Antibody structure determination using a combination of homology modeling, energy‐based refinement, and loop prediction

Zhu, Kai; Day, Tyler; Warshaviak, Dora Toledo; Murrett, Colleen S.; Friesner, Richard A.; Pearlman, David A.

doi:10.1002/prot.24551

Cited by 192 publications

(185 citation statements)

References 41 publications

(62 reference statements)

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“…This method has been shown to be effective in describing antibody CDR structures. 26 Thus, modeling of the antibody 001 CDR domains was employed. As seen in Fig.…”

Section: Resultsmentioning

confidence: 99%

Cysteinylation of a monoclonal antibody leads to its inactivation

McSherry

Ozaeta

et al. 2016

mAbs

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Post-translational modifications can have a signification effect on antibody stability. A comprehensive approach is often required to best understand the underlying reasons the modification affects the antibody's potency or aggregation state. Monoclonal antibody 001 displayed significant variation in terms of potency, as defined by surface plasmon resonance testing (Biacore), from lot to lot independent of any observable aggregation or degradation, suggesting that a post-translational modification could be driving this variability. Analysis of different antibody lots using analytical hydrophobic interaction chromatography (HIC) uncovered multiple peaks of varying size. Electrospray ionization mass spectrometry (ESI-MS) indicated that the antibody contained a cysteinylation post-translational modification in complementarity-determining region (CDR) 3 of the antibody light chain. Fractionation of the antibody by HIC followed by ESI-MS and Biacore showed that the different peaks were antibody containing zero, one, or two cysteinylation modifications, and that the modification interferes with the ability of the modified antibody arm to bind antigen. Molecular modeling of the modified region shows that this oxidation of an unpaired cysteine in the antibody CDR would block a potential antigen binding pocket, suggesting an inhibition mechanism.

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“…This method has been shown to be effective in describing antibody CDR structures. 26 Thus, modeling of the antibody 001 CDR domains was employed. As seen in Fig.…”

Section: Resultsmentioning

confidence: 99%

Cysteinylation of a monoclonal antibody leads to its inactivation

McSherry

Ozaeta

et al. 2016

mAbs

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“…The missing region of the AcrA structure was generated by using the SWISS-MODEL homology-modeling server and used to represent the whole structure of AcrA. The AcrA binding site was calculated by using the BioLuminate program in the Schrödinger suite (54). …”

Section: Methodsmentioning

confidence: 99%

AcrB-AcrA Fusion Proteins That Act as Multidrug Efflux Transporters

et al. 2016

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The AcrAB-TolC system in Escherichia coli is an intrinsic RND-type multidrug efflux transporter that functions as a tripartite complex of the inner membrane transporter AcrB, the outer membrane channel TolC, and the adaptor protein AcrA. Although the crystal structures of each component of this system have been elucidated, the crystal structure of the whole complex has not been solved. The available crystal structures have shown that AcrB and TolC function as trimers, but the number of AcrA molecules in the complex is now under debate. Disulfide chemical cross-linking experiments have indicated that the stoichiometry of AcrB-AcrA-TolC is 1:1:1; on the other hand, recent cryo-electron microscopy images of AcrAB-TolC suggested a 1:2:1 stoichiometry. In this study, we constructed 1:1-fixed AcrB-AcrA fusion proteins using various linkers. Surprisingly, all the 1:1-fixed linker proteins showed drug export activity under both acrAB-deficient conditions and acrAB acrEF double-pump-knockout conditions regardless of the lengths of the linkers. Finally, we optimized a shorter linker lacking the conformational freedom imparted by the AcrB C terminus. These results suggest that a complex with equal amounts of AcrA and AcrB is sufficient for drug export function. IMPORTANCEThe structure and stoichiometry of the RND-type multidrug exporter AcrB-AcrA-TolC complex are still under debate. Recently, electron microscopic images of the AcrB-AcrA-TolC complex have been reported, suggesting a 1:2:1 stoichiometry. However, we report here that the AcrB-AcrA 1:1 fusion protein is active for drug export under acrAB-deficient conditions and also under acrAB acrEF double-deficient conditions, which eliminate the aid of free AcrA and its close homolog AcrE, indicating that the AcrB-AcrA 1:1 stoichiometry is enough for drug export function. In addition, the AcrB-AcrA fusion protein can function without the aid of free AcrA. We believe that these results are very important for considering the structure and mechanism of AcrABTolC-mediated multidrug export.

