Mutations in Mycobacterium tuberculosis Rv0444c, the gene encoding anti-SigK, explain high level expression of MPB70 and MPB83 in Mycobacterium bovis

It has recently been advanced that Mycobacterium tuberculosis sigma factor K (SigK) positively regulates expression of the antigenic proteins MPB70 and MPB83. As expression of these proteins differs between M. tuberculosis (low) and Mycobacterium bovis (high), this study set out to determine whether M. bovis lacks a functional SigK repressor (anti-SigK). By comparing genes near sigK in M. tuberculosis H37Rv and M. bovis AF2122/97, we observed that Rv0444c, annotated as unknown function, had variable sequence in M. bovis. Analysis of in vitro mpt70/mpt83 expression and Rv0444c sequencing across M. tuberculosis complex (MTC) members revealed that high-level expression was associated with a mutated Rv0444c. Complementation of M. bovis bacillus Calmette-Guerin Russia, a high producer of MPB70/MPB83, with wild-type Rv0444c resulted in a significant decrease in mpb70/mpb83 expression. Conversely, a M. tuberculosis H37Rv mutant which expressed sigK but not Rv0444c manifested the M. bovis phenotype of high-level MPB70/MPB83 expression. Further support that Rv0444c encodes the anti-SigK was obtained by yeast two-hybrid studies, where the N-terminal region of Rv0444c-encoded protein interacted with SigK. Together these findings indicate that Rv0444c encodes the regulator of SigK (RskA) and mutations in this gene explain high-level MPT70/MPT83 expression by certain MTC members.     

Mycobacterium tuberculosis UsfX (Rv3287c) exhibits novel nucleotide binding and hydrolysis properties

The Mycobacterium tuberculosis UsfX protein is an anti-sigma factor which regulates its cognate sigma factor SigF. UsfX shares low sequence homology with other anti-sigma factors making it difficult to identify the nucleotide binding site and characterize its properties. We have identified that the NTP binding site occurs close to Trp106 and the area around the nucleotide binding site is predominantly negatively charged. UsfX binds to a variety of nucleotides unlike other reported anti-sigma factors and exhibits an unusual dual NTPase activity. In silico computational experiments have identified a XGSFS motif close to the nucleotide binding site for metal ion binding. This motif is analogous to the DXSXS motif reported earlier in the human integrin CR3 protein superfamily. Overall, the experiments suggest that the M. tuberculosis UsfX represents a distinct anti-sigma factor family with a novel nucleotide binding motif.     

Domain Structure of Virulence-associated Response Regulator PhoP of Mycobacterium tuberculosis: ROLE OF THE LINKER REGION IN REGULATOR-PROMOTER INTERACTION(S)

The PhoP and PhoR proteins from Mycobacterium tuberculosis form a highly specific two-component system that controls expression of genes involved in complex lipid biosynthesis and regulation of unknown virulence determinants. The several functions of PhoP are apportioned between a C-terminal effector domain (PhoPC) and an N-terminal receiver domain (PhoPN), phosphorylation of which regulates activation of the effector domain. Here we show that PhoPN, on its own, demonstrates PhoR-dependent phosphorylation. PhoPC, the truncated variant bearing the DNA binding domain, binds in vitro to the target site with affinity similar to that of the full-length protein. To complement the finding that residues spanning Met¹ to Arg¹³⁸ of PhoP constitute the minimal functional PhoPN, we identified Arg¹⁵⁰ as the first residue of the distal PhoPC domain capable of DNA binding on its own, thereby identifying an interdomain linker. However, coupling of two functional domains together in a single polypeptide chain is essential for phosphorylation-coupled DNA binding by PhoP. We discuss consequences of tethering of two domains on DNA binding and demonstrate that linker length and not individual residues of the newly identified linker plays a critical role in regulating interdomain interactions. Together, these results have implications for the molecular mechanism of transmission of conformation change associated with phosphorylation of PhoP that results in the altered DNA recognition by the C-terminal domain.     

