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.

中文翻译：

---

甲基转移酶 CheR 与其化学感受器底物结合，独立于其信号传导构象，但对它们进行差异性修饰。


甲基转移酶 CheR 对特定化学感受器谷氨酰残基的甲基化介导细菌趋化中的感觉适应和梯度传感。酶的作用是化学感受器信号传导构象的函数：激酶关闭受体比激酶开启受体更容易甲基化，这是适应和梯度传感机制的核心特征。不同的酶作用可以反映不同的结合、催化或两者。我们通过测量 CheR 与大肠杆菌天冬氨酸受体激酶关闭和激酶开启形式的结合进行了研究，Tar 删除了其 CheR 束缚、羧基末端五肽。这使得能够表征酶与底物受体体的低亲和力结合，否则会被与五肽的高亲和力相互作用所掩盖。我们通过使用生物层干涉测量法确定缔合和解离的动力学速率常数并根据这些值计算平衡常数来量化低亲和力蛋白质-蛋白质相互作用。无论焦油信号传导构象是通过配体占据还是适应性修饰而改变，两种信号传导构象在酶-受体结合的动力学或平衡参数方面几乎没有差异或没有差异。因此，甲基转移酶作用的差异并不反映结合的差异。相反，不同的信号传导构象必须具有共同的结合的主要决定因素。结合速率常数对 Deybe 长度依赖性的表征，Deybe 长度是静电影响的量度，暗示静电相互作用是常见的结合决定因素。 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开启化学感受器的差异作用并不是差异结合的结果，并且表明它反映了差异催化倾向。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。