The N-terminal region of XpsE is located at the interface of protein-protein interaction with itself

• Main Authors: Wan-Ling Chiang, 蔣婉鈴
• Other Authors: N-T Hu
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/19673603174214331629

碩士 === 國立中興大學 === 生物化學研究所 === 97 === The Xanthomonas campestris pv. campestris type II secretion system comprises of 12 protein components. Of all components, XpsE is the only cytoplasmic protein with weak ATPase activity. The N domain of XpsE (XpsEN) acts as the determinant in its association with XpsL. Two crystal structures of XpsEN have been resolved, an open and a closed form. They differ in the position of α1、α2 relative to α3 at their N-termini. A hydrophobic patch surrounded by residues 11, 25 and 39 was detected only in the closed form. It has been suggested to be involved in the interaction between XpsE and XpsL.. In this study I used two different thiol-specific hetero-bifunctional crosslinkers to examine if the three residues surrounding the hydrophobic patch interact directly with XpsL. Mutation of each residue into cysteine in a cys-less XpsE did not cause any effect in the normal function of XpsE for secretion. Each mutated XpsE, when expressed alone or co-expressed with MBP-XpsLN for isolating XpsE/MBP-XpsLN complex, was treated with the crosslinker SIA or SPDP and analyzed in SDS-polyacrylamide gel. Signals with apparent molecular weight of crosslinked dimer were detected in each case. Detailed analysis indicated that the crosslinked dimer originated from XpsE-XpsE crosslinking, not from XpsE-XpsLN crosslinking. Dimer-sized signal that was sensitive to reducing agent was detected in the control sample, into which no crosslinker was added. These observations implied disulfide bond formation between two cysteines located in neighboring XpsE. The proposition was confirmed by significant increase in the dimer signal when each mutated XpsE was incubated with the oxidizing agent Cu-phenanthroline. Analysis of size distribution of monomeric and oligomeric XpsE through size exclusion chromatography revealed that two mutated proteins XpsE(Cys-, V11C) and XpsE(Cys-, L39C), but not XpsE(Cys-, L25C), exhibited elution profiles with oligomer in higher proportion than that of the cys-less XpsE. It appears that XpsE hexamer was stabilized by L39C- or V11C-mediated dimer formation. In summary, the experimental evidences presented in this study suggested at least two residues surrounding the N-terminal hydrophobic patch observed in the closed form crystal structure of XpsEN, V11 and L39, are located at the interactive interface of two neighboring XpsE.