Lysine Acetyltransferases (KATs) in Disguise: Diseases Implications.

Acetylation is one of the key post-translational protein modifications catalysed by the protein lysine acetyltransferases (KATs). KATs catalyse the transfer of acetyl groups to the epsilon-amino groups of lysine residues in histones and non-histone proteins. Because of its wide range of target proteins, KATs regulate many biological processes, and their aberrant activities may underlie several human diseases, including cancer, asthma, Chronic Obstructive Pulmonary Disease (COPD), and neurological disorders. Unlike most of the histone modifying enzymes, such as lysine methyltransferases, KATs do not possess any conserved domain like SET domain of lysine methyltransferases. However, almost all the major families of KATs are found to be transcriptional coactivators or adaptor proteins, with defined catalytic domains, called canonical KATs. Over the past two decades, a few proteins have been discovered to possess intrinsic KAT activity but are not classical coactivators. We would like to categorize them as non-canonical KATs (NC-KATs). These NC-KATs include general transcription factors TAFII250, mammalian TFIIIC complex, and mitochondrial protein GCN5L1, etc. This review focuses on our understanding, as well as controversies regarding non-canonical KATs, where we compare the structural and functional similarities and dissimilarities of non-canonical KATs with the canonical KATs. This review also highlights the potential role of NC-KATs in health and diseases.

Estradiol overcomes adiponectin-resistance in diabetic mice by regulating skeletal muscle adiponectin receptor 1 expression.

Adiponectin and insulin resistance creates a vicious cycle that exacerbates type 2 diabetes. Earlier, we observed that female leptin receptor-deficient BLKS mice (BKS-db/db) were more sensitive to an adiponectin mimetic GTDF than males, which led us to explore if E2 plays a crucial role in modulation of adiponectin-sensitivity. Male but not female BKS-db/db mice were resistant to metabolic effects of globular adiponectin treatment. Male BKS-db/db displayed reduced skeletal muscle AdipoR1 protein expression, which was consequent to elevated polypyrimidine tract binding protein 1 (PTB) and miR-221. E2 treatment in male BKS-db/db, and ovariectomized BALB/c mice rescued AdipoR1 protein expression via downregulation of PTB and miR-221, and also directly increased AdipoR1 mRNA by its classical nuclear receptors. Estrogen receptor regulation via dietary or pharmacological interventions may improve adiponectin resistance and consequently ameliorate insulin resistance in type 2 diabetes.

Histone Chaperone Nucleophosmin Regulates Transcription of Key Genes Involved in Oral Tumorigenesis.

Nucleophosmin (NPM1) is a multifunctional histone chaperone that can activate acetylation-dependent transcription from chromatin templates in vitro. p300-mediated acetylation of NPM1 has been shown to further enhance its transcription activation potential. Acetylated and total NPM1 pools are increased in oral squamous cell carcinoma. However, the role of NPM1 or its acetylated form (AcNPM1) in transcriptional regulation in cells and oral tumorigenesis is not fully elucidated. Using ChIP-seq analyses, we provide the first genome-wide profile of AcNPM1 and show that AcNPM1 is enriched at transcriptional regulatory elements. AcNPM1 co-occupies marks of active transcription at promoters and DNase I hypersensitive sites at enhancers. In addition, using a high-throughput protein interaction profiling approach, we show that NPM1 interacts with RNA Pol II, general transcription factors, mediator subunits, histone acetyltransferase complexes, and chromatin remodelers. NPM1 histone chaperone activity also contributes to its transcription activation potential. Further, NPM1 depletion leads to decreased AcNPM1 occupancy and reduced expression of genes required for proliferative, migratory and invasive potential of oral cancer cells. NPM1 depletion also abrogates the growth of orthotopic tumors in mice. Collectively, these results establish that AcNPM1 functions as a coactivator during during RNA polymerase II-driven transcription and regulates the expression of genes that promote oral tumorigenesis.

