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Defense
Defense
Defense
Defense
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Defense
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Defense
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Defense

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  • 1.  
  • 2.
    • Tuberculosis (TB), one of the oldest recorded human afflictions, is a contagious disease caused by an obligate human pathogen Mycobacterium tuberculosis .
    • Interest in Mycobacterium tuberculosis due to:
    • Emergence of MDR and XDR strains.
    • Synergism with HIV.
    • Its ability to overcome host immune system and adapt a latent/persistent life style. About 1/3 rd of the world’s population is infected with Mycobacterium tuberculosis , but that doesn’t mean all are ill. (www.who.int/tb).
    • Persistent/latent phase is maintained via the altered response of cellular processes to the changed environment. One of the important way of responding to the altered environments is by changes in the patterns of gene expression of those genes whose products are required to combat those changes (Hecker et al ., 2001).
    • Regulation of gene expression: Transcription initiation is the major step in the regulation of gene expression in bacteria (Browning et al ., 2004).
    • Bacteria use their genetic material with great effectiveness to make right products at right time and in correct amounts. This is accomplished by the regulation at every step between gene and function and for the sake of economy the key step will be to regulate the initiation of RNA-transcript formation (Browning et al ., 2004).
  • 3. RNA polymerase (ββ’α 2 ω σ ) and its interactions at promoter. -35 (TTGACA), extended –10 (TGn) and -10 (TATAAT) elements are shown. (Adapted from Douglas F. Browning et al ., 2004) σ factors are essential for transcription initiation by virtue of their role in promoter recognition. Each of several sigma factors in cell is required for the transcription of a specific subset of genes operons. (Mooney., 2005).
  • 4.
    • σ factors:
    • Two phylogenetically distinct families of σ factors : σ 54 or σ 70 from Escherichia coli.
    • All eubacteria encode at least one σ factor copy of the σ 70 class.
    • Based on their structural and physiological roles, σ 70 related σ factors have been categorized into four groups.
    • Group 1 contains the principal σ factor.
    • Group 2 σ factors are found in limited number of species and can be differentiated structurally from the members of Group 1.
    • Group 3 σ factors comprise factors for evolutionarily related proteins with similar functions, such as heat shock, sporulation or flagellar biosynthesis. These are sometimes called as ‘alternative σ factors’.
    • Group 4 members also described as extracytoplasmic function (ECF) σ factors are often involved in the response to stress conditions, such as iron limitation, oxidative stress and surface stress. Moreover, several σ factors from this category are important for virulence.
    • (Raivio et al ., 2001; Bashyam et al ., 2004, Lonetto et al ., 1992, Buck et al ., 2000, Gruber et al ., 2003)
  • 5.
    • Regulation of gene expression through σ factors:
    • The alteration of gene expression through σ factors occurs through the competition between the
    • principal sigma factor and the alternative sigma factors for the RNA polymerase.
    • Alternative σ factors accumulate in response to specific signals, they compete with σ 70 for RNA
    • polymerase and enable it to recognize promoters that control genes that assist the cell in coping
    • with the altered conditions.
    • Regulation of alternative σ factors occurs at transcriptional, translational and post-translational level.
    • In many cases regulation is effected by anti-sigma factors which modulate the activity of
    • a σ factor independently of its transcription and translation. Many anti-sigma factors sequester their
    • cognate σ factor so that it is not free to combine with RNA polymerase (Hughes et al ., 1998).
    • Bacillus subtilis has extended the anti- σ factor control mechanism to three areas of gene regulation:
    • Sporulation, Energy Stress and Environmental stress (Dufour et al ., 1994, Alper et al ., 1996).
    • Gene duplication of a key regulatory module: the phosphate-antagonist-kinase module .
    • The fundamental processes involved in these regulatory networks are protein-protein interactions
    • and protein ligand especially nucleotide interactions.
    • Protein-nucleotide interactions apparently acts as a steric or electrostatic plug that alters the affinity
    • of a protein for its binding partners and facilitates partner switching cascades.
