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• W2267115418 abstract "Type IV pili (T4P) are ubiquitous bacterial cell surface structures, involved in processes such as twitching motility, biofilm formation, bacteriophage infection, surface attachment, virulence, and natural transformation. T4P are assembled by machinery that can be divided into the outer membrane pore complex, the alignment complex that connects components in the inner and outer membrane, and the motor complex in the inner membrane and cytoplasm. Here, we characterize the inner membrane platform protein PilC, the cytosolic assembly ATPase PilB of the motor complex, and the cytosolic nucleotide-binding protein PilM of the alignment complex of the T4P machinery of Myxococcus xanthus. PilC was purified as a dimer and reconstituted into liposomes. PilB was isolated as a monomer and bound ATP in a non-cooperative manner, but PilB fused to Hcp1 of Pseudomonas aeruginosa formed a hexamer and bound ATP in a cooperative manner. Hexameric but not monomeric PilB bound to PilC reconstituted in liposomes, and this binding stimulated PilB ATPase activity. PilM could only be purified when it was stabilized by a fusion with a peptide corresponding to the first 16 amino acids of PilN, supporting an interaction between PilM and PilN(1–16). PilM-N(1–16) was isolated as a monomer that bound but did not hydrolyze ATP. PilM interacted directly with PilB, but only with PilC in the presence of PilB, suggesting an indirect interaction. We propose that PilB interacts with PilC and with PilM, thus establishing the connection between the alignment and the motor complex. Type IV pili (T4P) are ubiquitous bacterial cell surface structures, involved in processes such as twitching motility, biofilm formation, bacteriophage infection, surface attachment, virulence, and natural transformation. T4P are assembled by machinery that can be divided into the outer membrane pore complex, the alignment complex that connects components in the inner and outer membrane, and the motor complex in the inner membrane and cytoplasm. Here, we characterize the inner membrane platform protein PilC, the cytosolic assembly ATPase PilB of the motor complex, and the cytosolic nucleotide-binding protein PilM of the alignment complex of the T4P machinery of Myxococcus xanthus. PilC was purified as a dimer and reconstituted into liposomes. PilB was isolated as a monomer and bound ATP in a non-cooperative manner, but PilB fused to Hcp1 of Pseudomonas aeruginosa formed a hexamer and bound ATP in a cooperative manner. Hexameric but not monomeric PilB bound to PilC reconstituted in liposomes, and this binding stimulated PilB ATPase activity. PilM could only be purified when it was stabilized by a fusion with a peptide corresponding to the first 16 amino acids of PilN, supporting an interaction between PilM and PilN(1–16). PilM-N(1–16) was isolated as a monomer that bound but did not hydrolyze ATP. PilM interacted directly with PilB, but only with PilC in the presence of PilB, suggesting an indirect interaction. We propose that PilB interacts with PilC and with PilM, thus establishing the connection between the alignment and the motor complex. Type IV pili (T4P) 3The abbreviations used are: T4Ptype IV piliT4PST4P systemT2SStype II secretion systemIMinner membraneOMouter membraneTEVtobacco etch virusMANT2′(3′)-O-(N-methylanthraniloyl) adenosine 5′-triphosphateTNP-ATP(2′-(or-3′)-O-(trinitrophenyl) adenosine 5′-triphosphateIPTGisopropyl 1-thio-β-d-galactopyranoside. are versatile surface structures that are important for various processes, including twitching motility, biofilm formation, bacteriophage infection, surface attachment, virulence, and natural transformation. T4P are found on the surfaces of a wide variety of Gram-positive and Gram-negative bacteria and archaea (1.Berry J.