[Fe-S] biogenesis and unusual assembly of the ISC scaffold complex in the Plasmodium falciparum mitochondrion.

The malaria parasite harbors two [Fe-S] biogenesis pathways of prokaryotic origin-the SUF and ISC systems in the apicoplast and mitochondrion, respectively. While the SUF machinery has been delineated, there is little experimental evidence on the ISC pathway. We confirmed mitochondrial targeting of Plasmodium falciparum ISC proteins followed by analyses of cysteine desulfurase, scaffold, and [Fe-S]-carrier components. PfIscU functioned as the scaffold in complex with the PfIscS-PfIsd11 cysteine desulfurase and could directly assemble [4Fe-4S] without prior [2Fe-2S] formation seen in other homologs. Small angle X-ray scattering and spectral studies showed that PfIscU, a trimer, bound one [4Fe-4S]. In a deviation from reported complexes from other organisms, the P. falciparum desulfurase-scaffold complex assembled around a PfIscS tetramer instead of a dimer, resulting in a symmetric hetero-hexamer [2× (2PfIscS-2PfIsd11-2PfIscU)]. PfIscU directly transferred [4Fe-4S] to the apo-protein aconitase B thus abrogating the requirement of intermediary proteins for conversion of [2Fe-2S] to [4Fe-4S] before transfer to [4Fe-4S]-recipients. Among the putative cluster-carriers, PfIscA2 was more efficient than PfNifU-like protein; PfIscA1 primarily bound iron, suggesting its potential role as a Fe2+ carrier/donor. Our results identify the core P. falciparum ISC machinery and reveal unique features compared with those in bacteria or yeast and human mitochondria.

Leprosy drug clofazimine activates peroxisome proliferator-activated receptor-γ and synergizes with imatinib to inhibit chronic myeloid leukemia cells.

Leukemia stem cells contribute to drug-resistance and relapse in chronic myeloid leukemia (CML) and BCR-ABL1 inhibitor monotherapy fails to eliminate these cells, thereby necessitating alternate therapeutic strategies for patients CML. The peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone downregulates signal transducer and activator of transcription 5 (STAT5) and in combination with imatinib induces complete molecular response in imatinib-refractory patients by eroding leukemia stem cells. Thiazolidinediones such as pioglitazone are, however, associated with severe side effects. To identify alternate therapeutic strategies for CML we screened Food and Drug Administration-approved drugs in K562 cells and identified the leprosy drug clofazimine as an inhibitor of viability of these cells. Here we show that clofazimine induced apoptosis of blood mononuclear cells derived from patients with CML, with a particularly robust effect in imatinib-resistant cells. Clofazimine also induced apoptosis of CD34+38- progenitors and quiescent CD34+ cells from CML patients but not of hematopoietic progenitor cells from healthy donors. Mechanistic evaluation revealed that clofazimine, via physical interaction with PPARγ, induced nuclear factor kB-p65 proteasomal degradation, which led to sequential myeloblastoma oncoprotein and peroxiredoxin 1 downregulation and concomitant induction of reactive oxygen species-mediated apoptosis. Clofazimine also suppressed STAT5 expression and consequently downregulated stem cell maintenance factors hypoxia-inducible factor-1α and -2α and Cbp/P300 interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2). Combining imatinib with clofazimine caused a far superior synergy than that with pioglitazone, with clofazimine reducing the half maximal inhibitory concentration (IC50) of imatinib by >4 logs and remarkably eroding quiescent CD34+ cells. In a K562 xenograft study clofazimine and imatinib co-treatment showed more robust efficacy than the individual treatments. We propose clinical evaluation of clofazimine in imatinib-refractory CML.

Mycobacterial protein tyrosine kinase, PtkA phosphorylates PtpA at tyrosine residues and the mechanism is stalled by the novel series of inhibitors.

