The allosteric mechanism of activation of antithrombin as an inhibitor of factor IXa and factor Xa: heparin-independent full activation through mutations adjacent to helix D.
Allosteric conformational changes in antithrombin induced by binding a specific heparin pentasaccharide result in very large increases in the rates of inhibition of factors IXa and Xa but not of thrombin. These are accompanied by CD, fluorescence, and NMR spectroscopic changes. X-ray structures show that heparin binding results in extension of helix D in the region 131-136 with coincident, and possibly coupled, expulsion of the hinge of the reactive center loop. To examine the importance of helix D extension, we have introduced strong helix-promoting mutations in the 131-136 region of antithrombin (YRKAQK to LEEAAE). The resulting variant has endogenous fluorescence indistinguishable from WT antithrombin yet, in the absence of heparin, shows massive enhancements in rates of inhibition of factors IXa and Xa (114- and 110-fold, respectively), but not of thrombin, together with changes in near- and far-UV CD and (1)H NMR spectra. Heparin binding gives only ∼3-4-fold further rate enhancement but increases tryptophan fluorescence by ∼23% without major additional CD or NMR changes. Variants with subsets of these mutations show intermediate activation in the absence of heparin, again with basal fluorescence similar to WT and large increases upon heparin binding. These findings suggest that in WT antithrombin there are two major complementary sources of conformational activation of antithrombin, probably involving altered contacts of side chains of Tyr-131 and Ala-134 with core hydrophobic residues, whereas the reactive center loop hinge expulsion plays only a minor additional role.
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607.
Alexey
A
Dementiev
Center for Molecular Biology of Oral Disease, University of Illinois at Chicago, Chicago, Illinois 60607.
Richard
R
Swanson
Center for Molecular Biology of Oral Disease, University of Illinois at Chicago, Chicago, Illinois 60607.
Ryan
R
Roth
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607.
Giulia
G
Isetti
Center for Molecular Biology of Oral Disease, University of Illinois at Chicago, Chicago, Illinois 60607.
Gonzalo
G
Izaguirre
Center for Molecular Biology of Oral Disease, University of Illinois at Chicago, Chicago, Illinois 60607.
Steven T
ST
Olson
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607. Electronic address: pgettins@uic.edu.
Peter G W
PGW
Gettins
D000494:Allosteric Regulation / Q000235:genetics; D000990:Antithrombin III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D015949:Factor IXa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015951:Factor Xa / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary
D000990:Antithrombin III; D015949:Factor IXa; D015951:Factor Xa
Antithrombin; Blood Coagulation Factors; Circular Dichroism (CD); Coagulation Factors; Enzyme Kinetics; Fluorescence; Heparin; Mutagenesis; Protease Inhibitor; Serine Protease