-L. Pelicic V. Exceptionally widespread nanomachines composed of type IV pilins: the prokaryotic Swiss Army knives.FEMS Microbiol. Rev. 2015; 39: 134-154Crossref PubMed Scopus (153) Google Scholar, 2.Pohlschroder M. Ghosh A. Tripepi M. Albers S.-V. Archaeal type IV pilus-like structures—evolutionarily conserved prokaryotic surface organelles.Curr. Opin. Microbiol. 2011; 14: 357-363Crossref PubMed Scopus (64) Google Scholar, 3.Melville S. Craig L. Type IV pili in Gram-positive bacteria.Microbiol. Mol. Biol. Rev. 2013; 77: 323-341Crossref PubMed Scopus (139) Google Scholar). T4P systems (T4PS) are related to type II secretion systems (T2SS), which are responsible for the secretion of proteins across the outer membrane of Gram-negative bacteria (4.Ayers M. Howell P.L. Burrows L.L. Architecture of the type II secretion and type IV pilus machineries.Future Microbiol. 2010; 5: 1203-1218Crossref PubMed Scopus (99) Google Scholar, 5.Hobbs M. Mattick J.S. Common components in the assembly of type 4 fimbriae, DNA transfer systems, filamentous phage and protein-secretion apparatus: a general system for the formation of surface-associated protein complexes.Mol. Microbiol. 1993; 10: 233-243Crossref PubMed Scopus (309) Google Scholar), bacterial competence systems that are involved in the uptake of DNA (6.Chen I. Dubnau D. DNA uptake during bacterial transformation.Nat. Rev. Microbiol. 2004; 2: 241-249Crossref PubMed Scopus (506) Google Scholar), and systems that are involved in the assembly of archaeal surface structures (7.Albers S.-V. Jarrell K.F. The archaellum: how archaea swim.Front. Microbiol. 2015; 6: 23Crossref PubMed Scopus (90) Google Scholar). type IV pili T4P system type II secretion system inner membrane outer membrane tobacco etch virus 2′(3′)-O-(N-methylanthraniloyl) adenosine 5′-triphosphate (2′-(or-3′)-O-(trinitrophenyl) adenosine 5′-triphosphate isopropyl 1-thio-β-d-galactopyranoside. T4P are highly dynamic structures that undergo cycles of extension and retraction (8.Merz A.J. So M. Sheetz M.P. Pilus retraction powers bacterial twitching motility.Nature. 2000; 407: 98-102Crossref PubMed Scopus (614) Google Scholar, 9.Skerker J.M. Berg H.C. Direct observation of extension and retraction of type IV pili.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 6901-6904Crossref PubMed Scopus (438) Google Scholar). During extensions, the T4P assembly ATPase PilB stimulates the extraction of pilin monomers from the inner membrane (IM) and their incorporation at the base of the pilus fiber. The fiber has a diameter of ∼6 nm and can extend up to several micrometers in length (10.Craig L. Li J. Type IV pili: paradoxes in form and function.Curr. Opin. Struct. Biol. 2008; 18: 267-277Crossref PubMed Scopus (209) Google Scholar). During retractions, the T4P disassembly ATPase PilT stimulates the removal of pilin monomers from the base of the pilus and their reinsertion into the IM (9.Skerker J.M. Berg H.C. Direct observation of extension and retraction of type IV pili.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 6901-6904Crossref PubMed Scopus (438) Google Scholar, 11.Morand P.C. Bille E. Morelle S. Eugène E. Beretti J.-L. Wolfgang M. Meyer T.F. Koomey M. Nassif X. Type IV pilus retraction in pathogenic Neisseria is regulated by the PilC proteins.EMBO J. 2004; 23: 2009-2017Crossref PubMed Scopus (94) Google Scholar). T4P retraction generates forces up to 150 piconewtons (12.Maier B. Potter L. So M. Long C.D. Seifert H.S. Sheetz M.P. Single pilus motor forces exceed 100 pN.