Phosphorylation and dephosphorylation are the key mechanisms for mycobacterial physiology and play critical roles in mycobacterial survival and in its pathogenesis. Mycobacteria evade host immune mechanism by inhibiting phagosome - lysosome fusion in which mycobacterial protein tyrosine phosphatase A (PtpA;TP) plays an indispensable role. Tyrosine kinase (PtkA;TK) activated by autophosphorylation; phosphorylates TP, which subsequently leads to increase in its phosphatase activity. The phosphorylated TP is secreted in phagosome of macrophage. In the present study, we have shown that the phosphorylation at two sites of TP; Y128 and Y129 are critical for TK-mediated phosphatase activity. The disruption of this interaction between TK and TP inhibits activation of later which further leads to the decrease in intracellular survival of mycobacteria. Furthermore, the proof of concept has been established using benzylbenzofurans and benzofuranamides, which inhibit the growth and intracellular survival of mycobacteria, associate with the functional sites of TP and contend with the TK. This binding was further restated by looking at the anchorage of protein-protein and the protein-inhibitor complexes in the homology-based structure models and by surface plasmon resonance analysis.

Biochemical characterization and novel inhibitor identification of Mycobacterium tuberculosis Endonuclease VIII 2 (Rv3297).

Nei2 (Rv3297) is a DNA Base Excision Repair (BER) glycosylase that is essential for survival of Mycobacterium tuberculosis in primates. We show that MtbNei2 is a bifunctional glycosylase that specifically acts on oxidized pyrimidine-containing single-stranded, double-stranded, 5'/3' fork and bubble DNA substrates. MtbNei2 possesses Uracil DNA glycosylase activity unlike E. coli Nei. Mutational studies demonstrate that Pro2 and Glu3 located in the active site are essential for glycosylase activity of MtbNei2. Mutational analysis demonstrated that an unstructured C-terminal zinc finger domain that was important for activity in E. coli Nei and Fpg, was not required for the glycosylase activity of MtbNei2. Lastly, we screened the NCI natural product compound database and identified three natural product inhibitors with IC50 values ranging between 41.8 µM-92.7 µM against MtbNei2 in in vitro inhibition assays. Surface Plasmon Resonance (SPR) experiments showed that the binding affinity of the best inhibitor, NSC31867, was 74 nM. The present results set the stage for exploiting this important target in developing new therapeutic strategies that target Mycobacterial BER.

Characterization of EccA3, a CbbX family ATPase from the ESX-3 secretion pathway of M. tuberculosis.

EccA family proteins are conserved components of ESX secretion pathways in M. tuberculosis H37Rv. Here, we report the characterization of EccA3 (Rv0282), a CbbX family AAA (ATPases Associated with diverse cellular Activities) protein from the ESX-3 pathway that is required for in vitro growth of mycobacteria, secretion of virulence factors, and acquisition of iron and zinc. EccA3 is a thermostable ATPase with a molecular weight of ~68kDa. It exists as a dodecamer in the apo form and associates as a hexamer in the presence of ATP. Its C-terminal region consists of a CbbX-like AAA-domain while the N-terminal region contains a tetratricopeptide repeat (TPR) domain with lower homology to other EccA-type proteins. Further, the C-terminal domain functions as the oligomerization domain and also exhibits ATPase activity. Mutational analysis, steady state kinetics and molecular docking studies identify R573 as the important 'sensor arginine' and R505 as an 'arginine finger' in EccA3. Dynamic fluorescence quenching experiments suggest that the N-terminal domain moves closer to the C-terminal domain upon ATP-binding. The ATP-dependent 'open-close' relative movements of the two domains might help EccA3 interaction and secretion of essential virulence factors.

Small molecule adiponectin receptor agonist GTDF protects against skeletal muscle atrophy.

