The heptameric structure of the flagellar regulatory protein FlrC is indispensable for ATPase activity and disassembled by cyclic-di-GMP
2020; Elsevier BV; Volume: 295; Issue: 50 Linguagem: Inglês
10.1074/jbc.ra120.014083
ISSN1083-351X
AutoresShrestha Chakraborty, M. Biswas, Sanjay Kumar Dey, Shubhangi Agarwal, Tulika Chakrabortty, Biplab Ghosh, J. Dasgupta,
Tópico(s)Bacterial biofilms and quorum sensing
ResumoThe bacterial enhancer-binding protein (bEBP) FlrC, controls motility and colonization of Vibrio cholerae by regulating the transcription of class-III flagellar genes in σ54-dependent manner. However, the mechanism by which FlrC regulates transcription is not fully elucidated. Although, most bEBPs require nucleotides to stimulate the oligomerization necessary for function, our previous study showed that the central domain of FlrC (FlrCC) forms heptamer in a nucleotide-independent manner. Furthermore, heptameric FlrCC binds ATP in "cis-mediated" style without any contribution from sensor I motif 285REDXXYR291 of the trans protomer. This atypical ATP binding raises the question of whether heptamerization of FlrC is solely required for transcription regulation, or if it is also critical for ATPase activity. ATPase assays and size exclusion chromatography of the trans-variants FlrCC-Y290A and FlrCC-R291A showed destabilization of heptameric assembly with concomitant abrogation of ATPase activity. Crystal structures showed that in the cis-variant FlrCC-R349A drastic shift of Walker A encroached ATP-binding site, whereas the site remained occupied by ADP in FlrCC-Y290A. We postulated that FlrCC heptamerizes through concentration-dependent cooperativity for maximal ATPase activity and upon heptamerization, packing of trans-acting Tyr290 against cis-acting Arg349 compels Arg349 to maintain proper conformation of Walker A. Finally, a Trp quenching study revealed binding of cyclic-di-GMP with FlrCC. Excess cyclic-di-GMP repressed ATPase activity of FlrCC through destabilization of heptameric assembly, especially at low concentration of protein. Systematic phylogenetic analysis allowed us to propose similar regulatory mechanisms for FlrCs of several Vibrio species and a set of monotrichous Gram-negative bacteria. The bacterial enhancer-binding protein (bEBP) FlrC, controls motility and colonization of Vibrio cholerae by regulating the transcription of class-III flagellar genes in σ54-dependent manner. However, the mechanism by which FlrC regulates transcription is not fully elucidated. Although, most bEBPs require nucleotides to stimulate the oligomerization necessary for function, our previous study showed that the central domain of FlrC (FlrCC) forms heptamer in a nucleotide-independent manner. Furthermore, heptameric FlrCC binds ATP in "cis-mediated" style without any contribution from sensor I motif 285REDXXYR291 of the trans protomer. This atypical ATP binding raises the question of whether heptamerization of FlrC is solely required for transcription regulation, or if it is also critical for ATPase activity. ATPase assays and size exclusion chromatography of the trans-variants FlrCC-Y290A and FlrCC-R291A showed destabilization of heptameric assembly with concomitant abrogation of ATPase activity. Crystal structures showed that in the cis-variant FlrCC-R349A drastic shift of Walker A encroached ATP-binding site, whereas the site remained occupied by ADP in FlrCC-Y290A. We postulated that FlrCC heptamerizes through concentration-dependent cooperativity for maximal ATPase activity and upon heptamerization, packing of trans-acting Tyr290 against cis-acting Arg349 compels Arg349 to maintain proper conformation of Walker A. Finally, a Trp quenching study revealed binding of cyclic-di-GMP with FlrCC. Excess cyclic-di-GMP