  • 6. RsbW: Anti-σ factor in B. subtilis stress response regulation (Adapted from Chien-Cheng et al ., 2003) RsbW is a kinase, and during normal exponential growth, RsbW inactivates RsbV by phosphorylation. RsbW is then free to bind σB and inhibit σB-dependent transcription. A drop in the levels of ATP forms one of the important stress signals and the cell responds by inhibiting RsbW activity by the dephosphorylation of RsbV and RsbV catalyzed release of σB from the RsbW-σB complex. (Alper et al., 1996) A separate regulatory mechanism allows induction of σB-dependent expression by stresses that have no direct effect on cell’s ATP levels. (Voelker et al., 1995, 1996)
  • 7.
    • Mycobacterium tuberculosis σF:
    • Mycobacterium tuberculosis encodes 13 σ factors falling into the all groups of σ 70 families.
    • σA is the primary σ factor which controls the expression of all the essential genes and has been
    • found to be essential for bacterial viability in Mycobacterium smegmatis (Gomez et al ., 1998).
    • σF has been has been found to be homologous to Bacillus subtilis stress-response sigma factor, σB,
    • as well as to its developmental σ factor, σF ( DeMaio et al ., 1997) .
    • Both stationary phase and antimicrobial exposure lead to σF induction:
    • 100-fold expression in late-stationary-phase compared to that in exponential growth. Expression was found to be induced by stresses like anaerobic metabolism/oxidative stress, nitrogen depletion, stationary phase growth and cold shock ( DeMaio et al ., 1997) .
    • The exposure of antibiotics like ethambutol, rifampin, streptomycin and cycloserine resulted in the
    • over-expression of σF with the level of expression being proportional to the amount of antibiotic added (Theresa et al ., 1997).
    • The expression of genes controlled by σF was analyzed by microarray analysis with wild type and σF mutant. In the mutant σF,
    • 187 genes were relatively under expressed in early stationary phase,
    • 277 in late stationary phase and only
    • 38 in exponential growth phase (Geiman et al ., 2004).
  • 8. Post-translational regulation of σ factor activity in Mycobacterium tuberculosis : Four anti- σ factors have been identified in the genome of M. tuberculosis ; RshA, RseA, RslA and UsfX. UsfX as the anti-σF factor debated since the identification of the σF operon. usfX (Rv3287c) is located upstream of sigF (Rv3286c) and the two are co-transcribed ( DeMaio et al ., 1997). UsfX has been found to inhibit σF -dependent transcription from σF promoter (Beaucher et al ., 2002). UsfX has been reported to be regulated by two antagonistic proteins: RsfA and RsfB. RsfA has been shown to respond to changes in redox potential, RsfB is believed to respond in phosphorylation dependent manner (Beaucher et al ., 2002). NO BIOCHEMICAL/STRUCTURAL INFORMATION ABOUT THESE PROTEINS: WHETHER UsfX HAS NUCLEOTIDE BINDING PROPERTIES AND KINASE PROPERTIES? IF ANY DO THEY HAVE A ROLE IN σF-UsfX INTERACTION? WHAT MAKES RsfB RESPOND TO CHANGES IN REDOX POTENTIAL AND HOW DOES THIS GOVERN UsfX-RsfA INTERACTION?
  • 9. CHARACTERIZATION OF UsfX Data base searches: Tuberculist (http://genolist.pasteur.fr/Tuberculist/) database annotates Rv3287c as rsbW because of its homology to anti-sigma factor Regulator of SigB W of Bacillus subtilis . Later was named as UsfX (Upstream of SigF) for because of the identification of the Rv3287c as upstream of SigF with the initiation codon of SigF starting within UsfX (Beaucher et al ., 2002). Gene organisation in the SigF operon. (Adapted from http://genolist.pasteur.fr/TubercuList/)
  • 10. Sequence Alignment Studies: Protein sequence homology searches of the UsfX using BLASTP against the available protein databases didn’t yield good scores for the alignment. Position specific iterated BLAST (PSI-BLAST) against the Protein Data Bank (PDB) and other data bases also didn’t provide good hits. PSI-BLAST against the non-redundant protein sequences (nr) database showed homologies with : COG 2172 (Anti-sigma regulatory factor, Serine/Threonine Protein Kinase). cd00075 (Histidine kinase-like ATPases). PRK 03660 (anti-sigma F factor) of Conserved Domain Database and similar clusters. Clusters Of Orthologous Groups (COGs) phylogenetically classifies proteins on the basis of the orthologous relations between them. COG2172 contains the cluster of anti-sigma regulatory factors/Serine-threonine protein kinases. A very low global sequence homology among the members from diverse backgrounds was observed. Mycobacteria UsfX sequences are well conserved with homology of up to 83 % for the respective proteins. Conclusively in spite of a low sequence homology UsfX is an anti-sigma factor with a common ancestral origin with the sequence characteristics conserved within the mycobacteria family.
