1 Structure, Sulfatide-binding Properties, and Inhibition of Platelet Aggregation by a Disabled-2-derived Peptide * Shuyan Xiao 1 , John J. Charonko 2 , Xiangping Fu 3 , Alireza Salmanzadeh 4 , Rafael V. Davalos 4 , Pavlos P. Vlachos 2 , Carla V. Finkielstein 3 , and Daniel G. S. Capelluto 1 From the 1 Protein Signaling Domains Laboratory, Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061, 2 Advanced Experimental Thermofluidics Engineering Research Laboratory, Department of Mechanical Engineering, Virginia Tech, Blacksburg, VA 24061, 3 Integrated Cellular Responses Laboratory, Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061, and 4 Bioelectromechanical Systems Laboratory, Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences and Department of Engineering Science and Mechanics, Virginia Tech, Blacksburg, VA 24061. * Running Title: Platelet aggregation inhibition by a Disabled-2-derived peptide. To whom correspondence should be addressed: Daniel G. S. Capelluto, Department of Biological Sciences, Virginia Tech, 1981 Kraft Drive, Room 2007, Blacksburg, VA 24061. Tel: +540-231-0974; Fax: +540-231-3414. E-mail address: capellut@vt.edu (D. G. S. Capelluto). Keywords: Disabled-2; sulfatides; NMR; platelet aggregation Background: Binding of Dab2 to sulfatides results in platelet aggregation inhibition. Results: The structure of a Dab2-derived peptide (SBM) embedded in dodecylphosphocholine micelles, characterization of its minimal functional sulfatide-binding site, and its inhibitory platelet aggregation activity was determined. Conclusion: An amphipathic helical region of Dab2 SBM binds sulfatides leading to platelet aggregation inhibition. Significance: Dab2 SBM may lead to the design of novel aggregatory inhibitors. ABSTRACT Disabled-2 (Dab2) targets membranes and triggers a wide range of biological events, including endocytosis and platelet aggregation. Dab2, through its phosphotyrosine-binding (PTB) domain, inhibits platelet aggregation by competing with fibrinogen for α IIb β 3 integrin receptor binding. We have recently shown that the N-terminal region, including the PTB domain (N-PTB), drives Dab2 to the platelet membrane surface by binding to sulfatides through two sulfatide-binding motifs, modulating the extent of platelet aggregation. The three-dimensional structure of a Dab2- derived peptide encompassing the sulfatide- binding motifs (SBM) has been determined in dodecylphosphocholine (DPC) micelles using NMR spectroscopy. Dab2 SBM contains two helices when embedded in micelles, reversibly binds to sulfatides with moderate affinity, lies parallel to the micelle surface, and, when added to a platelet mixture, reduces the number and size of sulfatide-induced aggregates. Overall, our findings identify and structurally characterize a minimal region in Dab2 that modulates platelet homotypic interactions, all of which provide the foundation for rational design of a new generation of anti-aggregatory low-molecular mass molecules for therapeutic purposes. Disabled-2 (Dab2) is a cargo-specific adaptor protein involved in endocytosis, cell- surface receptor turnover, and cell signaling (1-4). Expression of Dab2 is frequently lost in about 90% of all breast and ovarian cancers and its homozygous deletion has been identified in numerous tumors, pointing to a role for this http://www.jbc.org/cgi/doi/10.1074/jbc.M112.385609 The latest version is at JBC Papers in Press. Published on September 13, 2012 as Manuscript M112.385609 Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc. by guest on February 5, 2016 http://www.jbc.org/ Downloaded from by guest on February 5, 2016 http://www.jbc.org/ Downloaded from by guest on February 5, 2016 http://www.jbc.org/ Downloaded from