repressed ATPase activity of FlrCC through destabilization of heptameric assembly, especially at low concentration of protein. Systematic phylogenetic analysis allowed us to propose similar regulatory mechanisms for FlrCs of several Vibrio species and a set of monotrichous Gram-negative bacteria. Vibrio cholerae, the facultative human pathogen that causes diarrheal disease cholera, is highly motile by means of a single, polar sheathed flagellum. V. cholerae enters inside the human host through ingestion of contaminated food or water, adheres to the apical surface of the intestinal epithelial cell, and expresses virulence factors (1Holmgren J. Svennerholm A.-M. Mechanisms of disease and immunity in cholera: a review.J. Infect. Dis. 1977; 136: S105-S11210.1093/infdis/136.Supplement.S105Crossref PubMed Google Scholar, 2Taylor R.K. Miller V.L. Furlong D.B. Mekalanos J.J. Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin.Proc. Natl. Acad. Sci. U.S.A. 1987; 84 (2883655): 2833-283710.1073/pnas.84.9.2833Crossref PubMed Google Scholar). Motility and colonization of V. cholerae are prerequisites of producing the virulence factors and immune-resistant biofilms which, in turn, are governed by flagellar synthesis (3Yildiz F.H. Visick K.L. Vibrio biofilms: so much the same yet so different.Trends Microbiol. 2009; 17 (19231189): 109-11810.1016/j.tim.2008.12.004Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar, 4Syed K.A. Beyhan S. Correa N. Queen J. Liu J. Peng F. Satchell K.J.F. Yildiz F. Klose K.E. The Vibrio cholerae flagellar regulatory hierarchy controls expression of virulence factors.J. Bacteriol. 2009; 191 (19717600): 6555-657010.1128/JB.00949-09Crossref PubMed Scopus (112) Google Scholar). Expression of the proteins required to synthesize the functional flagellum of V. cholerae is regulated by a four-tiered transcriptional hierarchy (4Syed K.A. Beyhan S. Correa N. Queen J. Liu J. Peng F. Satchell K.J.F. Yildiz F. Klose K.E. The Vibrio cholerae flagellar regulatory hierarchy controls expression of virulence factors.J. Bacteriol. 2009; 191 (19717600): 6555-657010.1128/JB.00949-09Crossref PubMed Scopus (112) Google Scholar, 5Prouty M.G. Correa N.E. Klose K.E. The novel σ54- and σ28-dependent flagellar gene transcription hierarchy of Vibrio cholerae.Mol. Microbiol. 2001; 39 (11260476): 1595-160910.1046/j.1365-2958.2001.02348.xCrossref PubMed Scopus (158) Google Scholar, 6Klose K.E. Mekalanos J.J. Distinct roles of an alternative sigma factor during both free-swimming and colonizing phases of the Vibrio cholerae pathogenic cycle.Mol. Microbiol. 1998; 28 (9632254): 501-52010.1046/j.1365-2958.1998.00809.xCrossref PubMed Scopus (128) Google Scholar, 7Echazarreta M.A. Klose K.E. Vibrio flagellar synthesis.Front. Cell. Infect. Microbiol. 2019; 9 (31119103): 13110.3389/fcimb.2019.00131Crossref PubMed Scopus (21) Google Scholar). The class-I gene product and bacterial enhancer-binding protein (bEBP), FlrA activates σ54-dependent transcription of the class-II genes flrBC, which encode another bEBP, FlrC and its cognate kinase FlrB (4Syed K.A. Beyhan S. Correa N. Queen J. Liu J. Peng F. Satchell K.J.F. Yildiz F. Klose K.E. The Vibrio cholerae flagellar regulatory hierarchy controls expression of virulence factors.J. Bacteriol. 2009; 191 (19717600): 6555-657010.1128/JB.00949-09Crossref PubMed Scopus (112) Google Scholar, 5Prouty M.G. Correa N.E. Klose K.E. The novel σ54- and σ28-dependent flagellar gene transcription hierarchy of Vibrio cholerae.Mol. Microbiol. 2001; 39 (11260476): 1595-160910.1046/j.1365-2958.2001.02348.xCrossref PubMed Scopus (158) Google Scholar). Transcription of class-III flagellar genes, which encode important flagellar components like basal body hook and the flagellin FlaA are regulated by FlrC (4Syed K.A. Beyhan S. Correa N. Queen J. Liu J. Peng F. Satchell K.J.F. Yildiz F. Klose K.E. The Vibrio cholerae flagellar regulatory hierarchy controls expression of virulence factors.J. Bacteriol. 