Protein Science
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Protein Science (2006), 15:1893-1906. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplemental Research Data
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hantgan, R. R.
Right arrow Articles by Medved, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hantgan, R. R.
Right arrow Articles by Medved, L.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Integrin {alpha}IIbbeta3:ligand interactions are linked to binding-site remodeling

Roy R. Hantgan1, Mary C. Stahle1, John H. Connor1, David A. Horita1, Mattia Rocco2, Mary A. McLane3, Sergiy Yakovlev4 and Leonid Medved4

1 Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1019, USA
2 Istituto Nazionale per la Ricerca sul Cancro, Genova I-16132, Italy
3 University of Delaware, Newark, Delaware 19716, USA
4 University of Maryland School of Medicine, Baltimore, Maryland 21201, USA

(RECEIVED December 16, 2005; FINAL REVISION May 8, 2006; ACCEPTED May 11, 2006)

This study tested the hypothesis that high-affinity binding of macromolecular ligands to the {alpha}IIbbeta3 integrin is tightly coupled to binding-site remodeling, an induced-fit process that shifts a conformational equilibrium from a resting toward an open receptor. Interactions between {alpha}IIbbeta3 and two model ligands—echistatin, a 6-kDa recombinant protein with an RGD integrin-targeting sequence, and fibrinogen's {gamma}-module, a 30-kDa recombinant protein with a KQAGDV integrin binding site—were measured by sedimentation velocity, fluorescence anisotropy, and a solid-phase binding assay, and modeled by molecular graphics. Studying echistatin variants (R24A, R24K, D26A, D26E, D27W, D27F), we found that electrostatic contacts with charged residues at the {alpha}IIb/beta3 interface, rather than nonpolar contacts, perturb the conformation of the resting integrin. Aspartate 26, which interacts with the nearby MIDAS cation, was essential for binding, as D26A and D26E were inactive. In contrast, R24K was fully and R24A partly active, indicating that the positively charged arginine 24 contributes to, but is not required for, integrin recognition. Moreover, we demonstrated that priming—i.e., ectodomain conformational changes and oligomerization induced by incubation at 35°C with the ligand-mimetic peptide cHarGD—promotes complex formation with fibrinogen's {gamma}-module. We also observed that the {gamma}-module's flexible carboxy terminus was not required for {alpha}IIbbeta3 integrin binding. Our studies differentiate priming ligands, which bind to the resting receptor and perturb its conformation, from regulated ligands, where binding-site remodeling must first occur. Echistatin's binding energy is sufficient to rearrange the subunit interface, but regulated ligands like fibrinogen must rely on priming to overcome conformational barriers.

Keywords: integrins; ligands; conformation; analytical ultracentrifugation; solid phase binding; fluorescence anisotropy; molecular modeling


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?




HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2006 by The Protein Society.