title: Glycan Bound to the Selectin Low Affinity State Engages Glu-88 to Stabilize the High Affinity State under Force.
abstract: Selectin interactions with fucosylated glycan ligands mediate leukocyte rolling in the vasculature under shear forces. Crystal structures of P- and E-selectin suggest a two-state model in which ligand binding to the lectin domain closes loop 83-89 around the Ca2+coordination site, enabling Glu-88 to engage Ca2+and fucose. This triggers further allostery that opens the lectin/EGF domain hinge. The model posits that force accelerates transition from the bent (low affinity) to the extended (high affinity) state. However, transition intermediates have not been described, and the role of Glu-88 in force-assisted allostery has not been examined. Here we report the structure of the lectin and EGF domains of L-selectin bound to a fucose mimetic; that is, a terminal mannose on anN-glycan attached to a symmetry-related molecule. The structure is a transition intermediate where loop 83-89 closes to engage Ca2+and mannose without triggering allostery that opens the lectin/EGF domain hinge. We used three complementary assays to compare ligand binding to WT selectins and to E88D selectins that replaced Glu-88 with Asp. Soluble P-selectinE88D bound with an ∼9-fold lower affinity to PSGL-1, a physiological ligand, due to faster dissociation. Adhesion frequency experiments with a biomembrane force probe could not detect interactions of P-selectinE88D with PSGL-1. Cells expressing transmembrane P-selectinE88D or L-selectinE88D detached from immobilized ligands immediately after initiating flow. Cells expressing E-selectinE88D rolled but detached faster. Our data support a two-state model for selectins in which Glu-88 must engage ligand to trigger allostery that stabilizes the high affinity state under force.
authors.affiliation: From the Cardiovascular Biology Research Program and.
authors.forename: Padmaja
authors.initials: P
authors.lastname: Mehta-D'souza
authors.affiliation: From the Cardiovascular Biology Research Program and.
authors.forename: Arkadiusz G
authors.initials: AG
authors.lastname: Klopocki
authors.affiliation: From the Cardiovascular Biology Research Program and.
authors.forename: Vaheh
authors.initials: V
authors.lastname: Oganesyan
authors.affiliation: Crystallography Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104.
authors.forename: Simon
authors.initials: S
authors.lastname: Terzyan
authors.affiliation: From the Cardiovascular Biology Research Program and.
authors.forename: Timothy
authors.initials: T
authors.lastname: Mather
authors.affiliation: the Coulter Department of Biomedical Engineering.
authors.forename: Zhenhai
authors.initials: Z
authors.lastname: Li
authors.affiliation: From the Cardiovascular Biology Research Program and.
authors.forename: Sumith R
authors.initials: SR
authors.lastname: Panicker
authors.affiliation: the Coulter Department of Biomedical Engineering.|Woodruff School of Mechanical Engineering, and.|the Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332.
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhu
authors.affiliation: From the Cardiovascular Biology Research Program and rodger-mcever@omrf.org.|the Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104.
authors.forename: Rodger P
authors.identifier: 0000-0001-5343-2622
authors.initials: RP
authors.lastname: McEver
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D018360:Crystallography, X-Ray; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D019041:L-Selectin / Q000737:chemistry / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D008562:Membrane Glycoproteins; C084882:P-selectin ligand protein; D011134:Polysaccharides; D019041:L-Selectin; D018698:Glutamic Acid
keywords: carbohydrate-binding protein; cell adhesion; endothelial cell; glycobiology; inflammation; leukocyte