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 16012-16017Crossref PubMed Scopus (307) Google Scholar, 13.Clausen M. Jakovljevic V. Søgaard-Andersen L. Maier B. High-force generation is a conserved property of type IV pilus systems.J. Bacteriol. 2009; 191: 4633-4638Crossref PubMed Scopus (98) Google Scholar), pulling a cell forward, and making T4P systems the strongest molecular motors known. The rod-shaped cells of the δ-proteobacterium Myxococcus xanthus, assemble 5–10 T4P at the leading cell pole that extend and retract to generate cell movement in the direction of the long axis of cells (14.Kaiser D. Social gliding is correlated with the presence of pili in Myxococcus xanthus.Proc. Natl. Acad. Sci. U.S.A. 1979; 76: 5952-5956Crossref PubMed Scopus (389) Google Scholar). Occasionally, cells reverse their direction of movement (15.Blackhart B.D. Zusman D.R. “Frizzy” genes of Myxococcus xanthus are involved in control of frequency of reversal of gliding motility.Proc. Natl. Acad. Sci. U.S.A. 1985; 82: 8767-8770Crossref PubMed Scopus (186) Google Scholar). During a reversal, the T4P disassemble at the old leading pole and reassemble at the new leading cell pole (16.Sun H. Zusman D.R. Shi W. Type IV pilus of Myxococcus xanthus is a motility apparatus controlled by the frz chemosensory system.Curr. Biol. 2000; 10: 1143-1146Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 17.Bulyha I. Schmidt C. Lenz P. Jakovljevic V. Höne A. Maier B. Hoppert M. Søgaard-Andersen L. Regulation of the type IV pili molecular machine by dynamic localization of two motor proteins.Mol. Microbiol. 2009; 74: 691-706Crossref PubMed Scopus (116) Google Scholar, 18.Mignot T. Merlie Jr., J.P. Zusman D.R. Regulated pole-to-pole oscillations of a bacterial gliding motility protein.Science. 2005; 310: 855-857Crossref PubMed Scopus (96) Google Scholar). Differences in the timing between reversals result in net movement (19.Spormann A.M. Kaiser A.D. Gliding movements in Myxococcus xanthus.J. Bacteriol. 1995; 177: 5846-5852Crossref PubMed Google Scholar). M. xanthus is the model system for this type of T4P-dependent directional movement, but the biochemistry of the T4P assembly system in this organism has not been studied in detail. Similarly to other T4PS in Gram-negative bacteria, the T4PS of M. xanthus consists of 12 conserved proteins. The nomenclature for proteins of T4PS varies widely between organisms, even for highly conserved proteins. Here, we use the nomenclature used for the M. xanthus T4PS. The pilin PilA is added onto the pilus base of the T4PS after cleavage of the class III signal sequence of the pre-pilin by the pre-pilin peptidase (PilD) (20.Nunn D.N. Lory S. Product of the Pseudomonas aeruginosa gene pilD is a pre-pilin leader peptidase.Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 3281-3285Crossref PubMed Scopus (178) Google Scholar). The remaining 10 proteins of the T4PS form three inter-connected subcomplexes. The outer membrane (OM) subcomplex that serves as a conduit for the pilus across the OM and consists of the secretin (PilQ) (21.Burkhardt J. Vonck J. Averhoff B. Structure and function of PilQ, a secretin of the DNA transporter from the thermophilic bacterium Thermus thermophilus HB27.J. Biol. Chem. 2011; 286: 9977-9984Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), the peptidoglycan-binding protein (TsaP) (22.Siewering K. Jain S. Friedrich C. Webber-Birungi M.T. Semchonok D.A. Binzen I. Wagner A. Huntley S. Kahnt J. Klingl A. Boekema E.J. Søgaard-Andersen L. van der Does C. Peptidoglycan-binding protein TsaP functions in surface assembly of type IV pili.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: E953-E961Crossref PubMed Scopus (45) Google Scholar), and a pilotin (Tgl), which stimulates insertion of PilQ into the OM and/or PilQ oligomerization (23.Nudleman E. Wall D. Kaiser D. Polar assembly of the type IV pilus secretin in Myxococcus xanthus.Mol. Microbiol. 