NlpC/P60 domain‐containing proteins of Mycobacterium avium subspecies paratuberculosis that differentially bind and hydrolyze peptidoglycan

A subset of proteins containing NlpC/P60 domains are bacterial peptidoglycan hydrolases that cleave noncanonical peptide linkages and contribute to cell wall remodeling as well as cell separation during late stages of division. Some of these proteins have been shown to cleave peptidoglycan in Mycobacterium tuberculosis and play a role in Mycobacterium marinum virulence of zebra fish; however, there are still significant knowledge gaps concerning the molecular function of these proteins in Mycobacterium avium subspecies paratuberculosis (MAP). The MAP genome sequence encodes five NlpC/P60 domain‐containing proteins. We describe atomic resolution crystal structures of two such MAP proteins, MAP_1272c and MAP_1204. These crystal structures, combined with functional assays to measure peptidoglycan cleavage activity, led to the observation that MAP_1272c does not have a functional catalytic core for peptidoglycan hydrolysis. Furthermore, the structure and sequence of MAP_1272c demonstrate that the catalytic residues normally required for hydrolysis are absent, and the protein does not bind peptidoglycan as efficiently as MAP_1204. While the NlpC/P60 catalytic triad is present in MAP_1204, changing the catalytic cysteine‐155 residue to a serine significantly diminished catalytic activity, but did not affect binding to peptidoglycan. Collectively, these findings suggest a broader functional repertoire for NlpC/P60 domain‐containing proteins than simply hydrolases.     

Structural analysis of the elongation factor G protein from the low-temperature-adapted bacterium Arthrobacter globiformis SI55

The first structural analysis of elongation factor G (EF-G) from a cold-adapted bacterium is presented. EF-G is an essential protein involved in the elongation process during protein synthesis and is therefore thought to play a crucial role in the low-temperature adaptation of cold-adapted microorganisms. To define its importance, the EF-G gene (fus) from the psychrotolerant bacterium Arthrobacter globiformis SI55 was cloned and sequenced. The deduced primary structure of the elongation factor is composed of 700 amino acids with a predicted molecular mass of 77.4 kDa. A three-dimensional model of the protein was constructed based on the known crystal structures of structurally homologous proteins. Structural features that might potentially be important for activity and flexibility at low temperature were deduced by comparisons with models of the EF-G proteins from the closely related mesophiles Micrococcus luteus and Mycobacterium tuberculosis. These features include a loss in the number of salt bridges in intradomain and interdomain positions, increased solvent interactions mediated by greater charge and polarity on domain surfaces, loop insertions, loss of proline residues in loop structures, and an increase of hydrophobicity in core regions. Specific changes have also been identified in the catalytic domain (G domain) and sites of potential ribosome interaction, which may directly affect guanosine triphosphate (GTP) hydrolysis and elongation rates at low temperature. Received: September 20, 1999 / Accepted: December 2, 1999     

Single-molecule force spectroscopy of mycobacterial adhesin-adhesin interactions

The heparin-binding hemagglutinin (HBHA) is one of the few virulence factors identified for Mycobacterium tuberculosis. It is a surface-associated adhesin that expresses a number of different activities, including mycobacterial adhesion to nonphagocytic cells and microbial aggregation. Previous evidence indicated that HBHA is likely to form homodimers or homopolymers via a predicted coiled-coil region located within the N-terminal portion of the molecule. Here, we used single-molecule atomic-force microscopy to measure individual homophilic HBHA-HBHA interaction forces. Force curves recorded between tips and supports derivatized with HBHA proteins exposing their N-terminal domains showed a bimodal distribution of binding forces reflecting the formation of dimers or multimers. Moreover, the binding peaks showed elongation forces that were consistent with the unfolding of α-helical coiled-coil structures. By contrast, force curves obtained for proteins exposing their lysine-rich C-terminal domains showed a broader distribution of binding events, suggesting that they originate primarily from intermolecular electrostatic bridges between cationic and anionic residues rather than from specific coiled-coil interactions. Notably, similar homophilic HBHA-HBHA interactions were demonstrated on live mycobacteria producing HBHA, while they were not observed on an HBHA-deficient mutant. Together with the fact that HBHA mediates bacterial aggregation, these observations suggest that the single homophilic HBHA interactions measured here reflect the formation of multimers that may promote mycobacterial aggregation.     

