Plasmodium falciparum ZIP1 Is a Zinc-Selective Transporter with Stage-Dependent Targeting to the Apicoplast and Plasma Membrane in Erythrocytic Parasites.

Replication of the malarial parasite in human erythrocytes requires massive zinc fluxes, necessitating the action of zinc transporters across the parasite plasma and organellar membranes. Although genetic knockout studies have been conducted on a few "orphan" zinc transporters in Plasmodium spp., none of them have been functionally characterized. We used the recombinant Plasmodium falciparum Zrt-/Irt-like protein (PfZIP1) and specific antibodies generated against it to explore the subcellular localization, function, metal-ion selectivity, and response to cellular zinc levels. PfZIP1 expression was enhanced upon the depletion of cytosolic Zn2+. The protein transitioned from the processed to unprocessed form through blood stages, localizing to the apicoplast in trophozoites and to the parasite plasma membrane in schizonts and gametocytes, indicating stage-specific functional role. The PfZIP1 dimer mediated Zn2+ influx in proteoliposomes. It exhibited preferential binding to Zn2+ compared to Fe2+, with the selectivity for zinc being driven by a C-terminal histidine-rich region conserved only in primate-infecting Plasmodium species.

Mycobacterium tuberculosis Rv2324 is a multifunctional feast/famine regulatory protein involved in growth, DNA replication and damage control.

Feast/famine regulatory proteins (FFRPs) are multifunctional regulators. We show that Mtb Rv2324 is important for growth, survival, and countering DNA damage in Mycobacterium tuberculosis (Mtb). DNA-relaxation activity against linear and supercoiled substrates suggest its involvement in transcription activation, while its high affinity for recombination, replication and repair substrates suggest a role there too. Small-Angle-X-ray scattering supports the adoption of an 'open' quaternary association in response to amino-acid binding. Size-exclusion-chromatography and glutaraldehyde cross-linking identify the adoption of diverse oligomers modulated by amino-acid binding, and DNA interactions. We tested G52A, G101T and D104A mutants which correspond to highly conserved residues, distal to the DNA-binding site, and are important for amino acids binding. G101T exhibits increased DNA affinity, while G52A and D104A exhibit weak DNA-binding thereby suggesting that they mediate effector-binding, and DNA binding activities. Gain and loss-of-function studies show that Rv2324 overexpression promotes growth-rate, while its knock-down leads to retarded growth. Rv2324 down-regulation lowers Mtb survival inside resting and IFN-ϒ-activated macrophages. Rv2324 protects the pathogen from DNA damage, as evidenced by the reduction in the knockdown strain's survival following treatment with H2O2 and UV light. Overall, we show that Rv2324 plays a crucial role in regulating survival and growth of Mtb.

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.

Setomimycin as a potential molecule for COVID­19 target: in silico approach and in vitro validation.

COVID-19 pandemic caused by the SARS-CoV-2 virus has led to a worldwide crisis. In view of emerging variants time to time, there is a pressing need of effective COVID-19 therapeutics. Setomimycin, a rare tetrahydroanthracene antibiotic, remained unexplored for its therapeutic uses. Herein, we report our investigations on the potential of setomimycin as COVID-19 therapeutic. Pure setomimycin was isolated from Streptomyces sp. strain RA-WS2 from NW Himalayan region followed by establishing in silico as well as in vitro anti-SARS-CoV-2 property of the compound against SARS-CoV-2 main protease (Mpro). It was found that the compound targets Mpro enzyme with an IC50 value of 12.02 ± 0.046 µM. The molecular docking study revealed that the compound targets Glu166 residue of Mpro enzyme, hence preventing dimerization of SARS-CoV-2 Mpro monomer. Additionally, the compound also exhibited anti-inflammatory and anti-oxidant property, suggesting that setomimycin may be a viable option for application against COVID-19 infections.

Regulation of futile ligation during early steps of BER in M. tuberculosis is carried out by a ß-clamp-XthA-LigA tri-component complex.

The Class-II AP-endonuclease (XthA) is a mycobacterial DNA base excision repair (BER) pathway enzyme that functions in the initial steps. It acts on DNA substrates that contain abasic sites to create nicks with 3'-hydroxyl (OH) and 5'-deoxyribose phosphate (5'-dRP) moieties. The NAD+-dependent DNA ligase (LigA) is the terminal player in mycobacterial BER and seals such nicks efficiently. Here, we demonstrate that the Mtbß-clamp-MtbXthA complex that exists in the initial steps of BER engages with MtbLigA to form a novel tri-component BER complex. Size exclusion chromatography (SEC) experiments analysis show that the three proteins interact with equimolar stoichiometry. Small angle X-ray scattering (SAXS) analysis and associated studies reveal that the apo tri-component BER-complex adopts an extended conformation where MtbXthA is sandwiched between the Mtbß-clamp and MtbLigA. The studies support that in the apo-complex MtbXthA binds subsite-I of Mtbß-clamp through 239QLRFPKK245 motif and to MtbLigA by 104DGQPSWSGKP113 motif simultaneously. However, the complex adopts a less-extended conformation in the presence of substrate DNA, where MtbXthA interactions switch from predominantly subsite-I to subsite-II of the Mtbß-clamp. Overall, the novel tri-component complex prevents futile ligation activity of MtbLigA on the product of MtbXthA and ensures forward progression of the pathway and productive mycobacterial BER interactions.

