Horn James R, Sosnick Tobin R, Kossiakoff Anthony A
Department of Biochemistry and Molecular Biology, and Institute for Biophysical Dynamics, and Computation Institute, University of Chicago, Chicago, IL 60637, USA.
Proc Natl Acad Sci U S A. 2009 Feb 24;106(8):2559-64. doi: 10.1073/pnas.0809800106. Epub 2009 Feb 5.
The binding transition state (TS) is the rate-limiting step for transient molecular interactions. This important step in the molecular recognition process, however, is largely understood only at a qualitative level. To establish a more quantitative picture of the TS structure, we exploit a set of biophysical techniques that have provided major insights in protein folding applications. As a model system representing the large class of "weakly charged" protein-protein interactions, we examine the binding of a variety of human growth hormone (hGH) variants to the human growth hormone receptor (hGHR) and the human prolactin receptor (hPRLR). hGH variants were chosen to probe different features of the TS structure, based on their highly reengineered interfaces. Both Eyring and urea (m value) analyses suggest that the majority of binding surface burial occurs after TS. A comprehensive phi analysis showed that individual hGH interface residues do not contribute energetically to the stability of the TS, but there is a TS "hot spot" in the receptor. Zinc dependence studies that take advantage of an endogenous tetracoordinated interfacial metal binding demonstrate that surfaces of the molecules have attained a high orientational complementarity by the time the TS is reached. The model that best fits these data are that a "knobs-into-holes" process precisely aligns the two molecular interfaces in forming the TS structure. Surprisingly, most of the thermodynamic character of the binding reaction is focused in the fine-tuning process occurring after TS.
结合过渡态(TS)是瞬态分子相互作用的限速步骤。然而,分子识别过程中的这一重要步骤在很大程度上仅在定性层面上得到理解。为了建立更定量的TS结构图景,我们利用了一组生物物理技术,这些技术在蛋白质折叠应用中提供了重要见解。作为代表一大类“弱电荷”蛋白质 - 蛋白质相互作用的模型系统,我们研究了多种人生长激素(hGH)变体与人生长激素受体(hGHR)和人催乳素受体(hPRLR)的结合。基于其高度重新设计的界面,选择hGH变体来探测TS结构的不同特征。艾林分析和尿素(m值)分析均表明,大部分结合表面的掩埋发生在TS之后。全面的phi分析表明,单个hGH界面残基对TS的稳定性没有能量贡献,但受体中存在一个TS“热点”。利用内源性四配位界面金属结合的锌依赖性研究表明,在达到TS时,分子表面已达到高度的取向互补性。最符合这些数据的模型是,“旋钮插入孔”过程在形成TS结构时精确地对齐了两个分子界面。令人惊讶的是,结合反应的大多数热力学特征集中在TS之后发生的微调过程中。