Jiang M, Bourret R B, Simon M I, Volz K
Department of Microbiology and Immunology, University of Illinois, Chicago, Illinois 60612, USA.
J Biol Chem. 1997 May 2;272(18):11850-5. doi: 10.1074/jbc.272.18.11850.
An aspartate to lysine mutation at position 13 of the chemotaxis regulatory protein CheY causes a constitutive tumbly phenotype when expressed at high copy number in vivo even though the mutant protein is not phosphorylatable. These properties suggest that the D13K mutant adopts the active, signaling conformation of CheY independent of phosphorylation, so knowledge of its structure could explain the activation mechanism of CheY. The x-ray crystallographic structure of the CheY D13K mutant has been solved and refined at 2.3 A resolution to an R-factor of 14.3%. The mutant molecule shows no significant differences in backbone conformation when compared with the wild-type, Mg2+-free structure, but there are localized changes within the active site. The side chain of lysine 13 blocks access to the active site, whereas its epsilon-amino group has no bonding interactions with other groups in the region. Also in the active site, the bond between lysine 109 and aspartate 57 is weakened, and the solvent structure is perturbed. Although the D13K mutant has the inactive conformation in the crystalline form, rearrangements in the active site appear to weaken the overall structure of that region, potentially creating a metastable state of the molecule. If a conformational change is required for signaling by CheY D13K, then it most likely proceeds dynamically, in solution.
趋化性调节蛋白CheY第13位的天冬氨酸突变为赖氨酸，即使该突变蛋白不可磷酸化，但在体内以高拷贝数表达时仍会导致组成型翻滚表型。这些特性表明，D13K突变体独立于磷酸化作用而采用了CheY的活性信号传导构象，因此了解其结构可以解释CheY的激活机制。已解析出CheY D13K突变体的X射线晶体结构，并在2.3埃分辨率下将其精修至R因子为14.3%。与野生型无镁结构相比，突变体分子在主链构象上没有显著差异，但活性位点内存在局部变化。赖氨酸13的侧链阻碍了进入活性位点的通道，而其ε-氨基在该区域内与其他基团没有键合相互作用。同样在活性位点，赖氨酸109与天冬氨酸57之间的键被削弱，溶剂结构也受到扰动。尽管D13K突变体在晶体形式中具有非活性构象，但活性位点的重排似乎削弱了该区域的整体结构，可能使分子处于亚稳态。如果CheY D13K信号传导需要构象变化，那么它很可能在溶液中动态进行。
