Krishan Gopal Thakur

Structural and Biochemical Bases for the Redox Sensitivity of Mycobacterium tuberculosis RslA

An effective transcriptional response to redox stimuli is of particular importance for Mycobacterium tuberculosis, as it adapts to the environment of host alveoli and macrophages. The M. tuberculosis σ factor σL regulates the expression of genes involved in cell-wall and polyketide syntheses. σL interacts with the cytosolic anti-σ domain of a membrane-associated protein, RslA. Here we demonstrate that RslA binds Zn2+ and can sequester σL in a reducing environment. In response to an oxidative stimulus, proximal cysteines in the CXXC motif of RslA form a disulfide bond, releasing bound Zn2+. This results in a substantial rearrangement of the σL/RslA complex, leading to an 8-fold decrease in the affinity of RslA for σL. The crystal structure of the − 35-element recognition domain of σL, σ4L, bound to RslA reveals that RslA inactivates σL by sterically occluding promoter DNA and RNA polymerase binding sites. The crystal structure further reveals that the cysteine residues that coordinate Zn2+ in RslA are solvent exposed in the complex, thus providing a structural basis for the redox sensitivity of RslA. The biophysical parameters of σL/RslA interactions provide a template for understanding how variations in the rate of Zn2+ release and associated conformational changes could regulate the activity of a Zn2+-associated anti-σ factor.

Mycobacterium tuberculosis Keto-Mycolic Acid and Macrophage Nuclear Receptor TR4 Modulate Foamy Biogenesis in Granulomas: A Case of a Heterologous and Noncanonical Ligand-Receptor Pair

The cell wall of Mycobacterium tuberculosis is configured of bioactive lipid classes that are essential for virulence and potentially involved in the formation of foamy macrophages (FMs) and granulomas. Our recent work established crosstalk between M. tuberculosis cell wall lipids and the host lipid-sensing nuclear receptor TR4. In this study, we have characterized, identified, and adopted a heterologous ligand keto-mycolic acid from among M. tuberculosis lipid repertoire for the host orphan NR TR4. Crosstalk between cell wall lipids and TR4 was analyzed by transactivation and promoter reporter assays. Mycolic acid (MA) was found to transactivate TR4 significantly compared with other cell wall lipids. Among the MA, the oxygenated form, keto-MA, was responsible for transactivation, and the identity was validated by TR4 binding assays followed by TLC and nuclear magnetic resonance. Isothermal titration calorimetry revealed that keto-MA binding to TR4 is energetically favorable. This keto-MA–TR4 axis seems to be essential to this oxygenated MA induction of FMs and granuloma formation as evaluated by in vitro and in vivo model of granuloma formation. TR4 binding with keto-MA features a unique association of host nuclear receptor with a bacterial lipid and adds to the presently known ligand repertoire beyond dietary lipids. Pharmacologic modulation of this heterologous axis may hold promise as an adjunct therapy to frontline tuberculosis drugs.

Structural and Biophysical Studies on Two Promoter Recognition Domains of the Extra-cytoplasmic Function σ Factor σC from Mycobacterium tuberculosis

σ factors are transcriptional regulatory proteins that bind to the RNA polymerase and dictate gene expression. The extracytoplasmic function (ECF) σ factors govern the environment dependent regulation of transcription. ECF σ factors have two domains σ2 and σ4 that recognize the -10 and -35 promoter elements. However, unlike the primary σ factor σA, the ECF σ factors lack σ3, a region that helps in the recognition of the extended -10 element and σ1.1, a domain involved in the autoinhibition of σA in the absence of core RNA polymerase. Mycobacterium tuberculosis σC is an ECF σ factor that is essential for the pathogenesis and virulence of M. tuberculosis in the mouse and guinea pig models of infection. However, unlike other ECF σ factors, σC does not appear to have a regulatory anti-σ factor located in the same operon. We also note that M. tuberculosis σC differs from the canonical ECF σ factors as it has an N-terminal domain comprising of 126 amino acids that precedes the σC2 and σC4 domains. In an effort to understand the regulatory mechanism of this protein, the crystal structures of the σC2 and σC4 domains of σC were determined. These promoter recognition domains are structurally similar to the corresponding domains of σA despite the low sequence similarity. Fluorescence experiments using the intrinsic tryptophan residues of σC2 as well as surface plasmon resonance measurements reveal that the σC2 and σC4 domains interact with each other. Mutational analysis suggests that the Pribnow box-binding region of σC2 is involved in this interdomain interaction. Interaction between the promoter recognition domains in M. tuberculosis σC are thus likely to regulate the activity of this protein even in the absence of an anti-σ factor.