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“…Although repertoire-wide crystallographic studies are not feasible at present, advances in computational protein modeling have enabled the structure of antibodies having moderate-length CDR loops to be predicted accurately (23)(24)(25)(26). In particular, computational models using RosettaAntibody-predicted X-ray structures within 1-1.5 Å rmsd in framework and canonical loops and 2 Å in CDR-H3 loops (27).…”

Section: Significancementioning

confidence: 99%

Large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires

DeKosky

Lungu

Park

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Elucidating how antigen exposure and selection shape the human antibody repertoire is fundamental to our understanding of B-cell immunity. We sequenced the paired heavy-and light-chain variable regions (VH and VL, respectively) from large populations of single B cells combined with computational modeling of antibody structures to evaluate sequence and structural features of human antibody repertoires at unprecedented depth. Analysis of a dataset comprising 55,000 antibody clusters from CD19− IgM-naive B cells, >120,000 antibody clusters from CD19+ antigenexperienced B cells, and >2,000 RosettaAntibody-predicted structural models across three healthy donors led to a number of key findings: (i) VH and VL gene sequences pair in a combinatorial fashion without detectable pairing restrictions at the population level; (ii) certain VH:VL gene pairs were significantly enriched or depleted in the antigen-experienced repertoire relative to the naive repertoire; (iii) antigen selection increased antibody paratope net charge and solvent-accessible surface area; and (iv) public heavy-chain third complementarity-determining region (CDR-H3) antibodies in the antigen-experienced repertoire showed signs of convergent paired light-chain genetic signatures, including shared light-chain third complementarity-determining region (CDR-L3) amino acid sequences and/or Vκ,λ-Jκ,λ genes. The data reported here address several longstanding questions regarding antibody repertoire selection and development and provide a benchmark for future repertoire-scale analyses of antibody responses to vaccination and disease.antibody | B cell | immunology | high-throughput sequencing | computational modeling E ffective antigen recognition by the humoral immune system is predicated on the somatic generation of a large antibody repertoire that encompasses the sequence and conformational diversity to respond to a highly diversified set of antigens (1-3). Upon antigen challenge, naive B cells (NBCs) expressing unmutated antibodies capable of binding antigen with an affinity sufficient to initiate B-cell receptor (BCR) signaling may be stimulated to undergo somatic hypermutation (SHM) of the antibody genes. B cells expressing higher-affinity BCRs are better equipped to compete for antigen and thus receive signals that enable their preferential proliferation and further antibody sequence diversification in additional rounds of SHM. This process generates a repertoire of somatically mutated antibodies that, at the structural level, generally display decreased conformational flexibility (4, 5), slower antigen dissociation rates, and increased binding selectivity relative to the germline repertoire.Understanding the salient features of the human antibody repertoire is critical for immunology research (6, 7). Specifically, additional information is needed regarding how a history of pathogen and environmental exposure modulates the sequence and conformational properties of naive antibodies to yield a mature antibody repertoire that confers effective protection. Hi...

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Antibody structure determination using a combination of homology modeling, energy‐based refinement, and loop prediction

Cited by 192 publications

References 41 publications

Cysteinylation of a monoclonal antibody leads to its inactivation

Cysteinylation of a monoclonal antibody leads to its inactivation

AcrB-AcrA Fusion Proteins That Act as Multidrug Efflux Transporters

Large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires

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