  • 11. Cloning, Over-expression and Purification
  • 12. Nucleotide binding properties of UsfX Using Fluorophore labelled nucleotides didn’t give any significant results. Changes in the intrinsic trytophan fluorescence were monitored as function of nucleotide concentration. UsfX has a single tryptophan molecule: Trp-106. TITRATION CURVES
  • 13. HILL PLOTS
  • 14. The Kd values were determined from non-linear least-squares regression analysis of titration data using equation ΔF/ΔFmax=[Nucleotide]tot/(Kd +[ Nucleotide]tot ) The stoichiometry of binding was established from a linear version of the Hill equation, log(ΔF/ΔFmax–ΔF)=nlog[Nucleotide]-logK’ where n is the order of the binding reaction with respect to ligand concentration and K’is the concentration of nucleotide that yields 50% of ΔFmax. 0.97 1.6±0.1 205±5 1900±50 ADP 0.98 1.9±0.2 420±5 180±20 CTP 0.97 1.8±0.2 470±5 200±20 TTP 0.97 1.9±0.2 430±5 200±20 GTP 0.99 1.8±0.1 210±5 1300±50 ATP R 2 n H ΔF max(calc) K d (μM) Ligand
  • 15. In silico analysis of nucleotide binding In silico modeling and docking approaches used to evaluate the binding of nucleotides. The binding site was identified based on a comparison with the B. stearothermophilus SpoIIAB co-ordinates . The nucleotide binding site of 1L0O is proximal to the UsfX Trp106 in the superposition (FIG A). Using Trp106 as the docking centre we confirmed tryptophan is a part of the nucleotide binding site. All the four nucleotides viz., ATP, GTP, CTP and TTP docked at the same site with the nucleotides interacting primarily through their ring moieties.
  • 16. Nucleotide binding site is designed to accommodate a divalent ion also A D X S X S motif in the human integrin CR3 structure has been found to be involved in metal ion binding. Analysis of the UsfX model along with the sequence analysis lead to the identification of a conserved XGSFS motif in mycobacterial UsfX homologs where X is mainly a P or L.
  • 17. 0.45 1 (γ quencher /γ acrylamide ) eff 3.2 10.94 11.3 17.3 K SV (eff) (M -1 ) UsfX Trp UsfX Trp Parameter I - Acrylamide Probing the nucleotide binding site with solvent quenchers confirmed that binding site has an affinity for positively charged molecules. ATP binding in presence and absence of MgCl 2 . MgCl 2 increases the binding of ATP as is evident from enhanced decrease in % Δ F. Ionic quenchers provide information about the polarity of the environment surrounding the tryptophan in proteins. 3.5 times decrease in accessibility towards KI.
