XpsE Walker B motif中保留性胺基酸重要性之分析

Abstract

XpsE is the only cytoplasmic protein component of the Xanthomonas campestris pv. campestris type II secretion system. It exhibits weak ATPase activity. As suggested from previous in vitro studies, ATP binding to XpsE triggers its hexamerization as well as its association with the cytoplasmic N-terminal domain of the integral membrane protein XpsL. In order to determine the significance of major XpsE-XpsE interactive interface between the C1 domain of molecule A and the N2 domain of molecule B, C1moleculeA：N2moleculeB, three conserved residues were identified by aligning five proteins of the Type II/IV secretion NTPase family. They are R385, D387 and D389. Each was mutated to Ala, transformed into the xpsE-null mutant XC1723 and analyzed for their secretion function on starch plate. All three mutants XpsER385A, XpsED387A and XpsED389A were unable to resume secretion of α-amylase in XC1723. And they all interfered with normal secretion in the wild type strain XC1701. As revealed from size exclusion chromatography, of all three mutants, XpsED387A was the only one that displayed distinctly decreased level of hexamer. Furthermore, weaker association of XpsED387A with MBP-XpsLN was suggested by reduced level of XpsED387A in the XpsE/MBP-XpsLN complex formed by their co-expression in E. coli. In contrast, the MBP-XpsLN pull down assay, showed that both XpsED387A and XpsED389A exhibited XpsLN binding activity slightly weaker than that of the wild-type XpsE. To minimize contaminating ATPase, monomeric XpsE and its variants (except XpsED389A) were purified through affinity chromatography followed by anion exchange chromatography. Results from ATPase assay indicated that the ATP hydrolysis activity of XpsER385A and XpsED387A were reduced by four and five fold, respectively. In summary, D387 of XpsE appears to play significant role in keeping hexamer stable. Its deficiency in ATP hydrolysis may be an indirect effect. As a contrast, R385 may act as an “arginine finger” and be directly required for ATP hydrolysis by the neighboring XpsE molecule.