Lou Yuan-Chao, Wang Iren, Rajasekaran M, Kao Yi-Fen, Ho Meng-Ru, Hsu Shang-Te Danny, Chou Shan-Ho, Wu Shih-Hsiung, Chen Chinpan
Institute of Biomedical Sciences, Institute of Biological Chemistry, Academia Sinica, Taipei 115, Institute of Biochemistry and Agricultural Biotechnology Center, National Chung Hsing University, Taichung 40227, Taiwan, Republic of China.
Nucleic Acids Res. 2014 Apr;42(6):4080-93. doi: 10.1093/nar/gkt1345. Epub 2013 Dec 25.
Klebsiella pneumoniae PmrA is a polymyxin-resistance-associated response regulator. The C-terminal effector/DNA-binding domain of PmrA (PmrAC) recognizes tandem imperfect repeat sequences on the promoters of genes to induce antimicrobial peptide resistance after phosphorylation and dimerization of its N-terminal receiver domain (PmrAN). However, structural information concerning how phosphorylation of the response regulator enhances DNA recognition remains elusive. To gain insights, we determined the nuclear magnetic resonance solution structure of PmrAC and characterized the interactions between PmrAC or BeF3(-)-activated full-length PmrA (PmrAF) and two DNA sequences from the pbgP promoter of K. pneumoniae. We showed that PmrAC binds to the PmrA box, which was verified to contain two half-sites, 5'-CTTAAT-3' and 5'-CCTAAG-3', in a head-to-tail fashion with much stronger affinity to the first than the second site without cooperativity. The structural basis for the PmrAC-DNA complex was investigated using HADDOCK docking and confirmed by paramagnetic relaxation enhancement. Unlike PmrAC, PmrAF recognizes the two sites simultaneously and specifically. In the PmrAF-DNA complex, PmrAN may maintain an activated homodimeric conformation analogous to that in the free form and the interactions between two PmrAC molecules aid in bending and binding of the DNA duplex for transcription activation.
肺炎克雷伯菌PmrA是一种与多粘菌素耐药性相关的反应调节因子。PmrA的C端效应器/DNA结合结构域（PmrAC）识别基因启动子上的串联不完全重复序列，在其N端受体结构域（PmrAN）磷酸化和二聚化后诱导抗菌肽耐药性。然而，关于反应调节因子的磷酸化如何增强DNA识别的结构信息仍然难以捉摸。为了深入了解，我们确定了PmrAC的核磁共振溶液结构，并表征了PmrAC或BeF3(-)激活的全长PmrA（PmrAF）与肺炎克雷伯菌pbgP启动子的两个DNA序列之间的相互作用。我们表明，PmrAC与PmrA框结合，经证实该框包含两个半位点，5'-CTTAAT-3'和5'-CCTAAG-3'，以头对尾的方式结合，对第一个位点的亲和力比对第二个位点强得多，且无协同作用。使用HADDOCK对接研究了PmrAC-DNA复合物的结构基础，并通过顺磁弛豫增强进行了确认。与PmrAC不同，PmrAF同时特异性识别这两个位点。在PmrAF-DNA复合物中，PmrAN可能保持类似于游离形式的活化同二聚体构象，并且两个PmrAC分子之间的相互作用有助于DNA双链的弯曲和结合以进行转录激活。
