Biology Department, University of Utah, Salt Lake City, UT 84112, USA.
J Bacteriol. 2011 Dec;193(23):6452-60. doi: 10.1128/JB.05887-11. Epub 2011 Sep 30.
Chemoreceptors such as Tsr, the serine receptor, function in trimer-of-dimer associations to mediate chemotactic behavior in Escherichia coli. The two subunits of each receptor homodimer occupy different positions in the trimer, one at its central axis and the other at the trimer periphery. Residue N381 of Tsr contributes to trimer stability through interactions with its counterparts in a central cavity surrounded by hydrophobic residues at the trimer axis. To assess the functional role of N381, we created and characterized a full set of amino acid replacements at this Tsr residue. We found that every amino acid replacement at N381 destroyed Tsr function, and all but one (N381G) of the mutant receptors also blocked signaling by Tar, the aspartate chemoreceptor. Tar jamming reflects the formation of signaling-defective mixed trimers of dimers, and in vivo assays with a trifunctional cross-linking reagent demonstrated trimer-based interactions between Tar and Tsr-N381 mutants. Mutant Tsr molecules with a charged amino acid or proline replacement exhibited the most severe trimer formation defects. These trimer-defective receptors, as well as most of the trimer-competent mutant receptors, were unable to form ternary signaling complexes with the CheA kinase and with CheW, which couples CheA to receptor control. Some of the trimer-competent mutant receptors, particularly those with a hydrophobic amino acid replacement, may not bind CheW/CheA because they form conformationally frozen or distorted trimers. These findings indicate that trimer dynamics probably are important for ternary complex assembly and that N381 may not be a direct binding determinant for CheW/CheA at the trimer periphery.
化学感受器（如 Tsr，丝氨酸受体）以三聚体二聚体的形式发挥作用，介导大肠杆菌的趋化行为。每个受体同源二聚体的两个亚基占据三聚体的不同位置，一个位于中央轴，另一个位于三聚体外围。Tsr 的残基 N381 通过与中央腔中对应残基的相互作用，有助于三聚体的稳定性，该中央腔由三聚体轴上的疏水残基环绕。为了评估 N381 的功能作用，我们在该 Tsr 残基处创建并表征了一组完整的氨基酸替换。我们发现，N381 处的每个氨基酸替换都破坏了 Tsr 的功能，而且除了一个（N381G）之外，所有突变受体也阻断了天冬氨酸化学感受器 Tar 的信号转导。Tar 阻塞反映了信号缺陷的混合二聚体三聚体的形成，并且使用三功能交联试剂进行的体内测定表明 Tar 和 Tsr-N381 突变体之间存在基于三聚体的相互作用。带有带电荷氨基酸或脯氨酸替换的突变 Tsr 分子表现出最严重的三聚体形成缺陷。这些三聚体缺陷受体，以及大多数三聚体相容的突变受体，无法与 CheA 激酶和将 CheA 与受体控制偶联的 CheW 形成三元信号复合物。一些三聚体相容的突变受体，特别是那些带有疏水性氨基酸替换的受体，可能无法结合 CheW/CheA，因为它们形成构象冻结或扭曲的三聚体。这些发现表明三聚体动力学可能对三元复合物组装很重要，并且 N381 可能不是三聚体外围 CheW/CheA 的直接结合决定因素。