2006; 60: 16-29Crossref PubMed Scopus (92) Google Scholar, 24.Koo J. Tammam S. Ku S.-Y. Sampaleanu L.M. Burrows L.L. Howell P.L. PilF is an outer membrane lipoprotein required for multimerization and localization of the Pseudomonas aeruginosa type IV pilus secretin.J. Bacteriol. 2008; 190: 6961-6969Crossref PubMed Scopus (80) Google Scholar). The alignment subcomplex consists of the cytosolic actin-like ATP-binding protein (PilM) (25.Karuppiah V. Derrick J.P. Structure of the PilM-PilN inner membrane type IV pilus biogenesis complex from Thermus thermophilus.J. Biol. Chem. 2011; 286: 24434-24442Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), two bitopic IM proteins with large periplasmic domains and short cytosolic N termini (PilN and PilO), and an IM lipoprotein with a large periplasmic domain (PilP) (26.Georgiadou M. Castagnini M. Karimova G. Ladant D. Pelicic V. Large-scale study of the interactions between proteins involved in type IV pilus biology in Neisseria meningitidis: characterization of a subcomplex involved in pilus assembly.Mol. Microbiol. 2012; 84: 857-873Crossref PubMed Scopus (62) Google Scholar, 27.Sampaleanu L.M. Bonanno J.B. Ayers M. Koo J. Tammam S. Burley S.K. Almo S.C. Burrows L.L. Howell P.L. Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex.J. Mol. Biol. 2009; 394: 143-159Crossref PubMed Scopus (60) Google Scholar, 28.Li C. Wallace R.A. Black W.P. Li Y.Z. Yang Z. Type IV pilus proteins form an integrated structure extending from the cytoplasm to the outer membrane.PLoS ONE. 2013; 8: e70144Crossref PubMed Scopus (14) Google Scholar, 29.Tammam S. Sampaleanu L.M. Koo J. Sundaram P. Ayers M. Chong P.A. Forman-Kay J.D. Burrows L.L. Howell P.L. Characterization of the PilN, PilO and PilP type IVa pilus subcomplex.Mol. Microbiol. 2011; 82: 1496-1514Crossref PubMed Scopus (54) Google Scholar, 30.Tammam S. Sampaleanu L.M. Koo J. Manoharan K. Daubaras M. Burrows L.L. Howell P.L. PilMNOPQ from the Pseudomonas aeruginosa type IV pilus system form a transenvelope protein interaction network that interacts with PilA.J. Bacteriol. 2013; 195: 2126-2135Crossref PubMed Scopus (73) Google Scholar). Cytosolic PilM interacts with the short N terminus of PilN (25.Karuppiah V. Derrick J.P. Structure of the PilM-PilN inner membrane type IV pilus biogenesis complex from Thermus thermophilus.J. Biol. Chem. 2011; 286: 24434-24442Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 26.Georgiadou M. Castagnini M. Karimova G. Ladant D. Pelicic V. Large-scale study of the interactions between proteins involved in type IV pilus biology in Neisseria meningitidis: characterization of a subcomplex involved in pilus assembly.Mol. Microbiol. 2012; 84: 857-873Crossref PubMed Scopus (62) Google Scholar, 31.Friedrich C. Bulyha I. Søgaard-Andersen L. Outside-in assembly pathway of the type IV pilus system in Myxococcus xanthus.J. Bacteriol. 2014; 196: 378-390Crossref PubMed Scopus (56) Google Scholar). PilN and PilO interact directly and likely form heterodimers (27.Sampaleanu L.M. Bonanno J.B. Ayers M. Koo J. Tammam S. Burley S.K. Almo S.C. Burrows L.L. Howell P.L. Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex.J. Mol. Biol. 2009; 394: 143-159Crossref PubMed Scopus (60) Google Scholar, 31.Friedrich C. Bulyha I. Søgaard-Andersen L. Outside-in assembly pathway of the type IV pilus system in Myxococcus xanthus.J. Bacteriol. 2014; 196: 378-390Crossref PubMed Scopus (56) Google Scholar). The PilNO complex interacts with PilP (30.Tammam S. Sampaleanu L.M. Koo J. Manoharan K. Daubaras M. Burrows L.L. Howell P.L. PilMNOPQ from the Pseudomonas aeruginosa type IV pilus system form a transenvelope protein interaction network that interacts with PilA.J. Bacteriol. 