The conformational activation of antithrombin - A 2.85-angstrom structure of a fluorescein derivative reveals an electrostatic link between the hingeand heparin binding regions

Antithrombin is unique among the serpins in that it circulates in a native conformation that is kinetically inactive toward its target proteinase, fac tor Xa, Activation occurs upon binding of a specific pentasaccharide sequen ce found in heparin that results in a rearrangement of the reactive center loop removing constraints on the active center P1 residue. We determined th e crystal structure of an activated antithrombin variant, N135Q S380C-fluor escein (P14-fluorescein), in order to see how full activation is achieved i n the absence of heparin and how the structural effects of the substitution in the hinge region are translated to the heparin binding region. The crys tal structure resembles native antithrombin except in. the hinge and hepari n binding regions. The absence of global conformational change allows for i dentification of specific interactions, centered on Glu(381) (P13), that ar e responsible for maintenance of the solution equilibrium between the nativ e and activated forms and establishes the existence of an electrostatic lin k between the hinge region and the heparin binding region. A revised model for the mechanism of the allosteric activation of antithrombin is proposed.