2009; 191 (19717600): 6555-657010.1128/JB.00949-09Crossref PubMed Scopus (112) Google Scholar, 8Correa N.E. Klose K.E. Characterization of enhancer binding by the Vibrio cholerae flagellar regulatory protein FlrC.J. Bacteriol. 2005; 187 (15838043): 3158-317010.1128/JB.187.9.3158-3170.2005Crossref PubMed Scopus (22) Google Scholar). The anti-σ factor FlgM is secreted through the basal body-hook to allow σ28-dependent transcription of class-IV genes, which encode four additional flagellins and some of the motor components (4Syed K.A. Beyhan S. Correa N. Queen J. Liu J. Peng F. Satchell K.J.F. Yildiz F. Klose K.E. The Vibrio cholerae flagellar regulatory hierarchy controls expression of virulence factors.J. Bacteriol. 2009; 191 (19717600): 6555-657010.1128/JB.00949-09Crossref PubMed Scopus (112) Google Scholar, 6Klose K.E. Mekalanos J.J. Distinct roles of an alternative sigma factor during both free-swimming and colonizing phases of the Vibrio cholerae pathogenic cycle.Mol. Microbiol. 1998; 28 (9632254): 501-52010.1046/j.1365-2958.1998.00809.xCrossref PubMed Scopus (128) Google Scholar). Motility and biofilm formation of V. cholerae are further regulated by ubiquitous second messenger cyclic di-guanosine monophosphate (c-di-GMP) at the transcriptional level. Although the precise molecular mechanisms by which c-di-GMP affects motility in V. cholerae are less well understood, a high c-di-GMP level was found to inhibit the production and function of V. cholerae's single polar flagellum (9Conner J.G. Zamorano-Sánchez D. Park J.H. Sondermann H. Yildiz F.H. The ins and outs of cyclic di-GMP signaling in Vibrio cholerae.Curr. Opin. Microbiol. 2017; 36 (28171809): 20-2910.1016/j.mib.2017.01.002Crossref PubMed Scopus (51) Google Scholar, 10Beyhan S. Tischler A.D. Camilli A. Yildiz F.H. Transcriptome and phenotypic responses of Vibrio cholerae to increased cyclic di-GMP level.J. Bacteriol. 2006; 188 (16672614): 3600-361310.1128/JB.188.10.3600-3613.2006Crossref PubMed Scopus (138) Google Scholar, 11Srivastava D. Hsieh M.L. Khataokar A. Neiditch M.B. Waters C.M. Cyclic di-GMP inhibits Vibrio cholerae motility by repressing induction of transcription and inducing extracellular polysaccharide production.Mol. Microbiol. 2013; 90 (24134710): 1262-127610.1111/mmi.12432Crossref PubMed Scopus (81) Google Scholar, 12Shikuma N.J. Fong J.C.N. Yildiz F.H. Cellular levels and binding of c-di-GMP control subcellular localization and activity of the Vibrio cholerae transcriptional regulator VpsT.PLoS Pathog. 2012; 8 (22654664)e100271910.1371/journal.ppat.1002719Crossref PubMed Scopus (42) Google Scholar). Available evidences suggest that V. cholerae responds to an elevated level of c-di-GMP by increasing the transcription of the vps, eps, and msh genes and decreasing that of flagellar genes (12Shikuma N.J. Fong J.C.N. Yildiz F.H. Cellular levels and binding of c-di-GMP control subcellular localization and activity of the Vibrio cholerae transcriptional regulator VpsT.PLoS Pathog. 2012; 8 (22654664)e100271910.1371/journal.ppat.1002719Crossref PubMed Scopus (42) Google Scholar). This clearly indicates a distinctive mode of interactions of c-di-GMP with the bEBPs involved in exopolysaccaride production and flagellar synthesis. FlrC of V. cholerae is made of N-terminal response regulator (R) domain, central AAA+ ATPase domain, and C-terminal DNA-binding domain. Phosphorylation occurs in the R domain of FlrC by cognate kinase FlrB (8Correa N.E. Klose K.E. Characterization of enhancer binding by the Vibrio cholerae flagellar regulatory protein FlrC.J. Bacteriol. 