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.

Mycobacterium tuberculosis Endonuclease VIII 2 (Nei2) forms a prereplicative BER complex with DnaN: Identification, characterization, and disruption of complex formation.

Mycobacterium tuberculosis Nei2 (Rv3297) is a BER glycosylase that removes oxidized base lesions from ssDNA and replication fork-mimicking substrates. We show that Endonuclease VIII 2 (Nei2) forms a BER complex with the ß-clamp (DnaN, Rv0002) with a KD of 170 nM. The Nei2-ß-clamp interactions enhance Nei2's activities up to several folds. SEC analysis shows that one molecule of Nei2 binds to a single ß-clamp dimer. Nei2 interacts with subsites I and II of the ß-clamp via a noncanonical 223 QGCRRCGTLIAY239 Clamp Interacting Protein (CIP) motif in the C-terminal zinc-finger domain, which was previously shown by us to be dispensable for intrinsic Nei2 activity. The 12-mer peptide alone exhibited a KD of 10.28 nM, suggesting that the motif is a key mediator of Nei2-ß-clamp interactions. Finally, we identified inhibitors of Nei2-ß-clamp interactions using rational methods, in vitro disruption, and SPR assays after querying a database of natural products. We found that Tubulosine, Fumitremorgin C, Toyocamycin, and Aleuritic acid exhibit IC50 values of 94.47, 83.49, 109.7, and 71.49 µM, respectively. They act by disrupting Nei2-ß-clamp interactions and do not affect intrinsic Nei2 activity. Among other things, the present study gives insights into the role of Nei2 in bacterial prereplicative BER.

Phase III, Randomized, Double-blind, Placebo controlled trial of Efficacy, Safety and Tolerability of Antiviral drug Umifenovir vs Standard care of therapy in non-severe COVID-19 patients.

OBJECTIVE: To test efficacy, safety and tolerability of Umifenovir in non-severe COVID-19 adult patients. METHODS: We carried out randomized, double-blind, placebo-controlled, multicenter, phase III trials involving adult (18-75 years), non-severe COVID19 patients, randomized 1:1 on placebo or Umifenovir (800 mg BID, maximum 14 days) respectively along with standard-of-care. The primary endpoint for Asymptotic-mild patients was time to nasopharyngeal swab RT-PCR test negativity. For Moderate patients, the average change in the ordinal scale from the baseline scores on the eight-point WHO ordinal scale was assessed. RESULTS: 132 patients were recruited between 3rd October to 28th April 2021, of which 9 discontinued due to various reasons. In Mild-asymptomatic patients (n=82), we found that 73% patients in the Umifenovir arm were RT-PCR negative, while 40% patients in the placebo arm were negative (P=0.004) on day 5. However, in the moderate group (n=41), the WHO scores for the Umifenovir arm was not statistically significant (P=0.125 on day 3), while it was statistically significant in the Mild-asymptomatic group (P=0.019 on day 5). CONCLUSION: Umifenovir meets the primary and secondary endpoint criteria and exhibits statistically significant efficacy for Mild-asymptomatic patients. It is efficacious, safe and well-tolerated at the tested dosage of 800mg BID, maximum 14 days.

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.

Salt bridges at the subdomain interfaces of the adenylation domain and active-site residues of Mycobacterium tuberculosis NAD+-dependent DNA ligase A (MtbLigA) are important for the initial steps of nick-sealing activity.

NAD+-dependent DNA ligase (LigA) is the principal bacterial ligase and catalyses a multistep ligation reaction. The adenylation (AdD) domain at the N-terminus consists of subdomains 1a and 1b, where subdomain 1a is unique to LigA. Small-angle X-ray scattering and X-ray diffraction studies were used to probe changes in the relative spatial dispositions of the two subdomains during the adenylation reaction. Structural analyses of the inter-subdomain interactions of the AdD domain suggest that salt bridges formed by Glu22, Glu26 and Glu87 of subdomain 1a with Arg144, Arg315 and His240 of subdomain 1b play an important role in stabilizing the intermediate conformations of the two subdomains. E22A, E26A and E87A mutations reduce the in vitro activity by 89%, 64% and 39%, respectively, on a nicked DNA substrate, while they show no activity loss on a pre-adenylated DNA substrate, thus suggesting that the salt bridges are important in the initial steps of the ligation reaction. Furthermore, the E22A, E26A and E87A mutants exhibited extremely delayed growth in complementation assays involving the Escherichia coli GR501 strain, which harbours its own temperature-sensitive LigA. The H236A and H236Y mutants, which involve the residue that stacks against the adenine moiety of AMP, severely impact the activity and the ability to complement the growth-defective E. coli GR501 strain. Analysis of the K123A and K123R mutations in the active site rationalizes their total loss of activity and inability to rescue the growth-defective E. coli GR501 strain.

[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.

Structure based identification of first-in-class fragment inhibitors that target the NMN pocket of M. tuberculosis NAD+-dependent DNA ligase A.