Skeletal muscle atrophy is a debilitating response to several major diseases, muscle disuse and chronic steroid treatment for which currently no therapy is available. Since adiponectin signaling plays key roles in muscle energetics, we assessed if globular adiponectin (gAd) or the small molecule adiponectin mimetic 6-C-ß-D-glucopyranosyl-(2S,3S)-(+)-5,7,3',4'-tetrahydroxydihydroflavonol (GTDF) could ameliorate muscle atrophy. Both GTDF and gAd induced C2C12 myoblast differentiation. GTDF and gAd effectively prevented reduction in myotube area and suppressed the expressions of atrophy markers; atrogin-1 and muscle ring finger protein-1 (MuRF1) in models of steroid, cytokine and starvation -induced muscle atrophy. The protective effects of GTDF and gAd were routed through AMPK and AKT activation and thereby stimulation of PPAR gamma coactivator 1α and inhibition of forkhead box O transcription factors. Finally, GTDF and gAd mitigated dexamethasone-induced muscle atrophy in vivo. Together, our results demonstrate that activating adiponectin signaling may be an effective therapeutic strategy against skeletal muscle atrophy.

Single Amino Acid Substitutions at Specific Positions of the Heptad Repeat Sequence of Piscidin-1 Yielded Novel Analogs That Show Low Cytotoxicity and In Vitro and In Vivo Antiendotoxin Activity.

Piscidin-1 possesses significant antimicrobial and cytotoxic activities. To recognize the primary amino acid sequence(s) in piscidin-1 that could be important for its biological activity, a long heptad repeat sequence located in the region from amino acids 2 to 19 was identified. To comprehend the possible role of this motif, six analogs of piscidin-1 were designed by selectively replacing a single isoleucine residue at a d (5th) position or at an a (9th or 16th) position with either an alanine or a valine residue. Two more analogs, namely, I5F,F6A-piscidin-1 and V12I-piscidin-1, were designed for investigating the effect of interchanging an alanine residue at a d position with an adjacent phenylalanine residue and replacing a valine residue with an isoleucine residue at another d position of the heptad repeat of piscidin-1, respectively. Single alanine-substituted analogs exhibited significantly reduced cytotoxicity against mammalian cells compared with that of piscidin-1 but appreciably retained the antibacterial and antiendotoxin activities of piscidin-1. All the single valine-substituted piscidin-1 analogs and I5F,F6A-piscidin-1 showed cytotoxicity greater than that of the corresponding alanine-substituted analogs, antibacterial activity marginally greater than or similar to that of the corresponding alanine-substituted analogs, and also antiendotoxin activity superior to that of the corresponding alanine-substituted analogs. Interestingly, among these peptides, V12I-piscidin-1 showed the highest cytotoxicity and antibacterial and antiendotoxin activities. Lipopolysaccharide (12 mg/kg of body weight)-treated mice, further treated with I16A-piscidin-1, the piscidin-1 analog with the highest therapeutic index, at a single dose of 1 or 2 mg/kg of body weight, showed 80 and 100% survival, respectively. Structural and functional characterization of these peptides revealed the basis of their biological activity and demonstrated that nontoxic piscidin-1 analogs with significant antimicrobial and antiendotoxin activities can be designed by incorporating single alanine substitutions in the piscidin-1 heptad repeat.

The M. tuberculosis HAD phosphatase (Rv3042c) interacts with host proteins and is inhibited by Clofazimine.

Mycobacterium tuberculosis codes for a HAD-phosphatase, Rv3042c (MtSerB2), that has earlier been characterized as a metabolic enzyme. Here we demonstrate that MtSerB2 is secreted into the cytosol of infected macrophages and is found in bronchoalveolar lavage samples of tuberculosis patients. MtSerB2 induces significant cytoskeleton rearrangements through cofilin activation and affects the expression of genes that regulate actin dynamics. It specifically interacts with HSP90, HSP70 and HSP27 that block apoptotic pathways and not with other HSPs. It actively dephosphorylates MAPK-p38 and NF-kappa B p65 that play crucial roles in inflammatory and immune responses. This in turn leads to down-regulation of Interleukin 8, a chemotactic and inflammatory cytokine. Finally, during evaluation of inhibitors against MtSerB2 we found that Clofazimine, a drug being evaluated for XDR and MDR tuberculosis, inhibits MtSerB2 phosphatase activity and reverses the above effects and interactions with host proteins. Overall, the study identifies that MtSerB2 has new functions that might help the pathogen to evade the host's immune response.