  • 18. Differential binding of adenosine nucleotides: Different ΔF max and K d values for a denosine nucleotides point towards differential behaviour in the binding site. GTP which is a pyrimidine like ATP has values similar to purines. Therefore the binding mode adapted by ATP is different as compared to the other nucleotides. K sv is the Stern-Volmer constant which depicts the accessibility of tryptophan molecule towards solvent. ΔF/F 0 is the fraction of fluorescence energy transfer from tryptophan to ligand. ASA is the accessible surface area around Trp-106 in free and bound states. Conclusively the binding of nucleotides in the active site is through their ring moieties but the significance of this differential binding of NTPs and differential response that this binding can produce may have an in vivo role which needs to be probed further. 53 0.98 0.87 3.58 GTP - 0.44 0.19 8.9 ADP 101 0.43 0.17 8.6 ATP 144 - - 9.53 NIL ASA (Å 2 ) around Trp-106 (ΔF/F 0 ) max (ΔF/F 0 ) 500μM K SV(500μM) (M -1 ) Ligand
  • 19. NTPase activity of UsfX LEFT PANEL: ATPase activity (Above), GTPase activity (Below). RIGHT PANEL: ATPase assay in presence of unlabelled nucleotide competitors. Cold ATP was found to result in maximum decrease in the hydrolysis of the radio-labelled ATP. Both ATPase and GTPase activities found but ATPase activity almost 4 times GTPase activity. ATP is the preferred substrate inspite of GTP having higher affinity. No Kinase activity or auto-phosphorylation activity observed.
  • 20. ANALYSIS OF UsfX- σ F INTERACTION Cloning, Over-expression and Purification of σ F Protein was over-expressed only when cells were induced at an OD 600 >2 probably due to the toxicity of the protein. Protein was soluble only in buffer containing β -mercaptoethanol. β -mercaptoethanol produced reduced state of cysteine residue which would otherwise form intermolecular disulfide linkages giving rise to large aggregates.
  • 21. UsfX- σ F Interaction
    • No in vitro interaction observed between two proteins on non SDS-polyacrylamide gels.
    • Chemical cross-linking in presence of ATP
    • also didn’t form the complex.
    • Co-transforming the cells with the individual clones also didn’t result in complex formation.
    • A co-expression vector was developed were the two proteins would be co-transcribed and co-translated resulting in an in vivo assembled UsfX- σ F complex.
    • Purification of the complex was achieved by attaching an affinity 6X-Histidine tag to one of the proteins (SigF).
  • 22. In the gel filtration analysis the elution volume of the complex pointed towards a molecular weight of 67kDa for the complex which is 7kDa more that when we presume complex to be formed by the interaction of a dimer of UsfX with a monomer of SigF. From densitometric analysis the proteins were estimated to bind in a ratio of 1.75:1 for UsfX/SigF. Hence UsfX and SigF bind in a stoichiometric ratio of 2:1. 1.75/1 Complex 2.24 19 16 15.6kDa UsfX 1.28 21 16 28.76kDa SigF Calculated stoichiometry Observed Intensity/mass Observed Intensity Monomeric mass Protein
  • 23. The presence or absence of nucleotide did not have an apparent effect on the UsfX-SigF interaction so we probed the effect of SigF on UsfX-nucleotide interaction. Similar binding affinities for ATP and ADP confirms that the presence of a nucleotide in the nucleotide binding pocket of UsfX is not essential for UsfX-SigF interaction. There may be other in vivo factors essential for the formation of the UsfX-SigF complex. No significant change was observed in the nucleotide binding properties of UsfX which suggests that they bind with nearly same affinities in the apo UsfX and UsfX-SigF complex. The identical values of K d and ΔF max for UsfX and UsfX-SigF complex show the non-interference of the two ligands in binding of each other and makes us to believe that the location of the nucleotide binding site is apparently distal to the protein-protein interaction interface. Nucleotide binding properties of UsfX-SigF complex 215±10 205±10 1550±50 1900± 50 ADP 220±10 210±10 1250±50 1300± 50 ATP UsfX-SigF Complex UsfX UsfX-SigF Complex UsfX ΔF max(calc) K d (μM) Ligand
  • 24.
    • ANALYSIS OF UsfX- RsfA INTERACTION
    • RsfA (Rv 1365c) and RsfB (Rv 3687c) have been reported as anti-anti sigma factors of SigF that inhibit UsfX. RsfA acts under reducing conditions while as RsfB binds in an nucleotide dependent manner. Homologues of RsfB are found in all bacterial species.
    • Sequence analysis and Database searching:
    • Four cysteine residues in RsfA viz ., Cys73, Cys74, Cys86 and Cys109 out of which Cys73 and Cys109 form a disulphide bridge. To our knowledge there are no other known anti-anti-sigma factors of this class.
