title: A recurrent Gly43Asp substitution in coagulation Factor X rigidifies its catalytic pocket and impairs catalytic activity and intracellular trafficking.
abstract: Factor X (FX) deficiency is one of the most severe among recessively inherited coagulation disorders. The homozygous Gly222Asp mutation (Gly43Asp in the chymotrypsinogen numbering) on the FX gene was found in fifteen patients with severe FX deficiency (FX:C <1%). The Gly(43) residue is located at the highly conserved 42-58 residues region shared among all trypsinogen-like proteins. In vitro expression studies showed that the replacement of a neutral Gly by a charged Asp residue into the so-called loop-40 of the FX (comprising residues from 42 to 58) causes an impairment of its catalytic competence as well as a secretion defect. Steady state kinetic studies showed also a severe defect of activation by FVIIa of the FX43Asp. Surprisingly, molecular dynamics studies clearly indicated that the Gly43Asp mutation neither disrupts nor destabilizes the FXa native structure of the catalytic site. Rather, it makes it more rigid, by thickening the H-bonding network around its catalytic site. Altogether, the defects of FX43Asp explain the severe bleeding symptoms in the patients and outline the relevance of the plasticity in the FXa catalytic pocket for maintenance of its catalytic competence.
authors.affiliation: Department of Pathophysiology and Transplantation, University of Milan and Luigi Villa Foundation, Milan, Italy; A. Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy. Electronic address: marzia.menegatti@unimi.it.
authors.forename: Marzia
authors.initials: M
authors.lastname: Menegatti
authors.affiliation: Department of Chemistry and Biology, University of Salerno, Fisciano (SA), Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Vangone
authors.affiliation: Department of Pathophysiology and Transplantation, University of Milan and Luigi Villa Foundation, Milan, Italy; A. Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Palla
authors.affiliation: Department of Chemistry and Biology, University of Salerno, Fisciano (SA), Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Milano
authors.affiliation: Department of Chemistry and Biology, University of Salerno, Fisciano (SA), Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Cavallo
authors.affiliation: Department of Sciences and Technologies, University "Parthenope" of Naples, Centro Direzionale Isola C4, Naples, Italy.
authors.forename: Romina
authors.initials: R
authors.lastname: Oliva
authors.affiliation: Hemostasis Research Centre, Institute of Internal Medicine and Geriatrics, Catholic University School of Medicine, Rome, Italy.
authors.forename: Raimondo
authors.initials: R
authors.lastname: De Cristofaro
authors.affiliation: Department of Pathophysiology and Transplantation, University of Milan and Luigi Villa Foundation, Milan, Italy; A. Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.
authors.forename: Flora
authors.initials: F
authors.lastname: Peyvandi
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002384:Catalysis; D020134:Catalytic Domain; D005170:Factor X / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005171:Factor X Deficiency / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D009154:Mutation; D013329:Structure-Activity Relationship
chemical_list: D005170:Factor X
keywords: Amidolytic activity; Catalytic activity; Factor X Gly43Asp mutation; Factor X deficiency; Molecular dynamics