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Atomic basis for the species-specific inhibition of ?V integrins by monoclonal antibody 17E6 is revealed by the crystal structure of ?V?3 ectodomain-17E6 Fab complex.

ABSTRACT: The function-blocking, non-RGD-containing, and primate-specific mouse monoclonal antibody 17E6 binds the ?V subfamily of integrins. 17E6 is currently in phase II clinical trials for treating cancer. To elucidate the structural basis of recognition and the molecular mechanism of inhibition, we crystallized ?V?3 ectodomain in complex with the Fab fragment of 17E6. Protein crystals grew in presence of the activating cation Mn(2+). The integrin in the complex and in solution assumed the genuflected conformation. 17E6 Fab bound exclusively to the Propeller domain of the ?V subunit. At the core of ?V-Fab interface were interactions involving Propeller residues Lys-203 and Gln-145, with the latter accounting for primate specificity. The Propeller residue Asp-150, which normally coordinates Arg of the ligand Arg-Gly-Asp motif, formed contacts with Arg-54 of the Fab that were expected to reduce soluble FN10 binding to cellular ?V?3 complexed with 17E6. This was confirmed in direct binding studies, suggesting that 17E6 is an allosteric inhibitor of ?V integrins.

SUBMITTER: Mahalingam B

PROVIDER: S-EPMC4022854 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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Atomic basis for the species-specific inhibition of αV integrins by monoclonal antibody 17E6 is revealed by the crystal structure of αVβ3 ectodomain-17E6 Fab complex.

Mahalingam Bhuvaneshwari B Van Agthoven Johannes F JF Xiong Jian-Ping JP Alonso José Luis JL Adair Brian D BD Rui Xianliang X Anand Saurabh S Mehrbod Mehrdad M Mofrad Mohammad R K MR Burger Christa C Goodman Simon L SL Arnaout M Amin MA

The Journal of biological chemistry 20140401 20

The function-blocking, non-RGD-containing, and primate-specific mouse monoclonal antibody 17E6 binds the αV subfamily of integrins. 17E6 is currently in phase II clinical trials for treating cancer. To elucidate the structural basis of recognition and the molecular mechanism of inhibition, we crystallized αVβ3 ectodomain in complex with the Fab fragment of 17E6. Protein crystals grew in presence of the activating cation Mn(2+). The integrin in the complex and in solution assumed the genuflected ...[more]

PMID: 24692540

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Project description:BackgroundMany antibody crystal structures have been solved. Structural modeling programs have been developed that utilize this information to predict 3-D structures of an antibody based upon its sequence. Because of the problem of self-reference, the accuracy and utility of these predictions can only be tested when a new structure has not yet been deposited in the Protein Data Bank.MethodsWe have solved the crystal structure of the Fab fragment of RAC18, a protective anti-ricin mAb, to 1.9 Å resolution. We have also modeled the Fv structure of RAC18 using publicly available Ab modeling tools Prediction of Immunoglobulin Structures (PIGS), RosettaAntibody, and Web Antibody Modeling (WAM). The model structures underwent energy minimization. We compared results to the crystal structure on the basis of root-mean-square deviation (RMSD), template modeling score (TM-score), Z-score, and MolProbity analysis.FindingsThe crystal structure showed a pocket formed mainly by AA residues in each of the heavy chain complementarity determining regions (CDRs). There were differences between the crystal structure and structures predicted by the modeling tools, particularly in the CDRs. There were also differences among the predicted models, although the differences were small and within experimental error. No one modeling program was clearly superior to the others. In some cases, choosing structures based only on sequence homology to the crystallized Ab yielded RMSDs comparable to the models.ConclusionsMolecular modeling programs accurately predict the structure of most regions of antibody variable domains of RAC18. The hypervariable CDRs proved most difficult to model, particularly H chain CDR3. Because CDR3 is most often involved in contact with antigen, this defect must be considered when using models to identify potential contacts between antibody and antigen. Because this study represents only a single case, the results cannot be generalized. Rather they highlight the utility and limitations of modeling programs.

Dataset Information

Atomic basis for the species-specific inhibition of ?V integrins by monoclonal antibody 17E6 is revealed by the crystal structure of ?V?3 ectodomain-17E6 Fab complex.

Publications

Atomic basis for the species-specific inhibition of αV integrins by monoclonal antibody 17E6 is revealed by the crystal structure of αVβ3 ectodomain-17E6 Fab complex.

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