Identification of gallic acid based glycoconjugates as a novel tubulin polymerization inhibitors.

A novel class of gallic acid based glycoconjugates were designed and synthesized as potential anticancer agents. Among all the compounds screened, compound 2a showed potent anticancer activity against breast cancer cells. The latter resulted in tubulin polymerization inhibition and induced G2/M cell cycle arrest, generation of reactive oxygen species, mitochondrial depolarization and subsequent apoptosis in breast cancer cells. In addition, ultraviolet-visible spectroscopy and fluorescence quenching studies of the compound with tubulin confirmed direct interaction of compounds with tubulin. Molecular modeling studies revealed that it binds at the colchicine binding site in tubulin. Further, 2a also exhibited potent in vivo anticancer activity in LA-7 syngeneic rat mammary tumor model. Current data projects its strong candidature to be developed as anticancer agent.

Rv3868 (EccA1), an essential component of the Mycobacterium tuberculosis ESX-1 secretion system, is thermostable.

Rv3868 (EccA1) is an essential CbxX/CfqX-family ATPase of the Mycobacterium tuberculosis ESX-1 secretion system. Previously, we demonstrated that Rv3868 is composed of two domains; a regulatory N-terminal domain (NT-Rv3868) and an ATP binding C-terminal domain (CT-Rv3868). In the present report, chemical denaturation studies show that electrostatic interactions stabilize the Rv3868. Interestingly, Rv3868 has notable heat stability and retains about 50% of ATPase activity even at 60°C. The C-terminal domain was found to be important for the heat stability as demonstrated by both enzymatic activity assays and thermal denaturation experiments. Furthermore a structure-sequence analysis based on the content of charged and aliphatic amino acids rationalizes the higher propensity of Rv3868 for thermophilic characteristics.

Mechanism of inhibition of the ATPase domain of human topoisomerase IIα by 1,4-benzoquinone, 1,2-naphthoquinone, 1,4-naphthoquinone, and 9,10-phenanthroquinone.

The inhibition of human topoisomerase IIα (Hu-TopoIIα), a major enzyme involved in maintaining DNA topology, repair, and chromosome condensation/decondensation results in loss of genomic integrity. In the present study, the inhibition of ATPase domain of Hu-TopoIIα as a possible mechanism of genotoxicity of 1,4-benzoquinone (BQ), hydroquinone (HQ), naphthoquinone (1,2-NQ and 1,4-NQ), and 9,10-phenanthroquinone (9,10-PQ) was investigated. In silico modeling predicted that 1,4-BQ, 1,2-NQ, 1,4-NQ, and 9,10-PQ could interact with Ser-148, Ser-149, Asn-150, and Asn-91 residues of the ATPase domain of Hu-TopoIIα. Biochemical inhibition assays with the purified ATPase domain of Hu-TopoIIα revealed that 1,4-BQ is the most potent inhibitor followed by 1,4-NQ > 1,2-NQ > 9,10-PQ > HQ. Ligand-binding studies using isothermal titration calorimetry revealed that 1,4-BQ, HQ, 1,4-NQ, 1,2-NQ, and 9,10-PQ enter into four sequentially binding site models inside the domain. 1,4-BQ exhibited the strongest binding, followed by 1,4-NQ > 1,2-NQ > 9,10-PQ > HQ, as revealed by their average K(d) values. The cellular fate of such inhibition was further evidenced by an increase in the number of Hu-TopoIIα-DNA cleavage complexes in the human lung epithelial cells (BEAS-2B) using trapped in agarose DNA immunostaining (TARDIS) assay, which utilizes antibody specific for Hu-TopoIIα. Furthermore, the increase in γ-H2A.X levels quantitated by flow cytometry and visualized by immunofluorescence microscopy illustrated that accumulation of DNA double-strand breaks inside the cells can be attributed to the inhibition of Hu-TopoIIα. These findings collectively suggest that 1,4-BQ, 1,2-NQ, 1,4-NQ, and 9,10-PQ inhibit the ATPase domain and potentially result in Hu-TopoIIα-mediated clastogenic and leukemogenic events.

