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J Biol Chem, Vol. 275, Issue 20, 15377-15383, May 19, 2000

The Conformational Activation of Antithrombin
A 2.85-Å STRUCTURE OF A FLUORESCEIN DERIVATIVE REVEALS AN ELECTROSTATIC LINK BETWEEN THE HINGE AND HEPARIN BINDING REGIONS^*

James A. Huntington^§, Airlie McCoy, Klara J. Belzar, Xue Y. Pei, Peter G. W. Gettins^¶, and Robin W. Carrell

From the University of Cambridge, Department of Haematology, Wellcome Trust Centre for the Study of Molecular Mechanisms in Disease, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, United Kingdom and ^¶ Department of Biochemistry and Molecular Biology, University of Illinois, Chicago, Illinois 60612

Antithrombin is unique among the serpins in that it circulates in a native conformation that is kinetically inactive toward its target proteinase, factor Xa. Activation occurs upon binding of a specific pentasaccharide sequence found in heparin that results in a rearrangement of the reactive center loop removing constraints on the active center P1 residue. We determined the crystal structure of an activated antithrombin variant, N135Q S380C-fluorescein (P14-fluorescein), in order to see how full activation is achieved in the absence of heparin and how the structural effects of the substitution in the hinge region are translated to the heparin binding region. The crystal structure resembles native antithrombin except in the hinge and heparin binding regions. The absence of global conformational change allows for identification of specific interactions, centered on Glu³⁸¹ (P13), that are responsible for maintenance of the solution equilibrium between the native and activated forms and establishes the existence of an electrostatic link between the hinge region and the heparin binding region. A revised model for the mechanism of the allosteric activation of antithrombin is proposed.

The atomic coordinates and structure factors (codes 1dzg and 1dzh) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

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