2013; 195: 2126-2135Crossref PubMed Scopus (73) Google Scholar), and PilP interacts with PilQ (30.Tammam S. Sampaleanu L.M. Koo J. Manoharan K. Daubaras M. Burrows L.L. Howell P.L. PilMNOPQ from the Pseudomonas aeruginosa type IV pilus system form a transenvelope protein interaction network that interacts with PilA.J. Bacteriol. 2013; 195: 2126-2135Crossref PubMed Scopus (73) Google Scholar, 31.Friedrich C. Bulyha I. Søgaard-Andersen L. Outside-in assembly pathway of the type IV pilus system in Myxococcus xanthus.J. Bacteriol. 2014; 196: 378-390Crossref PubMed Scopus (56) Google Scholar, 32.Balasingham S.V. Collins R.F. Assalkhou R. Homberset H. Frye S.A. Derrick J.P. Tønjum T. Interactions between the lipoprotein PilP and the secretin PilQ in Neisseria meningitidis.J. Bacteriol. 2007; 189: 5716-5727Crossref PubMed Scopus (61) Google Scholar), thus putatively connecting components in the cytosol and IM to components in the OM. The IM motor subcomplex consists of the IM protein PilC (33.Takhar H.K. Kemp K. Kim M. Howell P.L. Burrows L.L. The platform protein is essential for type IV pilus biogenesis.J. Biol. Chem. 2013; 288: 9721-9728Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) and the associated cytosolic ATPases, which provide the energy for extension (PilB) and retraction (PilT), respectively (34.Jakovljevic V. Leonardy S. Hoppert M. Søgaard-Andersen L. PilB and PilT are ATPases acting antagonistically in type IV pilus function in Myxococcus xanthus.J. Bacteriol. 2008; 190: 2411-2421Crossref PubMed Scopus (105) Google Scholar). The T4PS in Gram-negative bacteria span both the IM and OM, whereas homologous systems in Gram-positive bacteria and in archaea only span the cytoplasmic membrane. Consistently, only the IM motor subcomplex is conserved in all T4PS. Moreover, the retraction ATPase PilT is only present in bacterial T4PS systems. Here, we investigate the IM protein PilC, the actin-like protein PilM, and the assembly ATPase PilB and analyze their function and association in the T4PS of M. xanthus. PilC is a polytopic IM protein that belongs to the GspF/PilC superfamily of IM proteins of T2SS and T4PS. Sequence analysis suggests that proteins of this family consist of two cytoplasmic domains with high sequence similarity, separated by two transmembrane domains and followed by a short C-terminal periplasmic extension. Here, we refer to M. xanthus PilC and homologs thereof in T4PS and T2SS as the IM platform protein (33.Takhar H.K. Kemp K. Kim M. Howell P.L. Burrows L.L. The platform protein is essential for type IV pilus biogenesis.J. Biol. Chem. 2013; 288: 9721-9728Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). To date, no structural data are available on a full-length protein of the GspF/PilC superfamily. The structure of the cytosolic N-terminal domain of Thermus thermophilus PilC revealed a dimeric helical bundle structure with the dimer formed by interactions between the fifth and sixth α-helix (35.Karuppiah V. Hassan D. Saleem M. Derrick J.P. Structure and oligomerization of the PilC type IV pilus biogenesis protein from Thermus thermophilus.Proteins. 2010; 78: 2049-2057Crossref PubMed Scopus (40) Google Scholar). The N-terminal domain of EpsF from the T2SS of Vibrio cholerae also crystallized as a helix bundle but showed a different dimer interface than T. thermophilus PilC (36.Collins R.F. Saleem M. Derrick J.P. Purification and three-dimensional electron microscopy structure of the Neisseria meningitidis type IV Pilus biogenesis protein PilG.J. Bacteriol. 