2005; 187 (15838043): 3158-317010.1128/JB.187.9.3158-3170.2005Crossref PubMed Scopus (22) Google Scholar). Previous studies by Klose and colleagues (6Klose K.E. Mekalanos J.J. Distinct roles of an alternative sigma factor during both free-swimming and colonizing phases of the Vibrio cholerae pathogenic cycle.Mol. Microbiol. 1998; 28 (9632254): 501-52010.1046/j.1365-2958.1998.00809.xCrossref PubMed Scopus (128) Google Scholar) delineated that a V. cholerae strain containing a deletion of flrC is nonmotile and also displays a modest colonization defect, whereas a strain expressing a hyperactive form of FlrC has an altered cell morphology (6Klose K.E. Mekalanos J.J. Distinct roles of an alternative sigma factor during both free-swimming and colonizing phases of the Vibrio cholerae pathogenic cycle.Mol. Microbiol. 1998; 28 (9632254): 501-52010.1046/j.1365-2958.1998.00809.xCrossref PubMed Scopus (128) Google Scholar, 8Correa N.E. Klose K.E. Characterization of enhancer binding by the Vibrio cholerae flagellar regulatory protein FlrC.J. Bacteriol. 2005; 187 (15838043): 3158-317010.1128/JB.187.9.3158-3170.2005Crossref PubMed Scopus (22) Google Scholar, 13Correa N.E. Lauriano C.M. McGee R. Klose K.E. Phosphorylation of the flagellar regulatory protein FlrC is necessary for Vibrio cholerae motility and enhanced colonization.Mol. Microbiol. 2000; 35 (10692152): 743-75510.1046/j.1365-2958.2000.01745.xCrossref PubMed Scopus (81) Google Scholar). They further showed that both inactive and constitutively active mutants of FlrC cause more severe colonization defects than a strain lacking FlrC entirely, which implies that both unphosphorylated and phosphorylated forms of FlrC are required for the colonization, and locking FlrC into either an active or an inactive state would send incorrect stimuli into this stepwise colonization process (6Klose K.E. Mekalanos J.J. Distinct roles of an alternative sigma factor during both free-swimming and colonizing phases of the Vibrio cholerae pathogenic cycle.Mol. Microbiol. 1998; 28 (9632254): 501-52010.1046/j.1365-2958.1998.00809.xCrossref PubMed Scopus (128) Google Scholar, 8Correa N.E. Klose K.E. Characterization of enhancer binding by the Vibrio cholerae flagellar regulatory protein FlrC.J. Bacteriol. 2005; 187 (15838043): 3158-317010.1128/JB.187.9.3158-3170.2005Crossref PubMed Scopus (22) Google Scholar, 13Correa N.E. Lauriano C.M. McGee R. Klose K.E. Phosphorylation of the flagellar regulatory protein FlrC is necessary for Vibrio cholerae motility and enhanced colonization.Mol. Microbiol. 2000; 35 (10692152): 743-75510.1046/j.1365-2958.2000.01745.xCrossref PubMed Scopus (81) Google Scholar). Although domain organization portrays FlrC as NtrC-type bEBP, this is atypical in many respects. The σ54-dependent activators usually bind to the enhancer elements located upstream of the RNAP-σ54 binding site and contact this complex at the promoter by DNA looping mechanism (14Xu H. Hoover T.R. Transcriptional regulation at a distance in bacteria.Curr. Opin. Microbiol. 2001; 4: 138-14410.1016/S1369-5274(00)00179-XCrossref PubMed Scopus (94) Google Scholar). In contrast, FlrC binds enhancer elements located downstream of the σ54-binding and transcriptional start sites of the flaA and flgK promoters (8Correa N.E. Klose K.E. Characterization of enhancer binding by the Vibrio cholerae flagellar regulatory protein FlrC.J. Bacteriol. 2005; 187 (15838043): 3158-317010.1128/JB.187.9.3158-3170.2005Crossref PubMed Scopus (22) Google Scholar). The same feature was observed in FleQ of Pseudomonas aeruginosa for flhA, fliE, and fliL genes where the authors argued for direct interaction between RNAP-σ54 and the activator without DNA looping (15Jyot J. Dasgupta N. Ramphal R. FleQ, the major flagellar gene regulator in Pseudomonas aeruginosa, binds to enhancer sites located either upstream or atypically downstream of the RpoN binding site.J. Bacteriol. 