    • Sequences were retrieved using RsfA as the query sequences and more than 100 anti-anti-sigma factor sequences with high E values (>10 -20 ) were selected for analysis.
    • Multiple sequence alignment revealed the existence of mycobacterium specific proteins with cysteine residues present in place of conserved serines (in RsfB and homologous anti-anti sigma factors). Two patterns of sequences were observed:
    • CC-(11)-C-(22)-C pattern found in all species.
    • SC-(11)-C-(22)-C found in only three species.
    • Apparently have evolved with the duplication of the parent gene and the proteins with the second pattern may be designed to perform through both redox and phosphate potential sensing pathways.
  • 25. Sequence co-variation in RsfA homologs Proteins performing similar functions usually possess similar structural features like topologies and folds. The sequence perturbations in the primary sequence in one part of the structure mostly leads to changes in the other parts to accommodate/compensate for these changes. The co-variation in the residues of the protein sequence allows for the maintenance of the overall structural integrity. Scoring of sequence alignment : Positional entropy or informational entropy gives estimates about scores of multiple sequence alignment where the calculated values are normalized for the Shannon’s entropy so that conserved sequences i.e , those having low entropy score 1 and divergent sequences i.e , a high entropy score 0. Positional entropies were calculated for the aligned sequences of anti-anti sigma factors from the whole set retrieved from the database searches and for those sequences retrieved from mycobacterial sources. The analysis was carried out using the following criteria: (i) Overall positional entropy of 1 i.e ., a common residue in all samples. (ii) Positional entropy of 1 with respect to cysteine or serine containing proteins but overall entropy less than 1 i.e ., representative of the groups at that particular position or a primary co-variant signifying that the variation has come along with the change in the primary active residue. (iii) Positional entropy of 1 with respect to one group but less than 1 for the other group at that position i.e ., a secondary co-variant for that particular group.
  • 26. Evolution of this class of anti-anti sigma factors has been accompanied by changes in the primary sequences which may be important for their functions under stress conditions. Group 1 describes the conserved residue in all the anti-anti sigma factor sequences. Group 2 can be described as the signature residues for their groups. Cys73 and Val63 of RsfA have replaced Ser61and Ile51 of RsfB respectively. The positional entropy at these positions within the group is 1 i.e ., the position is highly conserved over the whole sequence set. Group 3 describes the co-variations that have been brought by the introduction of cysteines in the sequence. The positions do not follow a conservation pattern in serine containing anti-anti sigma factors but does so in cysteine containing anti-sigma antagonists. So we can call them ‘structural co-variants’ . Cys109 is an example of ‘functional co-variant’ . It has to be essentially cysteine in redox sensor anti-anti sigma as it is the key to the formation of disulphide bond but is not conserved in serine containing antagonists.
  • 27. Cloning, Over-expression and Purification of RsfA BL21(DE3) Origami host cells were essential for protein solubility potentially due to the disulfide bond of RsfA as Origami strain is important for maintaining the disulphide bonds.
  • 28. UsfX-RsfA Interaction
    • DDT was found to be important for the interaction.
    • From gel filtration and densitometry UsfX and RsfA were found to interact in a 2:1 stoichiometry which was unlike the 2:2 reported for earlier systems.
  • 29. Reduction of the Cys73-Cys109 increases the hydrodynamic radius of the protein. Elution volume of the native protein corresponds to a Stokes radius (R S ) of 18.5Å while that of the disulphide bond reduced form is 22.1Å. Reduction of the disulphide bond might result in increased flexibility of the protein and allow for the enhanced hydrodynamic radius which might be necessary for interaction with UsfX. Effect of DTT on conformation of RsfA DTT is a reducing agent and mimics the increased reduced environment that cells encounter in the stress phase. RsfA binds to UsfX only when the disulphide bond is reduced. However, the actual significance of the disulphide bond in regulating its interactions with UsfX is not known.
