Abstract
In bacteria, the two-component signal-transduction system is the most common system for sensing environmental signals and transducing the information into the cell. The PmrA-PmrB two-component signal transduction system, responsible for sensing external stimuli of low Mg2+ and mild acidic conditions, can control the genes involved in lipopolysaccharide (LPS) modification and polymyxin resistance in pathogens. The small basic protein PmrD, a polymyxin B resistance protein, is the only protein known to be able to connect PmrA/PmrB and PhoP/PhoQ two-component systems. First, we found that the recombinant PmrD of Klebsiella penumoniae (KP-PmrD) initially contains both oxidized and reduced forms, and the reduced form is nearly converted into the oxidized form within two weeks in the air. The CD spectra revealed the secondary structure and the melting temperature are somewhat different between the two forms. Both mass analysis and NMR data confirmed the formation of a disulfide bridge at Cys17 and Cys35 for the oxidized form. Dramatic chemical-shift changes between the two forms were observed, further revealing their structural difference. A detailed comparison of secondary structures between the two forms showed that the major conformational difference is located at C-terminal □-helix. The existence of both reduced and oxidized forms has not been previously reported for any PmrD, and in vivo polymyxin B susceptibility assay of the site-directed mutagenesis on KP-PmrD reveals that the two cysteine, which form a disulfide bridge between Cys17-Cys35, is functional relevant. Second, we provided the NMR solution structure of PmrD in Klebsiella pneumoniae (KP-PmrD), which showed similar in vitro phosphotransfer activity to Salmonella PmrD. Structural comparisons and backbone dynamics data revealed that the C-terminal □-helix of KP-PmrD is shorter and much more flexible. To further study the molecular interactions involved in KP-PmrD and the N-terminal receiver domain of PmrA (PmrAN), we performed quantitative and qualitative SPR as well as NMR analyses both in the absence and presence of the phosphoryl analog beryllofluoride (BeF3-). The results demonstrated that KP-PmrD participates in specific interactions (KD = 1.74 ± 0.81 □M) with the PmrAN in the presence of BeF3-. Chemical shift perturbations and cross-saturation experiments defined a cluster of residues (Trp3, Leu26, Met28, Asp50, Ala51, and Ile65) forming a contiguous patch near loops 2, 4, and 6 of the □-barrel upon binding with the BeF3--activated PmrAN. Third, while several structures of receiver domain of response regulators have been solved, structures of regulatory domain in complex with its connector protein have remained elusive. We used the data-driven docking method HADDOCK and site-directed spin labeling approaches to proposed an interaction mode in which the N-terminal receiver domain of PmrA (PmrAN) interacts with KP-PmrD through the active site pockets, which is widely used by response regulatory domains for intermolecular protein/protein interactions with the histidine kinase. The results demonstrate that site-directed spin labeling could provide an independent biophysical method to confirm the complex model from HADDOCK and validate long-range distance information between corresponding regions of KP-PmrD/PmrAN complex structure. This study furthers the understanding of KP-PmrD polymyxin B resistance regulation in the PmrAB TCS and our results should provide insights into the structural basis of KP-PmrD for specific interaction with the phospho-PmrA in the PmrAB two-component system.