Relationship between mutation of serine residue at 315th position in M. tuberculosis catalase-peroxidase enzyme and Isoniazid susceptibility: An in silico analysis

Remarkable advances have been made in the drug therapy of tuberculosis. However much remains to be learned about the molecular and structural basis of drug resistance in Mycobacterium tuberculosis. It is known that, activation of Isoniazid (INH) is mediated by Mycobacterium tuberculosis catalase-peroxidase (MtBKatG) and mutation at position 315 (serine to threonine) leads to resistance. We have conducted studies on the drug resistance through docking and binding analysis supported by time-scale (∼1000 ps) and unrestrained all-atom molecular dynamics simulations of wild and mutant MtBKatG. The study showed conformational changes of binding residues. Mutant (S315T) showed high docking score and INH binding affinity as compared to wild enzyme. In molecular dynamics simulation, mutant enzyme exhibited less structure fluctuation at INH binding residues and more degree of fluctuation at C-terminal domain compared to wild enzyme. Our computational studies and data endorse that MtBKatG mutation (S315T) decrease the flexibility of binding residues and made them rigid by altering the conformational changes, in turn it hampers the INH activity. We ascertain from this work that, this study on structural mechanism of resistance development in Mycobacterium tuberculosis would lead to new therapeutics based on the result obtained in this study.     

Characterization of physical interaction between replication initiator protein DnaA and replicative helicase from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> H37Rv

In the pathogenic Mycobacterium tuberculosis H37Rv, the causative agent of tuberculosis, the genetic and biochemical mechanisms for initiation of DNA replication are largely unknown. In the present study, we have characterized the physical interactions between M. tuberculosis DnaA and DnaB using both in vivo methods, such as bacterial two-hybrid assays, and in vitro techniques, such as surface plasmon resonance (SPR) and Pull-down/Western blotting. The full-length N-terminus (1–206 residues) of DnaB was found to interact with DnaA, while the shorter N-terminal domain of DnaB (1–125 residues), which lacked the linker region, did not. Further SPR and electrophoretic mobility shift assays indicated that the N-terminus (1–206 residues) of DnaB also had a critical role in regulating DnaA complex formation at the origin of replication (OriC). This regulatory effect was not obviously observed for DNA substrates containing only two DnaA-boxes. This is the first report showing a physical interaction between DnaA and replicative helicase DnaB from M. tuberculosis and the role in subsequent DnaA-OriC interactions. The findings reported here further the understanding of the regulatory mechanisms for initiation of DNA replication in this important human pathogen.     

Functional insights by comparison of modeled structures of 18kDa small heat shock protein and its mutant in Mycobacterium leprae

In this work we are proposing Homology modeled structures of Mycobacterium leprae 18kDa heat shock protein and its mutant. The more closely related structure of the small heat shock protein (sHSP) belonging to the eukaryotic species from wheat sHSP16.9 and 16.3kDa ACR1 protein from Mycobacterium tuberculosis were used as template structures. Each model contains an N-terminal domain, alpha-crystalline domain and a C-terminal tail. The models showed that a single point mutation from serine to proline at 52^nd position causes structural changes. The structural changes are observed in N-terminal region and alpha-crystalline domains. Serine in 52^nd position is observed in β4 strand and Proline in 52^nd position is observed in loop. The number of residues contributing α helix at N-terminal region varies in both models. In 18S more number of residues is present in α helix when compared to 18P. The loop regions between β3 and β4 strands of both models vary in number of residues present in it. Number of residues contributing β4 strand in both models vary. β6 strand is absent in both models. Major functional peptide region of alpha crystalline domains of both models varies. These differences observed in secondary structures support their distinct functional roles. It also emphasizes that a point mutation can cause structural variation.     

Allostery and Conformational Dynamics in cAMP-binding Acyltransferases

Mycobacteria harbor unique proteins that regulate protein lysine acylation in a cAMP-regulated manner. These lysine acyltransferases from Mycobacterium smegmatis (KATms) and Mycobacterium tuberculosis (KATmt) show distinctive biochemical properties in terms of cAMP binding affinity to the N-terminal cyclic nucleotide binding domain and allosteric activation of the C-terminal acyltransferase domain. Here we provide evidence for structural features in KATms that account for high affinity cAMP binding and elevated acyltransferase activity in the absence of cAMP. Structure-guided mutational analysis converted KATms from a cAMP-regulated to a cAMP-dependent acyltransferase and identified a unique asparagine residue in the acyltransferase domain of KATms that assists in the enzymatic reaction in the absence of a highly conserved glutamate residue seen in Gcn5-related N-acetyltransferase-like acyltransferases. Thus, we have identified mechanisms by which properties of similar proteins have diverged in two species of mycobacteria by modifications in amino acid sequence, which can dramatically alter the abundance of conformational states adopted by a protein.