  • 30. Upper panel : Residues around Cys109 of RsfA where the Cys73-Cys109 disulphide bridge is maintained. The snapshot is at the beginning of the MD simulations (Left) and at the end of the MD run (Right). Lower panel: Snapshot of the same region at the beginning of the MD simulations where the Cys73-Cys109 disulphide bridge is broken ( Left ) and after the MD simulations. Rearrangement of the His107 and Phe104 side chains of UsfX takes place where the side chain of His107 comes close to Cys109 of RsfA. Interactions at the UsfX-RsfA interface
  • 31. Conclusively, A sigma factor (SigF), an anti-sigma factor (UsfX) and an anti-anti-sigma factor (RsfA) from M. tuberculosis H37Rv were cloned, over-expressed and purified in E. coli based expression systems. The anti-sigma factor, UsfX is a dimeric protein in solution with the monomeric molecular weight of about 15kDa. Two independent nucleotide binding sites were identified to be present in the UsfX dimer. Binds all the naturally occurring nucleotides which has not been reported from any other source. Adenosine nucleotides behave in a different manner in the binding pocket. May be has an in vivo role in transcription regulation? The nucleotide binding pocket like other nucleotide binding proteins is designed to accommodate divalent ions which enhances the binding of nucleotides. Although the nucleotide binding properties are like homologous proteins but the residues involved in the binding site are different from known proteins. The sequence is highly conserved within the mycobacteria family. An XGSFS motif can be identified in mycobacterial UsfX homologs which along with tryptophan form the core of the binding pocket. No kinase or autophosphorylation activity was observed in UsfX. But weak NTPase activity was observed with preference for ATP as the substrate.
  • 32. The sigma factor SigF is a monomer is solution with a molecular weight of about 28kDa. SigF-UsfX complex was co-purified using a co-expression vector system. No in vitro interaction could be observed between the two proteins even in the presence of the nucleotides. Some unknown factors/conditions apparently support the interaction. The two ligands: nucleotide and SigF didn’t produce any observable effect on the binding of one another. Thus binding of SigF to UsfX is independent of nucleotide binding. The anti-anti-sigma factor RsfA is a monomer is solution with a molecular weight of about 28kDa. Sequence analysis of RsfA points towards the structural and functional co-varience of the residues along with the functionally important Cys-Cys disulfide bridge in this mycobacterial specific class of anti-sigma factor antagonists. The Cys-Cys disulfide bridge serves a dual role: Acts as a sensor for the increased redox potential in the Stress phase and the reduction of this bond facilitates its interaction with UsfX which renders SigF free. Published results: M. tuberculosis UsfX (Rv3287c) exhibits novel nucleotide binding and hydrolysis properties. Biochemical and Biophysical Research Communications 375 (2008) 465–470. Interactions of the M. tuberculosis UsfX with the cognate sigma factor SigF and anti-anti-sigma factor RsfA. Biochimica et Biophysica Acta 1794 (2009) 541–553.
  • 33. Crystallization Crystals of M. tuberculosis UsfX.
    • Crystallization trials of SigF, UsfX, RsfA, UsfX-SigF complex and UsfX-RsfA complex were performed using vapor diffusion method in
    • Sparse matrix (Jancarik and Kim, 1991).
    • Crystal screen 2 (Jancarik and Kim., 1991; Cudney et al. , 1994).
    • Mixed precipitant screens (Mazeed et al. , 2003).
    • For crystallization of protein-protein complexes in addition to the above screening methods those described by Radaev. S and Sun., (2002) and Radaev. S et al ., (2006).
    • Both these methods involve using an enlarged screen around those crystallizing agents which have been reported for protein-protein complexes in Protein Data Bank.
    • For UsfX Alanine double mutant for Lysine residues was also used for crystallization trials. K2A concept : Where surface residues like lysine and arginine are believed to be entropically unfavorable for crystallization.
    • ANY OTHER THING WE COULD THINK, IMAGINE, VISUALISE, FANTASISE!
  • 34. Acknowledgement Dr. R. Ravishankar (My Ph.D mentor). Dr. Amoghanant Sahasrabudhe (CDRI). Dr. Shantunu Chaudhary (IGIB, New Delhi). Dr. J.V.Pratap. All the members of my own lab, Dr.J.V.Pratap’s group, MSB Division and CDRI fraternity. Special thanks to Dr. Sandeep K. Srivastava.
  • 35. THANKS

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