Structural basis for the redox sensitivity of the Mycobacterium tuberculosis SigK–RskA σ–anti-σ complex

The host-pathogen interactions in Mycobacterium tuberculosis infection are significantly influenced by redox stimuli and alterations in the levels of secreted antigens. The extracytoplasmic function (ECF) σ factor σ(K) governs the transcription of the serodominant antigens MPT70 and MPT83. The cellular levels of σ(K) are regulated by the membrane-associated anti-σ(K) (RskA) that localizes σ(K) in an inactive complex. The crystal structure of M. tuberculosis σ(K) in complex with the cytosolic domain of RskA (RskAcyto) revealed a disulfide bridge in the -35 promoter-interaction region of σ(K). Biochemical experiments reveal that the redox potential of the disulfide-forming cysteines in σ(K) is consistent with its role as a sensor. The disulfide bond in σ(K) influences the stability of the σ(K)-RskAcyto complex but does not interfere with σ(K)-promoter DNA interactions. It is noted that these disulfide-forming cysteines are conserved across homologues, suggesting that this could be a general mechanism for redox-sensitive transcription regulation.

Crystal structure of Mycobacterium tuberculosis CarD, an essential RNA polymerase binding protein, reveals a quasidomain‐swapped dimeric structural architecture

Mycobacterium tuberculosis (Mtb) CarD is an essential transcriptional regulator that binds RNA polymerase and plays an important role in reprogramming transcription machinery under diverse stress conditions. Here, we report the crystal structure of CarD at 2.3 Å resolution, that represents the first structural description of CarD/CdnL‐Like family of proteins. CarD adopts an overall bi‐lobed structural architecture where N‐terminal domain resembles ‘tudor‐like’ domain and C‐terminal domain adopts a novel five helical fold that lacks the predicted leucine zipper structural motif. The structure reveals dimeric state of CarD resulting from β‐strand swapping between the N‐terminal domains of each individual subunits. The structure provides crucial insights into the possible mode(s) of CarD/RNAP interactions. Proteins 2014; 82:879–884.

