Electrostatic steering and ionic tethering in the formation of thrombin-hirudin complexes: The role of the thrombin anion-binding exosite-I
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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DOI：
10.1021/bi0023549
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发表时间：
2001-04-24
期刊：
影响因子：
2.9
通讯作者：
Stone, SR
中科院分区：
文献类型：
--
作者：
Myles, T;Le Bonniec, BF;Stone, SR
凝血酶阴离子结合外位点-I（ABE-I）和水蛭素C-末端尾部之间的静电相互作用在凝血酶-水蛭素抑制剂复合物的形成中起重要作用，并作为凝血酶与其许多其他配体相互作用的模型。通过定点诱变研究了ABE-1中每个溶剂暴露的碱性残基（Arg（35）、Lys（36）、Arg（67）、Arg（73）、Arg（75）、Arg（77 a）、LyS（81）、LyS（109）、Lys（110）和Lys（149 e））在凝血酶-水蛭素抑制剂复合物形成中的静电操纵和离子束缚中的作用。每个残基对结合能（Δ G（B）（o））的贡献从1.9 kJ mol（-1）（Lys（110））变化到15.3 kJ mol（-1）（Arg（73）），并且与它们在凝血酶-水蛭素晶体结构中观察到的与水蛭素残基的相互作用基本一致[Rydel，T. J.，Tulinsky，A.，Bode，W.，和Huber，R.等人（1991）J. Mol. 221，583-601]。使用互补水蛭素突变体（h-D55 N、h-E57 Q和h-E58 Q）计算离子束缚中涉及的主要离子对相互作用的耦合能（Δ Δ G（int）（o））。h-Asp（55）/Arg（73）离子对具有协同作用（Δ Δ G（int）（o）2.4 kJ mol（-1））;然而，h-Asp（55）/Lys（149 e）的低耦合能（Δ Δ G（int）（o）0.6 kJ mol-L）和h-Glu（58）/ Arg（77 a）（Δ Δ G（int）（o）0.9 kJ mol（-1））表明这些不是主要的相互作用，正如晶体结构所预期的那样。有趣的是，分子间离子对h-Glu（57）/Arg（75）的耦合能很高。（Δ Δ G（int）（o），2.3 kJ mol（-1））和对于溶剂桥h-Glu（57）/Arg（77 a）（Delta DeltaG（int）（o），表明h-Glu（57）直接与凝血酶-水蛭素抑制剂复合物中的Arg（75）和Arg（77 a）相互作用。其余的ABE-I残基，不形成主要的接触，在拴水蛭素的C-末端尾部，使小，但集体重要的贡献，总的正静电场产生的ABE-I重要的静电转向。
Electrostatic interactions between the thrombin anion-binding exosite-I (ABE-I) and the hirudin C-terminal tail play an important role in the formation of the thrombin-hirudin inhibitor complex and serves as a model for the interactions of thrombin with its many other ligands. The role of each solvent exposed basic residue in ABE-I (Arg(35), Lys(36), Arg(67), Arg(73), Arg(75) Arg(77a), LyS(81), LyS(109), Lys(110), and Lys(149e)) in electrostatic steering and ionic tethering in the formation of thrombin-hirudin inhibitor complexes was explored by site directed mutagenesis. The contribution to the binding energy (DeltaG(b)(o)) by each residue varied from 1.9 kJ mol(-1) (Lys(110)) to 15.3 kJ mol(-1) (Arg(73)) and were in general agreement to their observed interactions 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 (Delta DeltaG(int)(o)) were calculated for the major ion-pair interactions involved in ionic tethering using complementary hirudin mutants (h-D55N, h-E57Q, and h-E58Q). Cooperativity was seen for the h-Asp(55)/Arg(73) ion pair (Delta DeltaG(int)(o) 2.4 kJ mol(-1)); however, low coupling energies for h-Asp(55)/Lys(149e) (Delta DeltaG(int)(o) 0.6 kJ mol-L) and h-Glu(58)/ Arg(77a) (Delta DeltaG(int)(o) 0.9 kJ mol(-1)) suggest these are not major interactions, as anticipated by the crystal structure. Interestingly, high coupling energies were seen for the intermolecular ion-pair h-Glu(57)/Arg(75) ( Delta DeltaG(int)(o), 2.3 kJ mol(-1)) and for the solvent bridge h-Glu(57)/Arg(77a) (Delta DeltaG(int)(o), 2.7 kJ mol(-1)) indicating that h-Glu(57) interacts directly with both Arg(75) and Arg(77a) in the thrombin-hirudin inhibitor complex. The remaining ABE-I residues that do not form major contacts in tethering the C-terminal tail of hirudin make small but collectively important contributions to the overall positive electrostatic field generated by ABE-I important in electrostatic steering.