Publications (21) View all
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Article: Structural and functional characterization of HP0377, a thioredoxin-fold protein from Helicobacter pylori.
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ABSTRACT: Maturation of cytochrome c is carried out in the bacterial periplasm, where specialized thiol-disulfide oxidoreductases provide the correct reduction of oxidized apocytochrome c before covalent haem attachment. HP0377 from Helicobacter pylori is a thioredoxin-fold protein that has been implicated as a component of system II for cytochrome c assembly and shows limited sequence similarity to Escherichia coli DsbC, a disulfide-bond isomerase. To better understand the role of HP0377, its crystal structures have been determined in both reduced and partially oxidized states, which are highly similar to each other. Sedimentation-equilibrium experiments indicate that HP0377 is monomeric in solution. HP0377 adopts a thioredoxin fold but shows distinctive variations as in other thioredoxin-like bacterial periplasmic proteins. The active site of HP0377 closely resembles that of E. coli DsbC. A reductase assay suggests that HP0377 may play a role as a reductase in the biogenesis of holocytochrome c553 (HP1227). Binding experiments indicate that it can form a covalent complex with HP0518, a putative L,D-transpeptidase with a catalytic cysteine residue, via a disulfide bond. Furthermore, physicochemical properties of HP0377 and its R86A variant have been determined. These results suggest that HP0377 may perform multiple functions as a reductase in H. pylori.Acta crystallographica. Section D, Biological crystallography 05/2013; 69(Pt 5):735-46. · 12.67 Impact Factor -
Article: Structural basis for the inhibition of Mycobacterium tuberculosisL,D-transpeptidase by meropenem, a drug effective against extensively drug-resistant strains.
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ABSTRACT: Difficulty in the treatment of tuberculosis and growing drug resistance in Mycobacterium tuberculosis (Mtb) are a global health issue. Carbapenems inactivate L,D-transpeptidases; meropenem, when administered with clavulanate, showed in vivo activity against extensively drug-resistant Mtb strains. LdtMt2 (Rv2518c), one of two functional L,D-transpeptidases in Mtb, is predominantly expressed over LdtMt1 (Rv0116c). Here, the crystal structure of N-terminally truncated LdtMt2 (residues Leu131-Ala408) is reported in both ligand-free and meropenem-bound forms. The structure of meropenem-inhibited LdtMt2 provides a detailed structural view of the interactions between a carbapenem drug and Mtb L,D-transpeptidase. The structures revealed that the catalytic L,D-transpeptidase domain of LdtMt2 is preceded by a bacterial immunogloblin-like Big_5 domain and is followed by an extended C-terminal tail that interacts with both domains. Furthermore, it is shown using mass analyses that meropenem acts as a suicide inhibitor of LdtMt2. Upon acylation of the catalytic Cys354 by meropenem, the `active-site lid' undergoes a large conformational change to partially cover the active site so that the bound meropenem is accessible to the bulk solvent via three narrow paths. This work will facilitate structure-guided discovery of L,D-transpeptidase inhibitors as novel antituberculosis drugs against drug-resistant Mtb.Acta crystallographica. Section D, Biological crystallography 03/2013; 69(Pt 3):420-31. · 12.67 Impact Factor -
Article: Mycobacterium tuberculosis Eis protein initiates suppression of host immune responses by acetylation of DUSP16/MKP-7.
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ABSTRACT: The intracellular pathogen Mycobacterium tuberculosis (Mtb) causes tuberculosis. Enhanced intracellular survival (Eis) protein, secreted by Mtb, enhances survival of Mycobacterium smegmatis (Msm) in macrophages. Mtb Eis was shown to suppress host immune defenses by negatively modulating autophagy, inflammation, and cell death through JNK-dependent inhibition of reactive oxygen species (ROS) generation. Mtb Eis was recently demonstrated to contribute to drug resistance by acetylating multiple amines of aminoglycosides. However, the mechanism of enhanced intracellular survival by Mtb Eis remains unanswered. Therefore, we have characterized both Mtb and Msm Eis proteins biochemically and structurally. We have discovered that Mtb Eis is an efficient N(ε)-acetyltransferase, rapidly acetylating Lys55 of dual-specificity protein phosphatase 16 (DUSP16)/mitogen-activated protein kinase phosphatase-7 (MKP-7), a JNK-specific phosphatase. In contrast, Msm Eis is more efficient as an N(α)-acetyltransferase. We also show that Msm Eis acetylates aminoglycosides as readily as Mtb Eis. Furthermore, Mtb Eis, but not Msm Eis, inhibits LPS-induced JNK phosphorylation. This functional difference against DUSP16/MKP-7 can be understood by comparing the structures of two Eis proteins. The active site of Mtb Eis with a narrow channel seems more suitable for sequence-specific recognition of the protein substrate than the pocket-shaped active site of Msm Eis. We propose that Mtb Eis initiates the inhibition of JNK-dependent autophagy, phagosome maturation, and ROS generation by acetylating DUSP16/MKP-7. Our work thus provides insight into the mechanism of suppressing host immune responses and enhancing mycobacterial survival within macrophages by Mtb Eis.Proceedings of the National Academy of Sciences 04/2012; 109(20):7729-34. · 9.68 Impact Factor -
Article: Structural and functional characterization of Helicobacter pylori DsbG.
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ABSTRACT: Dsb proteins play important roles in bacterial pathogenicity. To better understand the role of Dsb proteins in Helicobacter pylori, we have structurally and functionally characterized H. pylori DsbG (HP0231). The monomer consists of two domains connected by a helical linker. Two monomers associate to form a V-shaped dimer. The monomeric and dimeric structures of H. pylori DsbG show significant differences compared to Escherichia coli DsbG. Two polyethylene glycol molecules are bound in the cleft of the V-shaped dimer, suggesting a possible role as a chaperone. Furthermore, we show that H. pylori DsbG functions as a reductase against HP0518, a putative L,D-transpeptidase with a catalytic cysteine residue.FEBS letters 11/2011; 585(24):3862-7. · 3.54 Impact Factor -
Article: Crystal structures of YwqE from Bacillus subtilis and CpsB from Streptococcus pneumoniae, unique metal-dependent tyrosine phosphatases.
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ABSTRACT: Unique metal-dependent protein tyrosine phosphatases that belong to the polymerase and histindinol phosphatase (PHP) family are present in Gram-positive bacteria. They are distinct from the Cys-based, low-molecular-weight phosphotyrosine protein phosphatases (LMPTPs). Two representative members of the PHP family tyrosine phosphatases are YwqE from Bacillus subtilis and CpsB from Streptococcus pneumoniae. YwqE is involved in polysaccharide biosynthesis, bacterial DNA metabolism, and DNA damage response in B. subtilis. CpsB regulates capsular polysaccharide biosynthesis via tyrosine dephosphorylation of CpsD, its cognate tyrosine kinase, in S. pneumoniae. To gain insights into the active site and possible conformational changes of the metal-dependent tyrosine phosphatases from Gram-positive bacteria, we have determined the crystal structures of B. subtilis YwqE (in both the apo form and the phosphate-bound form) and S. pneumoniae CpsB (in the sulfate-bound form). Comparisons of the three structures reveal conformational plasticity of two active site loops. Furthermore, in both structures of the phosphate-bound YwqE and the sulfate-bound CpsB, the phosphate (or sulfate) ion is bound to a cluster of three metal ions in the active site, thus providing insight into the pre-catalytic state.Journal of Structural Biology 05/2011; 175(3):442-50. · 3.41 Impact Factor
