Mahn Joo Kim

Crystallization and preliminary X-ray analysis of native and selenomethionyl polymyxin resistance protein D fromE. Coli

Polymyxin B, a cationic antimicrobial peptide has been isolated from Paenibacillus polymyxa. The cationic peptide has polycationic, amphipathic and cyclic unique features. These characteristics of the cationic antimicrobial peptide are thought to contribute to the destruction of gram-negative bacteria by through an initial interaction with the negatively charged surface molecule lipopolysaccharide (LPS), which leads to self promoted uptake across the outer membrane into the negatively charged cytoplasmic membrane of the bacterium. This leads to membrane perturbation and probably translocation of the peptide across the membrane. Some gram-negative bacteria resistant to polymyxin B possess mechanism that modify the LPS through neutralization of its negative charge, which decreases the binding affinity of polymyxin B. Several of the genes regulated by PhoP/PhoQ and PmrA/ PmrB encode enzymes that modify either the acyl chain region or the phosphate group of LPS. In Salmonella, polymyxin B resistance is mainly controlled by the PmrA/PmrB two-component system (Figure 1). PmrA-activated genes encode periplasmic and cause the modification of the LPS. Transcription of PmrA-activated genes is not only promoted by high levels of extra-cytoplasmic Fe, which is sensed by the PmrB protein, also it is activated by the PhoP/PhoQ system in response to low levels of extra-cytoplasmic Mg. For activation of PmrA at the low level Mg condition not only requires the noncognate sensor PhoQ protein but also its cognate regulator PhoP, and the PhoP activated PmrD gene. The PmrD protein appears to control the PmrA/PmrB system at a post-transcriptional level. The PmrD protein activates the PmrA/PmrB system via a protection mechanism through dephosphorylation of PmrA. The protective activity of PmrD protein is suggestive of the behavior displayed by certain members of the 14-3-3 protein family in eukaryotes to prevent the phosphorylated state of CDC25. It has recently been reported that the differential regulation of homologous genes is the mechanism responsible for the divergence of Salmonella enterica and E. coli in their ability to make LPS modifications and mediate resistance to polymyxin B. In contrast to Salmonella, E. coli K-12 induces PmrA-activated gene transcription and polymyxin B resistance in response to Fe, but it makes no response to low levels of Mg. This discrepancy suggests that the structure of E.coli PmrD differs from Salmonella PmrD. In particular, the N-terminal region of PmrD, which differs between the E.coli and Salmonella proteins, is in part responsible for the distinct behaviors of the PmrD proteins (Figure 2). In the present report, we describe the crystallization and preliminary X-ray analysis of E. coli PmrD.