Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri.
Protein Sci. 2020 Feb;29(2):443-454. doi: 10.1002/pro.3760. Epub 2019 Nov 11.
Methylation of specific chemoreceptor glutamyl residues by methyltransferase CheR mediates sensory adaptation and gradient sensing in bacterial chemotaxis. Enzyme action is a function of chemoreceptor signaling conformation: kinase-off receptors are more readily methylated than kinase-on, a feature central to adaptational and gradient-sensing mechanisms. Differential enzyme action could reflect differential binding, catalysis or both. We investigated by measuring CheR binding to kinase-off and kinase-on forms of Escherichia coli aspartate receptor Tar deleted of its CheR-tethering, carboxyl terminus pentapeptide. This allowed characterization of the low-affinity binding of enzyme to the substrate receptor body, otherwise masked by high-affinity interaction with pentapeptide. We quantified the low-affinity protein-protein interactions by determining kinetic rate constants of association and dissociation using bio-layer interferometry and from those values calculating equilibrium constants. Whether Tar signaling conformations were shifted by ligand occupancy or adaptational modification, there was little or no difference between the two signaling conformations in kinetic or equilibrium parameters of enzyme-receptor binding. Thus, differential methyltransferase action does not reflect differential binding. Instead, the predominant determinants of binding must be common to different signaling conformations. Characterization of the dependence of association rate constants on Deybe length, a measure of the influence of electrostatics, implicated electrostatic interactions as a common binding determinant. Taken together, our observations indicate that differential action of methyltransferase on kinase-off and kinase-on chemoreceptors is not the result of differential binding and suggest it reflects differential catalytic propensity. Differential catalysis rather than binding could well be central to other enzymes distinguishing alternative conformations of protein substrates.
特定化学感受器谷氨酰残基的甲基化由甲基转移酶 CheR 介导，调节细菌趋化性中的感觉适应和梯度感应。酶的作用是化学感受器信号构象的函数：激酶关闭的受体比激酶打开的受体更容易被甲基化，这是适应和梯度感应机制的核心特征。不同的酶作用可能反映了不同的结合、催化或两者兼而有之。我们通过测量 CheR 与缺失其 CheR 连接羧基末端五肽的大肠杆菌天冬氨酸受体 Tar 的激酶关闭和激酶打开形式的结合来研究这一点。这使得可以对酶与底物受体体的低亲和力结合进行特征描述，否则由于与五肽的高亲和力相互作用而被掩盖。我们通过使用生物层干涉测量法确定结合的缔合和离解的动力学速率常数，并从这些值计算平衡常数，从而定量了低亲和力蛋白-蛋白相互作用。无论 Tar 信号构象是否因配体占据或适应修饰而发生变化，两种信号构象在酶-受体结合的动力学或平衡参数方面几乎没有差异。因此，差异甲基转移酶作用并不反映差异结合。相反，结合的主要决定因素必须与不同的信号构象共同存在。结合速率常数对 Deybe 长度（衡量静电影响的指标）的依赖性的特征表明，静电相互作用是共同的结合决定因素。综上所述，我们的观察结果表明，甲基转移酶对激酶关闭和激酶打开的化学感受器的差异作用不是由于差异结合所致，这表明它反映了差异的催化倾向。差异催化而不是结合很可能是其他酶区分蛋白质底物替代构象的核心。