Mycobacterium tuberculosis Rv2704 is a member of the YjgF/YER057c/UK114 family

M. tuberculosis Rv2704 is a member of the highly conserved YjgF/YER057c/UK114 protein superfamily. Homologues of this protein occur in eubacteria, archaea and eukaryotes. Proteins in this family are functionally diverse and are involved in variety of enzymatic and non-enzymatic functions. The high sequence and structural similarity between members of this protein family provides an intriguing dataset to rationalize the wide variety of functional roles. This protein family is an example of minimalistic changes leading to functional diversification. This feature is best exemplified by the three close homologues of YjgF proteins in mammals (human, rat and goat) with sequence identity better than 85%. These homologues perform different functions1-3. The diverse functions attributed to these proteins in this family include tumour antigen activity in the goat homologue1, translation inhibition in human and rat homologues (hp14.5 and rp14.5)2,3, endoribonuclease activity in rp14.54, calpain activation in the bovine homologue5, molecular chaperone activity in DUK1146 and involvement in the regulation of purine and removal of toxic metabolites in YjgF7,8 and isoleucine (YjgF, YER057c, Ibm1) biosynthetic pathways9-11. In addition, members of this protein family have also been shown to regulate mitochondrial maintenance (Ibm1) in yeast11. Proteins from the YjgF family in plants are involved in photosynthesis and chromoplastogenesis (CHRD)12. n nThe three dimensional structures of fifteen homologues of Rv2704 have been determined from bacteria, human, rat, goat and archaea7,13-18. These proteins share similar homotrimeric structures with the clefts between the monomeric subunits proposed to have some functional relevance7,15-17 (Supplementary Table I). The best characterized protein with the YjgF fold is chorismate mutase which typifies this α+β fold and trimeric quaternary association. However, this protein shares very little sequence similarity with the other members of this family that were subsequently structurally characterized17,18. Members of the Yjgf family could potentially bind various metabolites. This suggestion was based on crystal structures of some homologues bound to ligands like benzoate15, acetate17, 2-ketobutyrate7, 1,2-ethanediol7, propanoate7 and serine7. In these crystal structures, the ligands bound at the cleft between the monomeric subunits of the trimer. This prompted a comparative analysis of this intersubunit cleft and an examination of role of conserved residues lining the cleft7,15. No biological role could be assigned for these bound ligands except perhaps for 2-ketobutyrate where it was proposed that Yjgf may be involved in the removal of toxic products7. Even this functional implication, however, remains contestable with a recent report proposing that 2-ketobutyrate may not be the natural substrate for YjgF from Salmonella enterica9. These studies thus highlight the difficulties in structure-based functional assignment for proteins belonging to the YjgF family. n nM. tuberculosis Rv2704 lies in the same operon as the principal sigma (σ) factor, σA (Supplementary Figure 1). σ factors are transcriptional proteins that are often regulated by their interactions with an anti-σ factor located in the same operon. Rv2704 was thus examined for its role as a putative regulator for σA. While we could not detect any interaction between Rv2704 and σA in vitro, it is still likely that it could regulate σA activity in vivo, perhaps involving metabolite interactions. Here we present the crystal structure of Rv2704 at 1.93A resolution, solved using single isomorphous replacement with anomalous scattering (SIRAS). This protein is a trimer in solution with the overall structure similar to that of other YjgF homologues. The structure and biochemical features of Rv2704 could thus provide a template for more directed predictive methods for functional annotation in the YjgF family of proteins.

The intramolecular disulfide-stapled structure of laterosporulin, a class IId bacteriocin, conceals a human defensin-like structural module.

The growing emergence of antibiotic‐resistant bacteria has led to the exploration of naturally occurring defense peptides as antimicrobials. In this study, we found that laterosporulin (LS), a class IId bacteriocin, effectively kills active and nonmultiplying cells of both Gram‐positive and Gram‐negative bacteria. Fluorescence and electron microscopy suggest that growth inhibition occurs because of increased membrane permeability. The crystal structure of LS at 2.0 Å resolution reveals an all‐β conformation of this peptide, with four β‐strands forming a twisted β‐sheet. All six intrinsic cysteines are intramolecularly disulfide‐bonded, with two disulfides constraining the N terminus of the peptide and the third disulfide crosslinking the extreme C terminus, resulting in the formation of a closed structure. The significance of disulfides in maintaining the in‐solution peptide structure was confirmed by CD and fluorescence analyses. Despite a low overall sequence similarity, LS has disulfide connectivity [CI–CV, CII–CIV, and CIII–CVI] like that of β‐defensins and a striking architectural similarity with α‐defensins. Therefore LS presents a missing link between bacteriocins and mammalian defensins, and is also a potential antimicrobial lead, in particular against nonmultiplying bacteria.