2002; 184 (12218010): 5251-526010.1128/jb.184.19.5251-5260.2002Crossref PubMed Scopus (78) Google Scholar). However, the mechanistic details of binding RNAP-σ54 at the promoter is yet to be deciphered for FleQ or FlrC. Usually, oligomerization of bEBPs takes place through the AAA+ domain. Despite that, bEBPs display remarkable diversity in terms of oligomerization, ATP binding, and hydrolysis, mediated by different motifs within the AAA+ domain, which possibly play roles in directing the proteins toward specific functions (16Bush M. Dixon R. The role of bacterial enhancer binding proteins as specialized activators of σ54-dependent transcription.Microbiol. Mol. Biol. Rev. 2012; 76 (22933558): 497-52910.1128/MMBR.00006-12Crossref PubMed Scopus (178) Google Scholar, 17Chen B. Sysoeva T.A. Chowdhury S. Guo L. De Carlo S. Hanson J.A. Yang H. Nixon B.T. Engagement of arginine finger to ATP triggers large conformational changes in NtrC1 AAA+ ATPase for remodeling bacterial RNA polymerase.Structure. 2010; 18 (21070941): 1420-143010.1016/j.str.2010.08.018Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 18Sysoeva T.A. Chowdhury S. Guo L. Tracy Nixon B. Nucleotide-induced asymmetry within ATPase activator ring drives σ54–RNAP interaction and ATP hydrolysis.Genes Dev. 2013; 27 (24240239): 2500-251110.1101/gad.229385.113Crossref PubMed Scopus (30) Google Scholar, 19Joly N. Zhang N. Buck M. ATPase site architecture is required for self-assembly and remodeling activity of a hexameric AAA+ transcriptional activator.Mol. Cell. 2012; 47 (22789710): 484-49010.1016/j.molcel.2012.06.012Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 20Dey S. Biswas M. Sen U. Dasgupta J. Unique ATPase site architecture triggers cis-mediated synchronized ATP binding in heptameric AAA+-ATPase domain of flagellar regulatory protein FlrC.J. Biol. Chem. 2015; 290 (25688103): 8734-874710.1074/jbc.M114.611434Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar, 21Joly N. Buck M. Engineered interfaces of an AAA+-ATPase reveal a new nucleotide-dependent coordination mechanism.J. Biol. Chem. 2010; 285 (20197281): 15178-1518610.1074/jbc.M110.103150Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 22Rappas M. Schumacher J. Niwa H. Buck M. Zhang X. Structural basis of the nucleotide driven conformational changes in the AAA+ domain of transcription activator PspF.J. Mol. Biol. 2006; 357: 481-49210.1016/j.jmb.2005.12.052Crossref PubMed Scopus (69) Google Scholar). Generally, in the NtrC class of proteins, oligomerization is guided by Nt-dependent subunit remodeling from the inactive dimer to active hexa/heptamer (17Chen B. Sysoeva T.A. Chowdhury S. Guo L. De Carlo S. Hanson J.A. Yang H. Nixon B.T. Engagement of arginine finger to ATP triggers large conformational changes in NtrC1 AAA+ ATPase for remodeling bacterial RNA polymerase.Structure. 2010; 18 (21070941): 1420-143010.1016/j.str.2010.08.018Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 21Joly N. Buck M. Engineered interfaces of an AAA+-ATPase reveal a new nucleotide-dependent coordination mechanism.J. Biol. Chem. 2010; 285 (20197281): 15178-1518610.1074/jbc.M110.103150Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar, 22Rappas M. Schumacher J. Niwa H. Buck M. Zhang X. Structural basis of the nucleotide driven conformational changes in the AAA+ domain of transcription activator PspF.J. Mol. Biol. 2006; 357: 481-49210.1016/j.jmb.2005.12.052Crossref PubMed Scopus (69) Google Scholar). We have previously determined the crystal structure of the AAA+ domain of FlrC (FlrCC) in Nt-free (Fig. 1a) and AMP-PNP–bound (Fig. 