Role of electrostatic interactions in binding of thrombin to the fibrinogen γ' chain.
Role of electrostatic interactions in binding of thrombin to the fibrinogen γ' chain.
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静电相互作用在凝血酶与纤维蛋白原γ链结合中的作用。
DOI：
10.1021/bi2016519
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发表时间：
2012
期刊：
影响因子：
2.9
通讯作者：
Farrell,DavidH
中科院分区：
文献类型：
--
作者：
Alexander,KristineS;Fried,MichaelG;Farrell,DavidH
凝血酶通过阴离子结合外位点II与高阴离子纤维蛋白原γ′链结合。这种结合深刻地改变了凝血酶切割底物的能力，包括纤维蛋白原、因子VIII和PAR 1。然而，目前尚不清楚这种相互作用是否主要是由于γ′链和外位点II之间的一般静电互补性，或者是否存在关键的带电γ′链残基。因此，我们系统地确定了γ′链中带负电荷的氨基酸对凝血酶结合亲和力的贡献，无论是单独的还是共同的。表面等离子体共振结合实验使用具有带电至不带电氨基酸取代的固定化γ '链肽进行，即，Asp至Asn、Glu至Gln和pTyr至Tyr。单独地，不带电荷的氨基酸取代带电荷的氨基酸仅导致结合亲和力的微小变化，Asp 419 Asn取代的Kd的最大变化为0.440至0.705 μM。然而，预测接触凝血酶的保守β-转角中的所有三个带电氨基酸pTyr 418 Tyr、Asp 419 Asn和pTyr 422 Tyr的取代导致可测量结合的损失，所有侧翼带电氨基酸的取代也是如此。此外，随着NaCl浓度的增加，γ′链与凝血酶的结合强度呈剂量依赖性减弱，导致凝血酶与γ′链之间净损失3 ~ 4个离子对。因此，尽管γ′链中的每个电荷对整体结合亲和力的贡献是递增的，但组合电荷的集合在凝血酶-γ′链相互作用中起着重要作用。
Thrombin binds to the highly anionic fibrinogen γ′ chain through anion-binding exosite II. This binding profoundly alters thrombin’s ability to cleave substrates, including fibrinogen, factor VIII, and PAR1. However, it is unknown whether this interaction is due mainly to general electrostatic complementarity between the γ′ chain and exosite II or if there are critical charged γ′ chain residues involved. We therefore systematically determined the contribution of negatively charged amino acids in the γ′ chain, both individually and collectively, to thrombin binding affinity. Surface plasmon resonance binding experiments were performed using immobilized γ′ chain peptides with charged-to-uncharged amino acid substitutions, i.e., Asp to Asn, Glu to Gln, and pTyr to Tyr. Individually, the substitution of uncharged for charged amino acids resulted in only minor changes in binding affinity, with a maximal change inKdfrom 0.440 to 0.705 μM for the Asp419Asn substitution. However, substitution of all three charged amino acids in a conserved β-turn that is predicted to contact thrombin, pTyr418Tyr, Asp419Asn, and pTyr422Tyr, resulted in the loss of measurable binding, as did substitution of all the flanking charged amino acids. In addition, the binding of the γ′ chain to thrombin was weakened in a dose-dependent manner with increasing NaCl concentration, resulting in a net loss of three or four ion pairs between thrombin and the γ′ chain. Therefore, although each of the individual charges in the γ′ chain contributes only incrementally to the overall binding affinity, the ensemble of the combined charges plays a profound role in the thrombin−γ′ chain interactions.