Medicarpin, a legume phytoalexin, stimulates osteoblast differentiation and promotes peak bone mass achievement in rats: evidence for estrogen receptor ß-mediated osteogenic action of medicarpin.

Dietary isoflavones including genistein and daidzein have been shown to have favorable bone conserving effects during estrogen deficiency in experimental animals and humans. We have evaluated osteogenic effect of medicarpin (Med); a phytoalexin that is structurally related to isoflavones and is found in dietary legumes. Med stimulated osteoblast differentiation and mineralization at as low as 10⁻¹° M. Studies with signal transduction inhibitors demonstrated involvement of a p38 mitogen activated protein kinase-ER-bone morphogenic protein-2 pathway in mediating Med action in osteoblasts. Co-activator interaction studies demonstrated that Med acted as an estrogen receptor (ER) agonist; however, in contrast to 17ß-estradiol, Med had no uterine estrogenicity and blocked proliferation of MCF-7 cells. Med increased protein levels of ERß in osteoblasts. Selective knockdown of ERα and ERß in osteoblasts established that osteogenic action of Med is ERß-dependent. Female Sprague-Dawley (weaning) rats were administered Med at 1.0- and 10.0 mg.kg⁻¹ doses by gavage for 30 days along with vehicle control. Med treatment resulted in increased formation of osteoporgenitor cells in the bone marrow and osteoid formation (mineralization surface, mineral apposition/bone formation rates) compared with vehicle group. In addition, Med increased cortical thickness and bone biomechanical strength. In pharmacokinetic studies, Med exhibited oral bioavailability of 22.34% and did not produce equol. Together, our results demonstrate Med stimulates osteoblast differentiation likely via ERß, promotes achievement of peak bone mass, and is devoid of uterine estrogenicity. In addition, given its excellent oral bioavailability, Med can be potential osteogenic agent.

Bile acid receptor agonist GW4064 regulates PPARγ coactivator-1α expression through estrogen receptor-related receptor α.

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is induced in energy-starved conditions and is a key regulator of energy homeostasis. This makes PGC-1α an attractive therapeutic target for metabolic syndrome and diabetes. In our effort to identify new regulators of PGC-1α expression, we found that GW4064, a widely used synthetic agonist for the nuclear bile acid receptor [farnesoid X receptor (FXR)] strongly enhances PGC-1α promoter reporter activity, mRNA, and protein expression. This induction in PGC-1α concomitantly enhances mitochondrial mass and expression of several PGC-1α target genes involved in mitochondrial function. Using FXR-rich or FXR-nonexpressing cell lines and tissues, we found that this effect of GW4064 is not mediated directly by FXR but occurs via activation of estrogen receptor-related receptor α (ERRα). Cell-based, biochemical and biophysical assays indicate GW4064 as an agonist of ERR proteins. Interestingly, FXR disruption alters GW4064 induction of PGC-1α mRNA in a tissue-dependent manner. Using FXR-null [FXR knockout (FXRKO)] mice, we determined that GW4064 induction of PGC-1α expression is not affected in oxidative soleus muscles of FXRKO mice but is compromised in the FXRKO liver. Mechanistic studies to explain these differences revealed that FXR physically interacts with ERR and protects them from repression by the atypical corepressor, small heterodimer partner in liver. Together, this interplay between ERRα-FXR-PGC-1α and small heterodimer partner offers new insights into the biological functions of ERRα and FXR, thus providing a knowledge base for therapeutics in energy balance-related pathophysiology.