1b) states (20Dey S. Biswas M. Sen U. Dasgupta J. Unique ATPase site architecture triggers cis-mediated synchronized ATP binding in heptameric AAA+-ATPase domain of flagellar regulatory protein FlrC.J. Biol. Chem. 2015; 290 (25688103): 8734-874710.1074/jbc.M114.611434Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). In both cases FlrCC was found in the heptameric state demonstrating that, unlike the NtrC class of bEBPs, FlrC spontaneously forms heptamer without Nt-dependent subunit remodeling (PDB codes 4QHS and 4QHT) (20Dey S. Biswas M. Sen U. Dasgupta J. Unique ATPase site architecture triggers cis-mediated synchronized ATP binding in heptameric AAA+-ATPase domain of flagellar regulatory protein FlrC.J. Biol. Chem. 2015; 290 (25688103): 8734-874710.1074/jbc.M114.611434Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). The major presence of the heptameric species of FlrCC was also established in solution by size exclusion chromatography and dynamic light scattering (20Dey S. Biswas M. Sen U. Dasgupta J. Unique ATPase site architecture triggers cis-mediated synchronized ATP binding in heptameric AAA+-ATPase domain of flagellar regulatory protein FlrC.J. Biol. Chem. 2015; 290 (25688103): 8734-874710.1074/jbc.M114.611434Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). Although asymmetric split-ring hexamer was observed for NtrC1 to contact the RNAP-σ54 complex at promoter DNA (18Sysoeva T.A. Chowdhury S. Guo L. Tracy Nixon B. Nucleotide-induced asymmetry within ATPase activator ring drives σ54–RNAP interaction and ATP hydrolysis.Genes Dev. 2013; 27 (24240239): 2500-251110.1101/gad.229385.113Crossref PubMed Scopus (30) Google Scholar), heptameric FlrCC with a much wider central pore was presumed to be adequate for the same purpose without any serious ring distortion (20Dey S. Biswas M. Sen U. Dasgupta J. Unique ATPase site architecture triggers cis-mediated synchronized ATP binding in heptameric AAA+-ATPase domain of flagellar regulatory protein FlrC.J. Biol. Chem. 2015; 290 (25688103): 8734-874710.1074/jbc.M114.611434Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). In the NtrC class of bEBPs, such as NtrC1 and PspF, ATP binds in the protomer interface having predominant contacts with Walker A of the cis-protomer. Trans-acting arginine(s) belonging to the conserved "RXDXXXR" motif of sensor I acts as "R-finger" to stabilize the γ-phosphate of ATP (Fig. 1c) (17Chen B. Sysoeva T.A. Chowdhury S. Guo L. De Carlo S. Hanson J.A. Yang H. Nixon B.T. Engagement of arginine finger to ATP triggers large conformational changes in NtrC1 AAA+ ATPase for remodeling bacterial RNA polymerase.Structure. 2010; 18 (21070941): 1420-143010.1016/j.str.2010.08.018Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Conversely, a novel and solely cis-mediated ATP binding and hydrolysis occurs in the heptameric FlrCC, where γ-phosphate of ATP is stabilized by cis-acting Arg349 of sensor II (Fig. 1b). Although the 285REDXXYR291 motif of sensor I was conserved in FlrC and adjacent to the ATP-binding site in trans, no residue of this motif participates in binding ATP (Fig. 1b) (20Dey S. Biswas M. Sen U. Dasgupta J. Unique ATPase site architecture triggers cis-mediated synchronized ATP binding in heptameric AAA+-ATPase domain of flagellar regulatory protein FlrC.J. Biol. Chem. 2015; 290 (25688103): 8734-874710.1074/jbc.M114.611434Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). This atypical "cis-mediated" mode of ATP binding in FlrC necessitates further investigations on mechanistic relationships between heptamerization and ATPase activity. Because no residues of trans-protomer interact with ATP, FlrC should be capable of binding and hydrolyzing ATP even in its monomeric state. Our current study, therefore, investigated