Relating conformation to function in integrin α5β1.
Whether β1 integrin ectodomains visit conformational states similarly to β2 and β3 integrins has not been characterized. Furthermore, despite a wealth of activating and inhibitory antibodies to β1 integrins, the conformational states that these antibodies stabilize, and the relation of these conformations to function, remain incompletely characterized. Using negative-stain electron microscopy, we show that the integrin α5β1 ectodomain adopts extended-closed and extended-open conformations as well as a bent conformation. Antibodies SNAKA51, 8E3, N29, and 9EG7 bind to different domains in the α5 or β1 legs, activate, and stabilize extended ectodomain conformations. Antibodies 12G10 and HUTS-4 bind to the β1 βI domain and hybrid domains, respectively, activate, and stabilize the open headpiece conformation. Antibody TS2/16 binds a similar epitope as 12G10, activates, and appears to stabilize an open βI domain conformation without requiring extension or hybrid domain swing-out. mAb13 and SG/19 bind to the βI domain and βI-hybrid domain interface, respectively, inhibit, and stabilize the closed conformation of the headpiece. The effects of the antibodies on cell adhesion to fibronectin substrates suggest that the extended-open conformation of α5β1 is adhesive and that the extended-closed and bent-closed conformations are nonadhesive. The functional effects and binding sites of antibodies and fibronectin were consistent with their ability in binding to α5β1 on cell surfaces to cross-enhance or inhibit one another by competitive or noncompetitive (allosteric) mechanisms.
Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115;
Yang
Y
Su
Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115;
Wei
W
Xia
Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115;
Jing
J
Li
Rockefeller University, New York, NY 10065;
Thomas
T
Walz
Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom;
Martin J
MJ
Humphries
Max Planck Institute of Molecular Biomedicine, 48149 Muenster, Germany;
Dietmar
D
Vestweber
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), 28049 Madrid, Spain.
Carlos
C
Cabañas
Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115;
Chafen
C
Lu
Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; timothy.springer@childrens.harvard.edu.
Timothy A
TA
Springer
D004926:Escherichia coli; D005353:Fibronectins / Q000378:metabolism; D006801:Humans; D039081:Integrin alpha5beta1 / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D005353:Fibronectins; D039081:Integrin alpha5beta1