Dual Role of a Biosynthetic Enzyme, CysK, in Contact Dependent Growth Inhibition in Bacteria

Contact dependent growth inhibition (CDI) is the phenomenon where CDI+ bacterial strain (inhibitor) inhibits the growth of CDI−strain (target) by direct cell to cell contact. CDI is mediated by cdiBAI gene cluster where CdiB facilitates the export of CdiA, an exotoxin, on the cell surface and CdiI acts as an immunity protein to protect CDI+ cells from autoinhibition. CdiA-CT, the C-terminal region of the toxin CdiA, from uropathogenic Escherichia coli strain 536 (UPEC536) is a latent tRNase that requires binding of a biosynthetic enzyme CysK (O-acetylserine sulfyhydrylase) for activation in the target cells. CdiA-CT can also interact simultaneously with CysK and immunity protein, CdiI, to form a ternary complex in UPEC536. But the role of CysK in the ternary complex is not clear. We studied the hydrodynamic, thermodynamic and kinetic parameters of binary and ternary complexes using AUC, ITC and SPR respectively, to investigate the role of CysK in UPEC536. We report that CdiA-CT binds CdiI and CysK with nanomolar range affinity. We further report that binding of CysK to CdiA-CT improves its affinity towards CdiI by ~40 fold resulting in the formation of a more stable complex with over ~130 fold decrease in dissociation rate. Thermal melting experiments also suggest the role of CysK in stabilizing CdiA-CT/CdiI complex as Tm of the binary complex shifts ~10°C upon binding CysK. Hence, CysK acts a modulator of CdiA-CT/CdiI interactions by stabilizing CdiA-CT/CdiI complex and may play a crucial role in preventing autoinhibition in UPEC536. This study reports a new moonlighting function of a biosynthetic enzyme, CysK, as a modulator of toxin/immunity interactions in UPEC536 inhibitor cells.

Over-expression and purification strategies for recombinant multi-protein oligomers: A case study of Mycobacterium tuberculosis σ/anti-σ factor protein complexes

The function of a protein in a cell often involves coordinated interactions with one or several regulatory partners. It is thus imperative to characterize a protein both in isolation as well as in the context of its complex with an interacting partner. High resolution structural information determined by X-ray crystallography and Nuclear Magnetic Resonance offer the best route to characterize protein complexes. These techniques, however, require highly purified and homogenous protein samples at high concentration. This requirement often presents a major hurdle for structural studies. Here we present a strategy based on co-expression and co-purification to obtain recombinant multi-protein complexes in the quantity and concentration range that can enable hitherto intractable structural projects. The feasibility of this strategy was examined using the σ factor/anti-σ factor protein complexes from Mycobacterium tuberculosis. The approach was successful across a wide range of σ factors and their cognate interacting partners. It thus appears likely that the analysis of these complexes based on variations in expression constructs and procedures for the purification and characterization of these recombinant protein samples would be widely applicable for other multi-protein systems.

Facile fabrication of lipase to amine functionalized gold nanoparticles to enhance stability and activity

Among various techniques of immobilization, EDC/NHS cross linking is a simple and single step process for covalent coupling between enzymes and nanoparticles. Here we describe immobilization of lipase on amine functionalized gold nanoparticles (AuNPs-NH2) to attain enhanced activity and stability. To achieve a suitable orientation, it is necessary to understand the contribution of different functional groups on the enzymes surface. Therefore, the crystal structure of lipase was analyzed using a computational method (PyMOL) to find the exposed acidic amino acid residues that can be exploited for conjugation. Confirmation of conjugation (AuNP-NH2-lipase) was determined by various techniques such as agarose gel electrophoresis, zeta measurement, FTIR-spectroscopy and TEM. Further, catalytic parameters (Vmax, KM,app, Kcat, and Kcat/KM,app) have been studied to establish activity enhancement upon immobilization. The data also suggested that, AuNP-NH2-lipase has desirable improved parameters such as temperature and storage stability. The thermodynamic parameters for the kinetics of deactivation (, and ) of the AuNP-NH2-lipase and free lipase demonstrated better stability of the conjugate. CD and fluorescence spectroscopic studies revealed minor structural rearrangements in the enzyme upon conjugation. Thus the AuNP-NH2-lipase conjugate represents a novel enzyme preparation with attributes of high activity and stability that could be an attractive choice in diverse applications ranging from catalysis to diagnostics.