NAD+-dependent DNA ligase (LigA) is the essential replicative ligase in bacteria and differs from ATP-dependent counterparts like the human DNA ligase I (HligI) in several aspects. LigA uses NAD+ as the co-factor while the latter uses ATP. Further, the LigA carries out enzymatic activity with a single divalent metal ion in the active site while ATP-dependent ligases use two metal ions. Instead of the second metal ion, LigA have a unique NMN binding subdomain that facilitates the orientation of the ß-phosphate and NMN leaving group. LigA are therefore attractive targets for new anti-bacterial therapeutic development. Others and our group have earlier identified several LigA inhibitors that mainly bind to AMP binding site of LigA. However, no inhibitor is known to bind to the unique NMN binding subdomain. We initiated a fragment inhibitor discovery campaign against the M. tuberculosis LigA based on our co-crystal structure of adenylation domain with AMP and NMN. The study identified two fragments, 4-(4-fluorophenyl)-4,5,6,7-tetrahydro-3H imidazo[4,5-c] pyridine and N-(4-methylbenzyl)-1H-pyrrole-2-carboxamide, that bind to the NMN site. The fragments inhibit LigA with IC50 of 16.9 and 28.7 µM respectively and exhibit MIC of ~20 and 60 µg/ml against a temperature sensitive E. coli GR501 ligAts strain, rescued by MtbLigA. Co-crystal structures of the fragments with the adenylation domain of LigA show that they mimic the interactions of NMN. Overall, our results suggest that the NMN binding-site is a druggable target site for developing anti-LigA therapeutic strategies.

Structural-functional diversity of malaria parasite's PfHSP70-1 and PfHSP40 chaperone pair gives an edge over human orthologs in chaperone-assisted protein folding.

Plasmodium falciparum, the human malaria parasite harbors a metastable proteome which is vulnerable to proteotoxic stress conditions encountered during its lifecycle. How parasite's chaperone machinery is able to maintain its aggregation-prone proteome in functional state, is poorly understood. As HSP70-40 system forms the central hub in cellular proteostasis, we investigated the protein folding capacity of PfHSP70-1 and PfHSP40 chaperone pair and compared it with human orthologs (HSPA1A and DNAJA1). Despite the structural similarity, we observed that parasite chaperones and their human orthologs exhibit striking differences in conformational dynamics. Comprehensive biochemical investigations revealed that PfHSP70-1 and PfHSP40 chaperone pair has better protein folding, aggregation inhibition, oligomer remodeling and disaggregase activities than their human orthologs. Chaperone-swapping experiments suggest that PfHSP40 can also efficiently cooperate with human HSP70 to facilitate the folding of client-substrate. SPR-derived kinetic parameters reveal that PfHSP40 has higher binding affinity towards unfolded substrate than DNAJA1. Interestingly, the observed slow dissociation rate of PfHSP40-substrate interaction allows PfHSP40 to maintain the substrate in folding-competent state to minimize its misfolding. Structural investigation through small angle x-ray scattering gave insights into the conformational architecture of PfHSP70-1 (monomer), PfHSP40 (dimer) and their complex. Overall, our data suggest that the parasite has evolved functionally diverged and efficient chaperone machinery which allows the human malaria parasite to survive in hostile conditions. The distinct allosteric landscapes and interaction kinetics of plasmodial chaperones open avenues for the exploration of small-molecule based antimalarial interventions.

M. tuberculosis class II apurinic/ apyrimidinic-endonuclease/3'-5' exonuclease (XthA) engages with NAD+-dependent DNA ligase A (LigA) to counter futile cleavage and ligation cycles in base excision repair.

Class-II AP-endonuclease (XthA) and NAD+-dependent DNA ligase (LigA) are involved in initial and terminal stages of bacterial DNA base excision repair (BER), respectively. XthA acts on abasic sites of damaged DNA to create nicks with 3'OH and 5'-deoxyribose phosphate (5'-dRP) moieties. Co-immunoprecipitation using mycobacterial cell-lysate, identified MtbLigA-MtbXthA complex formation. Pull-down experiments using purified wild-type, and domain-deleted MtbLigA mutants show that LigA-XthA interactions are mediated by the BRCT-domain of LigA. Small-Angle-X-ray scattering, 15N/1H-HSQC chemical shift perturbation experiments and mutational analysis identified the BRCT-domain region that interacts with a novel 104DGQPSWSGKP113 motif on XthA for complex-formation. Isothermal-titration calorimetry experiments show that a synthetic peptide with this sequence interacts with MtbLigA and disrupts XthA-LigA interactions. In vitro assays involving DNA substrate and product analogs show that LigA can efficiently reseal 3'OH and 5'dRP DNA termini created by XthA at abasic sites. Assays and SAXS experiments performed in the presence and absence of DNA, show that XthA inhibits LigA by specifically engaging with the latter's BRCT-domain to prevent it from encircling substrate DNA. Overall, the study suggests a coordinating function for XthA whereby it engages initially with LigA to prevent the undesirable consequences of futile cleavage and ligation cycles that might derail bacterial BER.

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.