Crystallographic study of the Klebsiella pneumoniae RstA DNA binding domain complex with double-stranded DNA

• Main Authors: Li, Yi-Chuan, 李易撰
• Other Authors: Hsiao, Chwan-Deng
• Format: Others
• Language: en_US
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/jy83fy

博士 === 國立清華大學 === 生物資訊與結構生物研究所 === 102 === The RstA/RstB system is a bacterial two-component regulatory system consisting of the membrane sensor, RstB, and its cognate response regulator (RR) RstA. The RstA of Klebsiella pneumoniae (kpRstA) consists of an N-terminal receiver domain (RD, residues 1-119) and a C-terminal DNA-binding domain (DBD, residues 130-236). Phosphorylation of kpRstA induces dimerization, which allows two kpRstA DBDs to bind to a tandem repeat, called the RstA box, and regulate the expression of downstream genes. Here we report the crystal structure of the kpRstA DBD/RstA box DNA complex. The structure of the kpRstA DBD/RstA box complex suggests that the upstream and downstream RstA DBDs interact with the RstA box in a different way. Combine with the equilibrium binding studies supported by Dr. Tai-huang Huang’s lab. The ITC analysis data revealed the two protomers within the kpRstA dimer bind to the RstA box in a sequential manner. Taken together, our results suggest a binding model where dimerization of the kpRstA RDs provides the platform to allow the first kpRstA DBD protomer to anchor protein-DNA interaction, whereas the second protomer plays a key role in ensuring correct recognition of the RstA box. The PmrA/PmrB two-component system is the major regulator involved in the gene expression for lipopolysaccharides (LPS) modification in bacteria. PmrA is activated when the environment Fe3+, Al3+ and mild acidic environments. It activates genes including pbgPE, cptA and ugd can encode enzymes to change the composition of LPS. After modification, LPS can resist to polymyxin B and other host-derived antimicrobial peptides. The PmrA of Klebsiella pneumoniae (kpPmrA) is also consists of an N-terminal receiver domain (RD, residues 1-120) and a C-terminal DNA-binding domain (DBD, residues 126-226). Here we report the crystal structure of the kpPmrA/PmrA box DNA complex. Our intact response regulator complexed to DNA is asymmetric and reveals a novel heterodomain interface. A unique set of interactions between RD-DBD was observed. The asymmetric orientation of the PmrA dimer may play an important role in RNA polymerase recruitment through the upstream transactivation loop.