Electrostatic Steering and Ionic Tethering in the Formation of Thrombin-Hirudin Complexes: The Role of the Thrombin Anion-Binding Exosite-I

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• 作者：Timothy Myles ; Bernard F. Le Bonniec ; Andreas Betz ; and Stuart R. Stone
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：April 24, 2001
• 年：2001
• 卷：40
• 期：16
• 页码：4972 - 4979
• 全文大小：97K
• 年卷期：v.40,no.16(April 24, 2001)
• ISSN：1520-4995

文摘

Electrostatic interactions between the thrombin anion-binding exosite-I (ABE-I) and the hirudinC-terminal tail play an important role in the formation of the thrombin-hirudin inhibitor complex andserves as a model for the interactions of thrombin with its many other ligands. The role of each solventexposed basic residue in ABE-I (Arg³⁵, Lys³⁶, Arg⁶⁷, Arg⁷³, Arg⁷⁵, Arg^77a, Lys⁸¹, Lys¹⁰⁹, Lys¹¹⁰, and Lys^149e)in electrostatic steering and ionic tethering in the formation of thrombin-hirudin inhibitor complexeswas explored by site directed mutagenesis. The contribution to the binding energy (cribe/journals/bichaw/40/i16/eqn/bi0023549e10001.gif">) by each residuevaried from 1.9 kJ mol^-1 (Lys¹¹⁰) to 15.3 kJ mol^-1 (Arg⁷³) and were in general agreement to their observedinteractions with hirudin residues in the thrombin-hirudin crystal structure [Rydel, T. J., Tulinsky, A.,Bode, W., and Huber, R. (1991) J. Mol. Biol. 221, 583-601]. Coupling energies (cribe/journals/bichaw/40/i16/eqn/bi0023549e10002.gif">) werecalculated for the major ion-pair interactions involved in ionic tethering using complementary hirudinmutants (h-D55N, h-E57Q, and h-E58Q). Cooperativity was seen for the h-Asp⁵⁵/Arg⁷³ ion pair (cribe/journals/bichaw/40/i16/eqn/bi0023549e10003.gif">2.4 kJ mol^-1); however, low coupling energies for h-Asp⁵⁵/Lys^149e (cribe/journals/bichaw/40/i16/eqn/bi0023549e10004.gif"> 0.6 kJ mol^-1) and h-Glu⁵⁸/Arg^77a (cribe/journals/bichaw/40/i16/eqn/bi0023549e10005.gif"> 0.9 kJ mol^-1) suggest these are not major interactions, as anticipated by the crystalstructure. Interestingly, high coupling energies were seen for the intermolecular ion-pair h-Glu⁵⁷/Arg⁷⁵ (cribe/journals/bichaw/40/i16/eqn/bi0023549e10006.gif"> 2.3 kJ mol^-1) and for the solvent bridge h-Glu⁵⁷/Arg^77a (cribe/journals/bichaw/40/i16/eqn/bi0023549e10007.gif"> 2.7 kJ mol^-1) indicating thath-Glu⁵⁷ interacts directly with both Arg⁷⁵ and Arg^77a in the thrombin-hirudin inhibitor complex. Theremaining ABE-I residues that do not form major contacts in tethering the C-terminal tail of hirudinmake small but collectively important contributions to the overall positive electrostatic field generated byABE-I important in electrostatic steering.