Chemical Biology and Molecular Biophysics, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan.
J Struct Biol. 2010 Dec;172(3):319-30. doi: 10.1016/j.jsb.2010.06.007. Epub 2010 Jun 9.
In bacteria, the two-component system (TCS) is the most prevalent for sensing and transducing the environmental signals into the cell. In Salmonella, the small basic protein PmrD is found to protect phospho-PmrA and prolong the expression of PmrA-activated genes. In contrast, Escherichia coli PmrD fails to protect phospho-PmrA. Here, we show that Klebsiella pneumoniae PmrD (KP-PmrD) can inhibit the dephosphrylation of phospho-PmrA, and the interaction between KP-PmrD and the N-terminal receiver domain of PmrA (PmrA(N)) is much stronger in the presence than in the absence of the phosphoryl analog beryllofluoride (BeF(3)(-)) (K(D)=1.74 ± 0.81 μM vs. K(D)=236 ± 48 μM). To better understand the molecular interactions involved, the solution structure of KP-PmrD was found to comprise six β-strands and a flexible C-terminal α-helix. Amide chemical shift perturbations of KP-PmrD in complex with BeF(3)(-)-activated PmrA(N) suggested that KP-PmrD may undergo a certain conformational rearrangement on binding to activated PmrA(N). Saturation transfer experiments revealed the binding surface to be located on one face of the β-barrel. This finding was further verified by in vivo polymyxin B susceptibility assay of the mutants of KP-PmrD. The phospho-PmrA recognition surface of KP-PmrD, which involves two KP-PmrD proteins in complex with an activated-PmrA(N) dimer, is suggested to be a contiguous patch consisting of Trp3, Trp4, Ser23, Leu26, Glu27, Met28, Thr46, Leu48, Ala49, Asp50, Ala51, Arg52, Ile65, Asn67, Ala68, Thr69, His70, Tyr71, Ser73 and Glu74. Our study furthers the understanding of how PmrD protects phopho-PmrA in the PmrAB TCS.
在细菌中，双组分系统（TCS）是最常见的用于感应和转导环境信号到细胞中的系统。在沙门氏菌中，发现小碱性蛋白 PmrD 可保护磷酸化的 PmrA 并延长 PmrA 激活基因的表达。相比之下，大肠杆菌 PmrD 不能保护磷酸化的 PmrA。在这里，我们表明肺炎克雷伯氏菌 PmrD（KP-PmrD）可以抑制磷酸化的 PmrA 的去磷酸化，并且在存在氟化铍（BeF₃（-））的情况下，KP-PmrD 与 PmrA 的 N 端受体结构域（PmrA（N））之间的相互作用比不存在磷酸化类似物时要强得多（K(D)=1.74 ± 0.81 μM 对 K(D)=236 ± 48 μM）。为了更好地理解所涉及的分子相互作用，发现 KP-PmrD 的溶液结构由六个 β-折叠和一个灵活的 C 端 α-螺旋组成。在与 BeF₃（-）激活的 PmrA（N）复合物中，KP-PmrD 的酰胺化学位移扰动表明，KP-PmrD 可能在与激活的 PmrA（N）结合时发生一定的构象重排。饱和转移实验表明结合表面位于β桶的一个面上。这一发现通过 KP-PmrD 突变体的体内多粘菌素 B 敏感性测定进一步得到了验证。KP-PmrD 的磷酸化 PmrA 识别表面，涉及两个 KP-PmrD 蛋白与一个激活的 PmrA（N）二聚体复合物，被认为是一个连续的补丁，由 Trp3、Trp4、Ser23、Leu26、Glu27、Met28、Thr46、Leu48、Ala49、Asp50、Ala51、Arg52、Ile65、Asn67、Ala68、Thr69、His70、Tyr71、Ser73 和 Glu74 组成。我们的研究进一步了解了 PmrD 如何在 PmrAB TCS 中保护磷酸化的 PmrA。
