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Molecular Basis for the Dynamic Strength of the Integrin ₄ß₁/VCAM-1 Interaction

Xiaohui Zhang ^*, Susan E. Craig , Hishani Kirby , Martin J. Humphries  and Vincent T. Moy ^*

^* Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida, USA; Wellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences, University of Manchester, Manchester, United Kingdom; and Celltech Group, Slough, United Kingdom

Correspondence: Address reprint requests to Vincent T. Moy, Dept. of Physiology and Biophysics, University of Miami School of Medicine, 1600 N.W. 10th Ave., Miami, FL 33136. Tel.: 305-243-3201; Fax: 305-243-5931; E-mail: vmoy{at}newssun.med.miami.edu.

Intercellular adhesion mediated by integrin ₄ß₁ and vascular cell adhesion molecule-1 (VCAM-1) plays a crucial role in both the rolling and firm attachment of leukocytes onto the vascular endothelium. Essential to the ₄ß₁/VCAM-1 interaction is its mechanical strength that allows the complex to resist the large shear forces imposed by the bloodstream. Herein we employed single-molecule dynamic force spectroscopy to investigate the dynamic strength of the ₄ß₁/VCAM-1 complex. Our force measurements revealed that the dissociation of the ₄ß₁/VCAM-1 complex involves overcoming at least two activation potential barriers: a steep inner barrier and a more elevated outer barrier. The inner barrier grants the complex the tensile strength to withstand large pulling forces (>50 pN) and was attributed to the ionic interaction between the chelated Mg²⁺ ion at the N-terminal A-domain of the ß₁ subunit of ₄ß₁ and the carboxyl group of Asp-40 of VCAM-1 through the use of site-directed mutations. In general, additional mutations within the C-D loop of domain 1 of VCAM-1 suppressed both inner and outer barriers of the ₄ß₁/VCAM-1 complex, while a mutation at Asp-143 of domain 2 of VCAM-1 resulted in the suppression of the outer barrier, but not the inner barrier. In contrast, the outer barrier of ₄ß₁/VCAM-1 complex was stabilized by integrin activation. Together, these findings provide a molecular explanation for the functionally relevant kinetic properties of the ₄ß₁/VCAM-1 interaction.

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