Department of Chemistry, Columbia University, New York, NY 10027.
Department of Anesthesiology, Weill Cornell Medical College, New York, NY 10065.
Proc Natl Acad Sci U S A. 2019 Feb 5;116(6):2078-2085. doi: 10.1073/pnas.1811168116. Epub 2019 Jan 24.
Allosteric couplings underlie many cellular signaling processes and provide an exciting avenue for development of new diagnostics and therapeutics. A general method for identifying important residues in allosteric mechanisms would be very useful, but remains elusive due to the complexity of long-range phenomena. Here, we introduce an NMR method to identify residues involved in allosteric coupling between two ligand-binding sites in a protein, which we call chemical shift detection of allostery participants (CAP). Networks of functional groups responding to each ligand are defined through correlated NMR perturbations. In this process, we also identify allostery participants, groups that respond to both binding events and likely play a role in the coupling between the binding sites. Such residues exhibit multiple functional states with distinct NMR chemical shifts, depending on binding status at both binding sites. Such a strategy was applied to the prototypical ion channel KcsA. We had previously shown that the potassium affinity at the extracellular selectivity filter is strongly dependent on proton binding at the intracellular pH sensor. Here, we analyzed proton and potassium binding networks and identified groups that depend on both proton and potassium binding (allostery participants). These groups are viewed as candidates for transmitting information between functional units. The vital role of one such identified amino acid was validated through site-specific mutagenesis, electrophysiology functional studies, and NMR-detected thermodynamic analysis of allosteric coupling. This strategy for identifying allostery participants is likely to have applications for many other systems.
变构偶联是许多细胞信号转导过程的基础，为开发新的诊断和治疗方法提供了令人兴奋的途径。一种用于识别变构机制中重要残基的通用方法将非常有用，但由于长程现象的复杂性，这种方法仍然难以捉摸。在这里，我们引入了一种 NMR 方法来识别蛋白质中两个配体结合位点之间变构偶联涉及的残基，我们称之为变构偶联参与者的化学位移检测 (CAP)。通过相关的 NMR 扰动来定义响应每个配体的功能基团网络。在这个过程中，我们还确定了变构偶联体，即那些响应两个结合事件并可能在结合位点之间的偶联中发挥作用的基团。这些残基根据两个结合位点的结合状态表现出具有不同 NMR 化学位移的多个功能状态。这种策略被应用于典型的离子通道 KcsA。我们之前已经表明，细胞外选择性过滤器的钾亲和力强烈依赖于细胞内 pH 传感器的质子结合。在这里，我们分析了质子和钾结合网络，并确定了依赖于质子和钾结合的基团（变构偶联体）。这些基团被视为在功能单元之间传递信息的候选者。通过定点突变、电生理学功能研究和 NMR 检测的变构偶联热力学分析，验证了其中一个鉴定出的氨基酸的重要作用。这种识别变构偶联体的策略可能适用于许多其他系统。
