Structural Studies of Human RNase 7 and lebsiella pneumoniae Phosphatase Wzb
Date Issued
2009
Date
2009
Author(s)
Huang, Yu-Chie
Abstract
Chapter 1ibonucleases are found widely within living organisms and are thought to play important roles in RNA metabolism, angiogenesis, neurotoxicity, antitumor and antimicrobial activity. In human ribonucleases, antimicrobial activity was found in RNase 3, RNase 5 and RNase 7. The mechanisms of antimicrobial activities remain unclear although they possess similar properties, e.g., high isoelectric point and net positive charge. It is therefore interesting to investigate whether the positively charged residues or epitopes are responsible for the bactericidal activity. Here, we report on the role of cationic residues of human RNase 7 (hRNase 7) in its antimicrobial activity. We found that hRNase 7 is stable in the range of pH 3.5~9.5 and its structure is thermally and chemically stable with a Tm value of 66.4 ℃ and a Cm value of 3.27 M denatured by guanine hydrochloride. NMR structure of hRNase 7 shows that the 22 positively charged residues (18 Lys, 4 Arg) are distributed into three clusters on the surface. Residues in the first cluster including Lys1, Lys3, Lys111 and Lys112, are quite flexible and located near the N-terminus. On the contrary, the other two clusters, one with residues Lys32/Lys35 and the other with Lys96/Arg97/Lys100, are all located on secondary structure regions and are quite rigid. Mutagenesis studies confirmed that residues in the flexible cluster are critical for the bactericidal activity rather than the catalytic residues, such as His15, Lys38, His123, and residues in the other two positively charged clusters. Together, we suggest that hRNase 7 binds to bacterial membrane primarily by using the flexible residues, Lys1, Lys3, Lys111, and Lys112, in the first positively charged cluster and then renders the membrane permeable.hapter 2he low molecular weight protein tyrosine phosphatases (LMW-PTPs) in bacteria are ubiquitous regulators of tyrosine phosphorylation. Despite their abundance, the spectrum of functions of LMW-PTPs in prokaryote has yet to be elucidated. Several studies have demonstrated a role of LMW-PTPs in CPS/EPS synthesis and export, as well as in stress resistance. The target protein, LMW-PTPs Wzb, studied in this thesis is from the K. pneumoniae NTUH2044 strain clinically isolated at National Taiwan University Hospital (NTUH). It contains the conserved signature motif of CX5RS known as the P-loop or PTPloop, and an unique Asp residue situated on the DPY-loop of the LMW-PTPs. Our previous study has found that catalytic activity on Wzb_C9S mutant is dramatically reduced or even not seen at all. To ascertain the importance of the conversed residue Cys9 in the catalytic mechanism of Wzb, we expressed and purified unlabeled and isotopically labeled Wzb and Wzb_C9S mutant for structural study. Due to the lack of several cross peaks in NMR spectral of Wzb, we could not determine its structure. In comparison, Wzb_C9S gave much better NMR quality and its 3D NMR solution structure containing a canonical topology of LMW-PTPs with a central four-stranded parallel beta-sheet and five alpha-helices has been solved. Superimposition of 2D HSQC spectra of Wzb and Wzb_C9S reveals that the unobserved cross peaks and cross peaks exhibiting shift changes in Wzb are mostly located in the P-loop and its nearby regions. Backbone dynamics study showed that the residues in the P-loop are moderately rigid. In addition, both Tm and Cm values in Wzb_C9S are higher than those in Wzb. Taken together, we conclude there is a conformational change, especially in the P-loop, between Wzb and Wzb_C9S, with the P-loop in Wzb_C9S apparently more rigid. Due to the change of the rigidity in the P-loop, the Ser9 in C9S mutant is unable to act as a nucleophile to attack the phosphate group, as Cys9 does in Wzb.
Subjects
human RNase 7
antimicrobial activity
Klebsiella pneumoniae
LMW-PTP
CPS, LPS
NMR structure
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