Cordier F, Wang C, Grzesiek S, Nicholson L K
Biozentrum der Universität Basel, Klingelbergstrasse 70, Basel, CH-4058, Switzerland.
J Mol Biol. 2000 Dec 8;304(4):497-505. doi: 10.1006/jmbi.2000.4274.
Changes in the molecular conformation of proteins can result from a variety of perturbations, and can play crucial roles in the regulation of biological activity. A new solution NMR method has been applied to monitor ligand-induced changes in hydrogen bond geometry in the chicken c-Src SH3 domain. The structural response of this domain to ligand binding has been investigated by measuring trans-hydrogen bond (15)N-(13)C' scalar couplings in the free state and when bound to the high affinity class I ligand RLP2, containing residues RALPPLPRY. A comparison between hydrogen bonds in high resolution X-ray structures of this domain and those observed via (h3)J(NC') couplings in solution shows remarkable agreement. Two backbone-to-side-chain hydrogen bonds are observed in solution, and each appears to play a role in stabilization of loop structure. Reproducible ligand-induced changes in trans-hydrogen bond scalar couplings are observed across the domain that translate into changes in hydrogen bond length ranging between 0.02 to 0.12 A. The observed changes can be rationalized by an induced fit mechanism in which hydrogen bonds across the protein participate in a compensatory response to forces imparted at the protein-ligand interface. Upon ligand binding, mutual intercalation of the two Leu-Pro segments of the ligand between three aromatic side-chains protruding from the SH3 surface wedges apart secondary structural elements within the SH3 domain. This disruption is transmitted in a domino-like effect across the domain through networks of hydrogen bonded peptide planes. The unprecedented resolution obtained demonstrates the ability to characterize subtle structural rearrangements within a protein upon perturbation, and represents a new step in the endeavor to understand how hydrogen bonds contribute to the stabilization and function of biological macromolecules.
蛋白质分子构象的变化可由多种干扰因素引起，并在生物活性调节中发挥关键作用。一种新的溶液核磁共振方法已被应用于监测鸡源c-Src SH3结构域中配体诱导的氢键几何结构变化。通过测量该结构域在游离状态下以及与高亲和力I类配体RLP2（包含残基RALPPLPRY）结合时的反式氢键（15）N-（13）C'标量耦合，研究了该结构域对配体结合的结构响应。该结构域的高分辨率X射线结构中的氢键与溶液中通过（h3）J（NC'）耦合观察到的氢键之间的比较显示出显著的一致性。在溶液中观察到两个主链到侧链的氢键，并且每个氢键似乎都在环结构的稳定中发挥作用。在整个结构域中观察到可重复的配体诱导的反式氢键标量耦合变化，这转化为氢键长度在0.02至0.12埃之间的变化。观察到的变化可以通过诱导契合机制来解释，其中蛋白质中的氢键参与对蛋白质-配体界面施加的力的补偿响应。配体结合后，配体的两个亮氨酸-脯氨酸片段在从SH3表面突出的三个芳香族侧链之间相互嵌入，从而将SH3结构域内的二级结构元件楔开。这种破坏通过氢键连接的肽平面网络以多米诺骨牌效应在整个结构域中传递。所获得的前所未有的分辨率证明了在受到干扰时表征蛋白质内细微结构重排的能力，并且代表了在理解氢键如何有助于生物大分子的稳定和功能方面迈出的新一步。
