Department of Environmental Protection, Estación Experimental del Zaidín, CSIC, 18008 Granada, Spain.
J Biol Chem. 2010 Jul 23;285(30):23126-36. doi: 10.1074/jbc.M110.110403. Epub 2010 May 24.
We report the identification of McpS as the specific chemoreceptor for 6 tricarboxylic acid (TCA) cycle intermediates and butyrate in Pseudomonas putida. The analysis of the bacterial mutant deficient in mcpS and complementation assays demonstrate that McpS is the only chemoreceptor of TCA cycle intermediates in the strain under study. TCA cycle intermediates are abundantly present in root exudates, and taxis toward these compounds is proposed to facilitate the access to carbon sources. McpS has an unusually large ligand-binding domain (LBD) that is un-annotated in InterPro and is predicted to contain 6 helices. The ligand profile of McpS was determined by isothermal titration calorimetry of purified recombinant LBD (McpS-LBD). McpS recognizes TCA cycle intermediates but does not bind very close structural homologues and derivatives like maleate, aspartate, or tricarballylate. This implies that functional similarity of ligands, such as being part of the same pathway, and not structural similarity is the primary element, which has driven the evolution of receptor specificity. The magnitude of chemotactic responses toward these 7 chemoattractants, as determined by qualitative and quantitative chemotaxis assays, differed largely. Ligands that cause a strong chemotactic response (malate, succinate, and fumarate) were found by differential scanning calorimetry to increase significantly the midpoint of protein unfolding (T(m)) and unfolding enthalpy (DeltaH) of McpS-LBD. Equilibrium sedimentation studies show that malate, the chemoattractant that causes the strongest chemotactic response, stabilizes the dimeric state of McpS-LBD. In this respect clear parallels exist to the Tar receptor and other eukaryotic receptors, which are discussed.
我们报告了 McpS 作为假单胞菌中 6 种三羧酸（TCA）循环中间产物和丁酸盐的特异性化学感受器的鉴定。分析 mcpS 缺陷的细菌突变体和互补测定表明，McpS 是研究菌株中 TCA 循环中间产物的唯一化学感受器。TCA 循环中间产物在根分泌物中大量存在，向这些化合物的趋化作用被认为有助于获得碳源。McpS 具有异常大的配体结合域（LBD），在 InterPro 中未注释，预计包含 6 个螺旋。通过纯化重组 LBD（McpS-LBD）的等温滴定量热法确定了 McpS 的配体谱。McpS 识别 TCA 循环中间产物，但不结合非常接近的结构类似物和衍生物，如马来酸、天冬氨酸或三羟丁酸盐。这意味着配体的功能相似性，如作为同一途径的一部分，而不是结构相似性是主要因素，这推动了受体特异性的进化。通过定性和定量趋化性测定确定的这些 7 种趋化剂的趋化性反应幅度差异很大。通过差示扫描量热法发现，引起强烈趋化性反应的配体（马来酸、琥珀酸和富马酸）显著增加了 McpS-LBD 蛋白展开的中点（T(m)) 和展开焓（DeltaH)。平衡沉降研究表明，引起最强趋化性反应的趋化剂马来酸稳定了 McpS-LBD 的二聚体状态。在这方面，与 Tar 受体和其他真核受体存在明显的相似之处，对此进行了讨论。