2007; 189: 6389-6396Crossref PubMed Scopus (30) Google Scholar). Moreover, the Neisseria meningitidis PilC homolog has been described by analytical ultracentrifugation and single particle analysis to form dimers and tetramers in solution (36.Collins R.F. Saleem M. Derrick J.P. Purification and three-dimensional electron microscopy structure of the Neisseria meningitidis type IV Pilus biogenesis protein PilG.J. Bacteriol. 2007; 189: 6389-6396Crossref PubMed Scopus (30) Google Scholar). The exact function of PilC remains under discussion. Although a pilG mutant, the pilC ortholog in N. meningitidis, does not assemble pili, the pilG/pilT double mutant assembles pili (37.Carbonnelle E. Helaine S. Nassif X. Pelicic V. A systematic genetic analysis in Neisseria meningitidis defines the Pil proteins required for assembly, functionality, stabilization and export of type IV pili.Mol. Microbiol. 2006; 61: 1510-1522Crossref PubMed Scopus (150) Google Scholar). By contrast, both the pilC and the pilC/pilT double mutants in Pseudomonas aeruginosa are non-piliated (33.Takhar H.K. Kemp K. Kim M. Howell P.L. Burrows L.L. The platform protein is essential for type IV pilus biogenesis.J. Biol. Chem. 2013; 288: 9721-9728Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The PilB ATPase is a member of the secretion ATPase superfamily, a subgroup of RecA/Rad51-like motors (39.Iyer L.M. Leipe D.D. Koonin E.V. Aravind L. Evolutionary history and higher order classification of AAA+ ATPases.J. Struct. Biol. 2004; 146: 11-31Crossref PubMed Scopus (632) Google Scholar, 40.Planet P.J. Kachlany S.C. DeSalle R. Figurski D.H. Phylogeny of genes for secretion NTPases: identification of the widespread tadA subfamily and development of a diagnostic key for gene classification.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 2503-2508Crossref PubMed Scopus (166) Google Scholar, 41.Shin D.S. Pellegrini L. Daniels D.S. Yelent B. Craig L. Bates D. Yu D.S. Shivji M.K. Hitomi C. Arvai A.S. Volkmann N. Tsuruta H. Blundell T.L. Venkitaraman A.R. Tainer J.A. Full-length archaeal Rad51 structure and mutants: mechanisms for RAD51 assembly and control by BRCA2.EMBO J. 2003; 22: 4566-4576Crossref PubMed Scopus (220) Google Scholar). Crystal structures of T2SS assembly ATPases (42.Abendroth J. Murphy P. Sandkvist M. Bagdasarian M. Hol W.G. The x-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae.J. Mol. Biol. 2005; 348: 845-855Crossref PubMed Scopus (82) Google Scholar, 43.Robien M.A. Krumm B.E. Sandkvist M. Hol W.G. Crystal structure of the extracellular protein secretion NTPase EpsE of Vibrio cholerae.J. Mol. Biol. 2003; 333: 657-674Crossref PubMed Scopus (103) Google Scholar, 44.Yamagata A. Tainer J.A. Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism.EMBO J. 2007; 26: 878-890Crossref PubMed Scopus (77) Google Scholar, 45.Chen Y. Shiue S.-J. Huang C.-W. Chang J.-L. Chien Y.-L. Hu N.-T. Chan N.-L. Structure and function of the XpsE N-terminal domain, an essential component of the Xanthomonas campestris type II secretion system.J. Biol. Chem. 2005; 280: 42356-42363Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), T4P retraction ATPases (46.Misic A.M. Satyshur K.A. Forest K.T. P. aeruginosa PilT structures with and without nucleotide reveal a dynamic type Iv pilus retraction motor.J. Mol. Biol. 2010; 400: 1011-1021Crossref PubMed Scopus (69) Google Scholar, 47.Satyshur K.A. Worzalla G.A. Meyer L.S. Heiniger E.K. Aukema K.G. Misic A.M. Forest K.T. Crystal structures of the Pilus retraction motor PilT suggest large domain movements and subunit cooperation drive motility.Structure. 2007; 15: 363-376Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), and the motor ATPase of the archaellum (the archaeal T4P-like motility system) (48.Reindl S. Ghosh A. Williams G.J. Lassak K. Neiner T. Henche A.