if the heptameric structure of FlrC is required for binding the RNAP-σ54 complex at promoter, or it is decisive for ATPase activity as well. To address this issue, we have used three variants where cis-acting Arg349 of sensor II and two trans-acting residues Tyr290 and Arg291 (belonging to 285REDXXYR291 motif) of sensor I were replaced by Ala. Interestingly, all three variants, FlrCC-R349A, FlrCC-Y290A, and FlrCC-R291A showed impaired ATPase activity with destabilization of the heptameric state in varying degrees. Through the crystal structures of FlrCC-R349A and FlrCC-Y290A, we have addressed the molecular basis of the aforesaid structural and functional changes. Our observations suggest that heptamerization coupled with optimal conformation of Walker A is essential for efficient ATP binding and hydrolysis by FlrC. FlrCC forms heptamer through concentration-dependent positive cooperativity leading to maximal ATPase activity. Of the two key bEBPs involved in flagellar synthesis of V. cholerae, c-di-GMP abrogates interactions of FlrA with the promoters of the flrBC operon, leading to reduced expression of the downstream flagellar genes (11Srivastava D. Hsieh M.L. Khataokar A. Neiditch M.B. Waters C.M. Cyclic di-GMP inhibits Vibrio cholerae motility by repressing induction of transcription and inducing extracellular polysaccharide production.Mol. Microbiol. 2013; 90 (24134710): 1262-127610.1111/mmi.12432Crossref PubMed Scopus (81) Google Scholar). However, the role of c-di-GMP in regulation of FlrC was as yet unexplored. Our study has revealed for the first time that high concentrations of c-di-GMP repress ATPase activity of FlrCC by destabilizing heptameric assembly. Based on database and phylogenetic analyses we have further envisaged existence of such mechanisms in several other Vibrio species and a set of monotrichous Gram-negative bacteria. FlrCC and the variants were purified as His6-tagged proteins and tested for ATP hydrolysis using Malachite green assay as per the protocol described earlier (20Dey S. Biswas M. Sen U. Dasgupta J. Unique ATPase site architecture triggers cis-mediated synchronized ATP binding in heptameric AAA+-ATPase domain of flagellar regulatory protein FlrC.J. Biol. Chem. 2015; 290 (25688103): 8734-874710.1074/jbc.M114.611434Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar, 23Dey S. Dasgupta J. Purification, crystallization and preliminary X-ray analysis of the AAA+ σ54 activator domain of FlrC from Vibrio cholerae.Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2013; 69 (23832212): 800-80310.1107/S1744309113015613Crossref PubMed Scopus (2) Google Scholar, 24Geladopoulos T.P. Sotiroudis T.G. Evangelopoulos A.E. A malachite green colorimetric assay for protein phosphatase activity.Anal. Biochem. 1991; 192 (1646572): 112-11610.1016/0003-2697(91)90194-XCrossref PubMed Scopus (316) Google Scholar). Time course ATPase assays were performed where reaction mixtures containing 2.5 μm FlrCC (or variants) and 0.1 mm ATP (Sigma Aldrich) were incubated at 298 K for different time periods from 1 to 20 min (Fig. 1d). The release of Pi was measured at 630 nm upon incubation with Malachite green. Released Pi from each reaction was quantified by comparing with a Pi standard curve prepared using KH2PO4 (Fig. 1e). Time course experiments showed that the amount of Pi produced by FlrCC as a result of ATP hydrolysis increased approximately linearly with time for the first 5 min before it slowed down (Fig. 1d). However, ATPase activity of all three variants was drastically low throughout the time course (Fig. 1d). Concentrations of ATP inside the bacterial host may elevate up to 1 mm under certain conditions (25Buckstein M.H. He J. Rubin H. Characterization of nucleotide pools as a function of physiological state in Escherichia coli.J. Bacteriol. 