1 Atomic basis for the species-specific inhibition of αV integrins by mAb 17E6 is revealed by the crystal structure of αVβ3 ectodomain-17E6 Fab complex* Bhuvaneshwari Mahalingam 1 , Johannes F. Van Agthoven 1 , Jian-Ping Xiong 1 , José Luis Alonso 2 , Brian D. Adair 1 , Xianliang Rui 2 , Saurabh Anand 2 , Mehrdad Mehrbod 3 , Mohammad R. K. Mofrad 3 , Christa Burger 4 , Simon L Goodman 5 , and M. Amin Arnaout 1, 2 From the 1 Structural Biology Program, 2 Leukocyte Biology and Inflammation Program, Departments of Medicine and Developmental & Regenerative Biology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, 02129; 3 Departments of Bioengineering and Mechanical Engineering, University of California, Berkeley, CA, 94720; 4,5 Merck KGaA, 3 Discovery Technologies, Molecular Pharmacology, 5 Therapeutic Innovation Platform, Oncology, Darmstadt 64271, Germany. *Running head: Crystal structure of αVβ3-17E6 Fab complex To whom correspondence should be addressed: M. Amin Arnaout, Massachusetts General Hospital, 149 13th Street, Charlestown, MA, 02129, Tel: 617-726-5663; Email: aarnaout1@mgh.harvard.edu Keywords : Integrins, Fibronectin, Crystal structure, Molecular dynamics, Cancer therapy Background: 17E6, a primate-specific mouse mAb that inhibits αV integrins, is in a phase II trial for treating cancer. Results: We determined crystal structure of the αVβ3-17E6 Fab complex, revealing the molecular basis of 17E6 specificity and function. Conclusion: 17E6 is an allosteric inhibitor of fibronectin-integrin interaction. Significance: The defined 17E6 epitope may help in developing novel therapeutics targeting related regions in other integrins. ABSTRACT The function-blocking, non-RGD containing and primate-specific mouse monoclonal antibody 17E6 binds the αV subfamily of integrins. 17E6 is currently in phase 2 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 conformation. 17E6 Fab bound exclusively to the Propeller domain of the αV subunit. At the core of αV-Fab interface were interactions involving Propeller residues Lys203 and Gln145, the latter accounting for primate specificity. The Propeller residue Asp150, which normally coordinates Arg of the ligand Arg-Gly-Asp motif, formed contacts with Arg54 of the Fab that were expected to reduce soluble FN10 binding to cellular αVβ3 complexed with 17E6. This was confirmed in direct binding studies, suggesting that 17E6 is an allosteric inhibitor of αV integrins. Integrins are α/β heterodimeric adhesion receptors that transmit biochemical and mechanical signals bidirectionally across the plasma membrane, thus serving as key communication molecules between the extracellular environment and the cytoskeleton http://www.jbc.org/cgi/doi/10.1074/jbc.M113.546929 The latest version is at JBC Papers in Press. Published on April 1, 2014 as Manuscript M113.546929 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.