-L. Albers S.-V. Tainer J.A. Insights on FlaI functions in archaeal motor assembly and motilify from structures, conformations and genetics.Mol. Cell. 2013; 49: 1069-1082Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) revealed that these proteins consist of a conserved C-terminal ATPase domain and a much more variable N-terminal domain. These crystal structures and other data also revealed that PilB-like ATPases function as hexamers and that conformational changes upon hydrolysis of ATP most likely drive the extrusion or retraction of the pilus (48.Reindl S. Ghosh A. Williams G.J. Lassak K. Neiner T. Henche A.-L. Albers S.-V. Tainer J.A. Insights on FlaI functions in archaeal motor assembly and motilify from structures, conformations and genetics.Mol. Cell. 2013; 49: 1069-1082Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 49.Lu C. Turley S. Marionni S.T. Park Y.-J. Lee K.K. Patrick M. Shah R. Sandkvist M. Bush M.F. Hol W.G. Hexamers of the type II secretion ATPase GspE from Vibrio cholerae with increased ATPase activity.Structure. 2013; 21: 1707-1717Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Several studies have suggested an interaction between PilB and PilC homologs. Localization and stability of the assembly ATPase PilB of P. aeruginosa have been shown to depend on PilC. Co-purification and co-localization experiments revealed an interaction between PilC and PilB (33.Takhar H.K. Kemp K. Kim M. Howell P.L. Burrows L.L. The platform protein is essential for type IV pilus biogenesis.J. Biol. Chem. 2013; 288: 9721-9728Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 50.Chiang P. Habash M. Burrows L.L. Disparate subcellular localization patterns of Pseudomonas aeruginosa type IV pilus ATPases involved in twitching motility.J. Bacteriol. 2005; 187: 829-839Crossref PubMed Scopus (95) Google Scholar). Yeast two-hybrid analysis of the T2SS of Erwinia chrysanthemi revealed interactions between GspF (PilC homolog), GspE (PilB homolog), and GspL (PilM-N homolog), but co-immunoprecipitation of GspF with GspE required the presence of GspL (51.Py B. Loiseau L. Barras F. An inner membrane platform in the type II secretion machinery of Gram-negative bacteria.EMBO Rep. 2001; 2: 244-248Crossref PubMed Scopus (127) Google Scholar). In the T2SS of V. cholerae, structural data suggest that the ATPase EpsE interacts and forms a complex with the N-terminal cytosolic domain of the PilM-N homolog EpsL (42.Abendroth J. Murphy P. Sandkvist M. Bagdasarian M. Hol W.G. The x-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae.J. Mol. Biol. 2005; 348: 845-855Crossref PubMed Scopus (82) Google Scholar), resulting in a stimulation of the ATPase activity (52.Camberg J.L. Johnson T.L. Patrick M. Abendroth J. Hol W.G. Sandkvist M. Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids.EMBO J. 2007; 26: 19-27Crossref PubMed Scopus (73) Google Scholar). The structure of T. thermophilus PilM has been solved and is most similar to the actin-like ATPase FtsA (25.Karuppiah V. Derrick J.P. Structure of the PilM-PilN inner membrane type IV pilus biogenesis complex from Thermus thermophilus.J. Biol. Chem. 2011; 286: 24434-24442Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), BfpC (a PilM homolog) of the T4PS of Escherichia coli, and the cytoplasmic domains of GspL (a PilM-N homolog) of the T2SS of V. cholerae (53.Yamagata A. Milgotina E. Scanlon K. Craig L. Tainer J.A. Donnenberg M.S. Structure of an essential type IV pilus biogenesis protein provides insights into pilus and type II secretion systems.J. Mol. Biol. 2012; 419: 110-124Crossref PubMed Scopus (23) Google Scholar, 54.Abendroth