title: Directed evolution to probe protein allostery and integrin I domains of 200,000-fold higher affinity.
abstract: Understanding allostery may serve to both elucidate mechanisms of protein regulation and provide a basis for engineering active mutants. Herein we describe directed evolution applied to the integrin alpha(L) inserted domain for studying allostery by using a yeast surface display system. Many hot spots for activation are identified, and some single mutants exhibit remarkable increases of 10,000-fold in affinity for a physiological ligand, intercellular adhesion molecule-1. The location of activating mutations traces out an allosteric interface in the interior of the inserted domain that connects the ligand binding site to the alpha7-helix, which communicates allostery to neighboring domains in intact integrins. The combination of two activating mutations (F265S/F292G) leads to an increase of 200,000-fold in affinity to intercellular adhesion molecule-1. The F265S/F292G mutant is potent in antagonizing lymphocyte function-associated antigen 1-dependent lymphocyte adhesion, aggregation, and transmigration.
authors.affiliation: The CBR Institute for Biomedical Research and Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: Moonsoo
authors.initials: M
authors.lastname: Jin
authors.forename: Gang
authors.initials: G
authors.lastname: Song
authors.forename: Christopher V
authors.initials: CV
authors.lastname: Carman
authors.forename: Yong-Sung
authors.initials: YS
authors.lastname: Kim
authors.forename: Nathan S
authors.initials: NS
authors.lastname: Astrof
authors.forename: Motomu
authors.initials: M
authors.lastname: Shimaoka
authors.forename: Dane K
authors.initials: DK
authors.lastname: Wittrup
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Springer
mesh_terms: D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001665:Binding Sites; D019020:Directed Molecular Evolution; D016023:Integrins / Q000737:chemistry* / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D020349:Surface Plasmon Resonance
chemical_list: D016023:Integrins; D011506:Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D018799:Intercellular Adhesion Molecule-1