILLING lines are available in the hexaploid wheat variety ‘Cadenza’ but are not yet available for Chinese Spring. Moreover, results from recent experiments to edit a gene within the Ph1 locus suggest that CRISPR may be a better choice for deleting the TaDmc1-D1 gene. Like Ltp1, the Ph1 locus, on wheat chromosome 5B, is scored by its deletion phenotype. This locus, which affects both chromosome pairing and crossover, contains the major wheat meiotic crossover gene ZIP4. When zip4 CRISPR deletion mutants were compared with the ph1b deletion mutant , so that high-temperature treatment may have coincided with temperature-sensitive stages of meiosis or premeiosis to a greater or lesser extent.In ttemi1 mutants carry many duplications and deletions in addition to those we have identified on 5DL, particularly since the ttmei1 mutant plants were derived from seed irradiated with the higher dose (300 Gy) of gamma irradiation. We have determined through KASP genotyping that Dmc1-D1 has not been duplicated or inserted elsewhere in the genome, but it is possible that ttmei1 mutants carry a background deletion that has not yet been detected that contains a different meiotic recombination gene affecting high temperature tolerance that is segregating with the ttmei1 deletion. However, all 11 of the ttemi1 mutants scored at high temperatures exhibited reduced crossover, suggesting that the ttemi1 deletion is associated with reduced high temperature tolerance. We are currently conducting further experiments to understand this variation at high temperature. We are also backcrossing ttmei1 mutants with Chinese Spring wild-type plants to remove background deletions.However, it is also likely that In the future, breeders face the challenge of producing crops with increasing resilience to environmental stress whilst producing ever higher yields  temperature tolerance alleles at this locus.Heat tolerance is thought to be a complex trait in plants and is likely to be under the control of multiple genes  on chromosome 5A, TraesCS5B02G131900 (TaDmc1-B1) on 5B and TraesCS5D02G141200 (TaDmc1-D1) on 5D in different tissue types, based on the 876 RNASeq samples available in the wheat expression browser (http://www.wheat-expression.com). TaDmc1 expression levels are higher in meiotically active tissues than non-meiotic tissues. In meiotic anthers, expression levels of TaDmc1-D1 are higher than those of TaDmc1-A1 and TaDmc1-B1 (PDF 484 kb)Gene expression patterns of the three hexaploid wheat Suppl. Fig. 2Dmc1 homeologs: TraesCS5A02G133000 (TaDmc1-A1) on chromosome 5A, TraesCS5B02G131900 (TaDmc1-B1) on 5B and TraesCS5D02G141200 (TaDmc1-D1) on 5D. SNPs are shown in red text on a grey background (PDF 103 kb)Multiple alignment of the coding sequences (CDs) of the three hexaploid wheat Suppl. Fig. 3Dmc1 homeologs: TraesCS5A02G133000 (TaDmc1-A1) on chromosome 5A, TraesCS5B02G131900 (TaDmc1-B1) on 5B and TraesCS5D02G141200 (TaDmc1-D1) on 5D (DOCX 21 kb)Multiple alignment of the DNA sequences of the three hexaploid wheat Suppl. Fig. 4Dmc1 homeologs: TraesCS5A02G133000 (TaDmc1-A1) on chromosome 5A, TraesCS5B02G131900 (TaDmc1-B1) on 5B and TraesCS5D02G141200 (TaDmc1-D1) on 5D. The promoter regions include the 1500 nucleotides downstream of the start codon of each of the TaDmc1 homeologs. Note the large insertion mutation (163 nt in size) at position 430 downstream of the start codon of TaDmc1-B1 (PDF 165 kb)Multiple sequence alignment of the promoter regions of the three hexaploid wheat Suppl. Fig. 5Dmc1 DNA sequences from T. aestivum and its diploid and tetraploid ancestors, T. urartu (AA), Ae. tauschii (DD) and T. dicoccoides (AABB); note the large insertion of 294 bp in the B-genome copy of the T. aestivum gene (DOCX 33 kb)Multiple alignment of Suppl. Fig. 6T. aestivum and its diploid and tetraploid ancestors (as predicted in Ensembl Plants) (DOCX 982 kb)DMC1 transcripts in Suppl. Fig. 7T. aestivum (Ta) and its diploid and tetraploid ancestors: the A-genome donor, T. urartu (Tu_A), the D-genome donor, Ae. tauschii (Aet_D) and the AABB genome progenitor, T. dicoccoides (Td_A and Td_B). Ae tauschii and T. dicoccoides have multiple transcripts of their Dmc1 genes due to alternative splicing variants, but this alignment only includes transcripts that are most similar to those of T. aestivum. Numbers after the dots refer to the transcript number. Large red triangle indicates the single amino-acid substitution in TaDMC1-D1 that may confer low temperature tolerance in wheat. Note the conservation of this substitution in the ancestral D-genome of Ae. tauschii. Small blue triangles show the positions of three other amino-acid substitutions in TaDMC1-B1, and a small green triangle shows the position of a substitution in TaDMC1-A1 that is also conserved in the ancestral copies (RTF 101 kb)Multiple alignment of DMC1 amino-acid sequences from Suppl. Table 1ttmei1) mutant plants. The mean numbers of univalents, ring and rod bivalents, trivalents, tetravalents and pentavalents were scored along with chiasma frequency scored as single or double crossovers. Standard deviation (SD) and standard error (SE) are shown, as well as the maximum (max) and minimum (min) number of chromosomes with a particular conformation (PDF 675 kb)The effects of three different temperature treatments on meiotic metaphase I chromosomes of individual Chinese Spring (CS) wild-type and 22-F5 (Suppl. Table 2Dmc1 deletion region in 876 samples from different tissue types (XLSX 246 kb)Expression values (TPM) of all genes inside the Suppl. Table 3Dmc1 sequence identity matrices. a) Percentages of nucleotide sequence identity for the three TaDmc1 homeologs: TraesCS5A02G133000 (TaDmc1-A1) on chromosome 5A, TraesCS5B02G131900 (TaDmc1-B1) on 5B and TraesCS5D02G141200 (TaDmc1-D1) on 5D; b) percentages of amino-acid sequence identity for DMC1 proteins in wheat , barley (HvDMC1) and rice (OsDMC1A and OsDMC1B); c) percentages of nucleotide sequence identity for Dmc1 homeologs in T. aestivum (TaDmc1) and its progenitor genomes: T. urartu (TuDmc1-A), Ae. tauschii (AetDmc1-D) and T. dicoccoides A genome (TdDmc1-A) and B genome (TdDmc1-B); d) percentages of amino-acid sequence identity for DMC1 proteins in T. aestivum (TaDMC1) and its progenitor genomes: T. urartu (TuDMC1-A), Ae. tauschii (AetDMC1-D) and T. dicoccoides A genome (TdDMC1-A) and B genome (TdDMC1-B). Two different splice variants each are shown for TdDMC1-A, TdDMC1-B and AetDMC1-D. Note that of all the amino-acid sequences of T. aestivum and its progenitors, T. urartu is the most divergent (XLSX 14 kb)Below is the link to the electronic supplementary material.