J. Bagdasarian M. Sandkvist M. Hol W.G. The structure of the cytoplasmic domain of EpsL, an inner membrane component of the type II secretion system of Vibrio cholerae: an unusual member of the actin-like ATPase superfamily.J. Mol. Biol. 2004; 344: 619-633Crossref PubMed Scopus (53) Google Scholar). Despite the actin-like structure of PilM, the protein was purified and crystallized as a monomer, and no polymerization or ATP hydrolysis has been observed (25.Karuppiah V. Derrick J.P. Structure of the PilM-PilN inner membrane type IV pilus biogenesis complex from Thermus thermophilus.J. Biol. Chem. 2011; 286: 24434-24442Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). However, PilM has been reported to bind ATP (25.Karuppiah V. Derrick J.P. Structure of the PilM-PilN inner membrane type IV pilus biogenesis complex from Thermus thermophilus.J. Biol. Chem. 2011; 286: 24434-24442Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The short and highly conserved cytosolic N terminus of PilN has been reported to interact with PilM (26.Georgiadou M. Castagnini M. Karimova G. Ladant D. Pelicic V. Large-scale study of the interactions between proteins involved in type IV pilus biology in Neisseria meningitidis: characterization of a subcomplex involved in pilus assembly.Mol. Microbiol. 2012; 84: 857-873Crossref PubMed Scopus (62) Google Scholar), and indeed crystallization of T. thermophilus PilM was only successful in the presence of the N terminus of PilN, demonstrating that PilM binds to the N terminus of PilN (25.Karuppiah V. Derrick J.P. Structure of the PilM-PilN inner membrane type IV pilus biogenesis complex from Thermus thermophilus.J. Biol. Chem. 2011; 286: 24434-24442Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). In addition to the interaction with PilN, PilM has also been suggested to interact with PilC and PilB. Similar to T2SS of E. chrysanthemi (51.Py B. Loiseau L. Barras F. An inner membrane platform in the type II secretion machinery of Gram-negative bacteria.EMBO Rep. 2001; 2: 244-248Crossref PubMed Scopus (127) Google Scholar), in the T2SS of P. aeruginosa, co-expression of XcpY (PilM-N homolog) and XcpR (PilB homolog) is stabilized by XcpS (PilC homolog) suggesting the formation of a ternary complex via direct or indirect association of the three proteins (55.Robert V. Filloux A. Michel G.P. Subcomplexes from the Xcp secretion system of Pseudomonas aeruginosa.FEMS Microbiol. Lett. 2005; 252: 43-50Crossref PubMed Scopus (30) Google Scholar, 56.Arts J. de Groot A. Ball G. Durand E. El Khattabi M. Filloux A. Tommassen J. Koster M. Interaction domains in the Pseudomonas aeruginosa type II secretory apparatus component XcpS (GspF).Microbiology. 2007; 153: 1582-1592Crossref PubMed Scopus (32) Google Scholar). To examine how PilC, PilB, and PilM interact to stimulate T4P assembly, we overexpressed and purified PilC, PilB, and PilM of the T4PS of M. xanthus and characterized the proteins and their interactions with each other. Primers, plasmids, and strains used in this study are listed in TABLE 1, TABLE 2, TABLE 3, respectively. Cloning was performed in E. coli DH5α. If no template is named, PCRs were performed on chromosomal DNA of M. xanthus strain DK1622 (14.Kaiser D. Social gliding is correlated with the presence of pili in Myxococcus xanthus.Proc. Natl. Acad. Sci. U.S.A. 1979; 76: 5952-5956Crossref PubMed Scopus (389) Google Scholar). To generate a" @default.
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• W2267115418 title "The Type IV Pilus Assembly ATPase PilB of Myxococcus xanthus Interacts with the Inner Membrane Platform Protein PilC and the Nucleotide-binding Protein PilM" @default.
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