title: Conformational equilibria and intrinsic affinities define integrin activation.
abstract: We show that the three conformational states of integrin α5β1have discrete free energies and define activation by measuring intrinsic affinities for ligand of each state and the equilibria linking them. The 5,000-fold higher affinity of the extended-open state than the bent-closed and extended-closed states demonstrates profound regulation of affinity. Free energy requirements for activation are defined with protein fragments and intact α5β1On the surface of K562 cells, α5β1is 99.8% bent-closed. Stabilization of the bent conformation by integrin transmembrane and cytoplasmic domains must be overcome by cellular energy input to stabilize extension. Following extension, headpiece opening is energetically favored. N-glycans and leg domains in each subunit that connect the ligand-binding head to the membrane repel or crowd one another and regulate conformational equilibria in favor of headpiece opening. The results suggest new principles for regulating signaling in the large class of receptors built from extracellular domains in tandem with single-span transmembrane domains.
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.|Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.|Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Su
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.|Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Xia
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.|Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Qin
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester, UK.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Humphries
authors.affiliation: Max-Planck-Institute of Molecular Biomedicine, Münster, Germany.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Vestweber
authors.affiliation: Centro de Biología Molecular Severo Ochoa (CSIC-UAM) and Departamento de Microbiología I Facultad de Medicina UCM, Madrid, Spain.
authors.forename: Carlos
authors.identifier: 0000-0002-8767-060X
authors.initials: C
authors.lastname: Cabañas
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA.|Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
authors.forename: Chafen
authors.initials: C
authors.lastname: Lu
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA, USA timothy.springer@childrens.harvard.edu.|Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
authors.forename: Timothy A
authors.identifier: 0000-0001-6627-2904
authors.initials: TA
authors.lastname: Springer
mesh_terms: D002460:Cell Line; D006801:Humans; D039081:Integrin alpha5beta1 / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D013816:Thermodynamics
chemical_list: D039081:Integrin alpha5beta1
keywords: N‐glycan; affinity; conformation; integrin; thermodynamics