Exploring the positional importance of aromatic residues and lysine in the interactions of peptides with the Plasmodium falciparum Hsp70-1.

Plasmodium falciparum harbors an essential relict plastid called the apicoplast that is involved in several important biosynthetic processes. Over 500 nuclear encoded proteins are imported into the organelle that is now recognized as an important therapeutic target. These proteins contain an N-terminal transit peptide sequence essential for apicoplast targeting during which the P. falciparum Hsp70-1 plays an important role. In the present study, we have focused on the in vitro interactions of PfHsp70-1 with synthetic peptides endowed with transit peptide like features. The peptides exhibit higher affinity for PfHsp70-1 in the presence of ADP compared to ATP. The results highlight the positional importance of selected residues in the designed peptides for affinity. They suggest that better peptide affinity for the protein requires a Lys at second position, retention of aromatic residue at the last position, and absence of acidic residues at any position in the transit peptides. Overall, the present work is the first in vitro fluorescence-based study of PfHsp70-1 with peptides possessing transit peptide-like features.

Interactions of the M. tuberculosis UsfX with the cognate sigma factor SigF and the anti-anti sigma factor RsfA.

M. tuberculosis employs an exquisite cascade consisting of the cognate anti-sigma factor UsfX and anti-anti sigma factors RsfA and RsfB to regulate the functions of the alternate sigma factor SigF. We have purified these proteins to characterize their molecular properties and interactions with UsfX. UsfX forms a stable complex with SigF that could be purified only after co-expressing the proteins in E.coli. Formation of the complex is nucleotide independent and apparently requires unknown in vivo factors. Fluorescence spectroscopy experiments suggest that the nucleotide binding sites of UsfX are distal to the protein-protein interaction interface. RsfA is a novel anti-anti sigma factor whose binding to UsfX is triggered by the reduction of an intrachain disulphide bond between Cys73-Cys109. The reduction is accompanied by an increase in the hydrodynamic radius of the protein. The UsfX-RsfA complex exhibits a novel stoichiometry of 2:1 compared to the 2:2 stoichiometry reported for other anti-anti-sigma factors. The role of the disulphide bond in complex formation was explored using molecular dynamics simulations. These studies support specific conformational changes that occur upon reduction of the Cys73-Cys109 bond of RsfA. This leads to a rearrangement that increases the interactions of a conserved His107 of UsfX with Cys109 of RsfA.

Characterization of Rv3868, an essential hypothetical protein of the ESX-1 secretion system in Mycobacterium tuberculosis.

Rv3868, a conserved hypothetical protein of the ESAT-6 secretion system of Mycobacterium tuberculosis, is essential for the secretion of at least four virulence factors. Each protein chain is approximately 63 kDa and assembles into a hexamer. Limited proteolysis demonstrates that it consists of two domains joined by a linker. The N-terminal domain is a compact, helical domain of approximately 30 kDa and apparently functions to regulate the ATPase activity of the C-terminal domain and the oligomerization. The nucleotide binding site is situated in the C-terminal domain, which exhibits ATP-dependent self-association. It is also the oligomerization domain. Dynamic fluorescence quenching studies demonstrate that the domain is proximal to the C terminus in the apoprotein and exhibits a specific movement upon ATP binding. In silico modeling of the domains suggests that Arg-429 of a neighboring subunit forms a part of the binding site upon oligomerization. Mutational analysis of binding site residues demonstrates that the Arg-429 functions as the important "sensor arginine" in AAA-ATPases. Protein NMR experiments involving CFP-10 and activity assays rule out a general chaperone-like function for Rv3868. On the other hand, ATP-dependent "open-close" movements of the individual domains apparently enable it to interact and transfer energy to co-proteins in the ESX-1 pathway.