2008; 190 (17965154): 718-72610.1128/JB.01020-07Crossref PubMed Scopus (218) Google Scholar, 26Bhate M.A.P. Molnar K.A.S. Goulian M. Degrado W.F. Signal transduction in histidine kinases: insights from new structures.Structure. 2015; 23 (25982528): 981-99410.1016/j.str.2015.04.002Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Therefore, we measured ATPase activities of FlrCC and variants upon elevating ATP concentrations to 0.3 and 0.5 mm as well (Fig. 1f). Considering the linearity pattern of the time course graph in Fig. 1d, release of Pi by the proteins were measured after 1, 3, and 5 min incubation (Fig. 1f). Negligible Pi release by the variants compared with FlrCC further established inertness of the variants in terms of ATPase activity (Fig. 1f). Because trans-acting Tyr290 and Arg291 of sensor I have no direct interaction with ATP (Fig. 1b), drastic reduction in ATP hydrolysis upon mutation of these residues to Ala was truly thought provoking. These results indicated that Tyr290 and neighboring residue Arg291 of trans protomer have indirect yet definite roles in the ATPase activity of FlrC. The oligomeric states of FlrCC and its variants were compared by size exclusion chromatography (SEC) using Superdex 200 increase column 10/300) (Fig. 2, a–e). Peak II of Fig. 2a having an elution volume of 12.13 ml was indicative of the heptamer of FlrCC where peak I denoted a higher molecular weight aggregate. In contrast, FlrCC-Y290A eluted at 15.75 ml exclusively as a monomeric species (Fig. 2, b and e). During SEC experiments, 100 μl of each protein was loaded in the column. Concentrations of FlrCC and FlrCC-R291A were 360 μm. Although FlrCC-Y290A was initially 360 μm, its exclusive existence as monomeric species intended us to elevate its concentration to 500 μm, which produced the same result (Fig. 2b). Exclusive existence as a monomeric species of FlrCC-Y290A in SEC was further validated through dynamic light scattering experiments (Fig. 2f). FlrCC-R291A and FlrCC-R349A, however, eluted as a mixture of heptameric and monomeric species (Fig. 2, c–e). Although the heptameric state was prevalent for FlrCC-R291A, a majority of FlrCC-R349A was found as a monomeric species (Fig. 2, c–e). It should be noted that due to an aggregation problem, the concentration of FlrCC-R349A could not be increased beyond 158 μm (which was less than that of FlrCC-R291A). Prevalence of the monomeric species of FlrCC-R349A probably occurred at that concentration after ∼60-fold dilution during SEC (Fig. 2, d and e). Because of a higher concentration, FlrCC-R291A could maintain more heptameric species, even after similar dilution (Fig. 2, c and e). Crystal structures of FlrCC-R349A and FlrCC-Y290A were determined up to 3.1 and 3.45 Å, respectively. Very similar unit cell dimensions, space group (P212121), and Mathew's coefficient of the variants to that of FlrCC (apo or AMP-PNP bound) suggested similar assembly. We, therefore, calculated the initial electron density map of the respective variant using the apo coordinates of FlrCC (PDB code 4QHS) after rigid body refinement. Because FlrCC-Y290A and FlrCC-R349A showed complete/major existence as monomeric species in SEC, the heptameric species in their crystal structures suggest that these variant proteins were forced to form heptamer at supersaturation. Interestingly, the crystals of FlrCC-Y290A were short-lived and disappeared in 10-16 h. Nonetheless, the diffraction data were satisfactory and the structures of FlrCC-R349A and FlrCC-Y290A were refined up to Rwork of 19.03% (Rfree = 25.08%) and 18.77% (Rfree = 23.41%), respectively. Data collection and refinemen
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