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Secretion Superfamily ATPases Swing Big

2007; Elsevier BV; Volume: 15; Issue: 3 Linguagem: Inglês

10.1016/j.str.2007.02.003

1878-4186

Savvas N. Savvides,

Enzyme Structure and Function

In this issue of Structure, Satyshur et al., 2007Satyshur K.A. Worzalla G. Meyer L.S. Heiniger E.K. Aukema K.G. Misic A.M. Forest K.T. Structure. 2007; 15 (this issue): 363-376Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar present crystallographic snapshots of the bacterial type IV pilus retraction motor PilT and propose a general model for pilus retraction consistent with a growing consensus that secretion superfamily ATPases are dynamic hexameric assemblies. In this issue of Structure, Satyshur et al., 2007Satyshur K.A. Worzalla G. Meyer L.S. Heiniger E.K. Aukema K.G. Misic A.M. Forest K.T. Structure. 2007; 15 (this issue): 363-376Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar present crystallographic snapshots of the bacterial type IV pilus retraction motor PilT and propose a general model for pilus retraction consistent with a growing consensus that secretion superfamily ATPases are dynamic hexameric assemblies. Near the turn of the new millennium, a cascade of reports on the structural biology of bacterial secretion systems sparked an unprecedented interest in dissecting the macromolecular machineries responsible for mediating protein/DNA trafficking out of cells and the biogenesis of supramolecular assemblies such as pili and flagellae (Remaut and Waksman, 2004Remaut H. Waksman G. Curr. Opin. Struct. Biol. 2004; 14: 161-170Crossref PubMed Scopus (51) Google Scholar). One of the hallmarks of these molecular trafficking systems is the coupling of ATP binding and hydrolysis to energize their assembly and to drive substrate translocation. Indeed, all known bacterial and archaeal secretion systems employ essential NTPases (predominantly ATPases) of which a major subset are the type II/IV secretion NTPases, broadly known as the “secretion superfamily ATPases.” These encompass traffic ATPases participating in bacterial type IV secretion systems (T4SS) and motor NTPases driving bacterial and archaeal type II secretion systems (T2SS), bacterial type IV pilus biogenesis, and the assembly of archaeal flagellae (Planet et al., 2001Planet P.J. Kachlany S.C. DeSalle R. Figurski D.H. Proc. Natl. Acad. Sci. USA. 2001; 98: 2503-2508Crossref PubMed Scopus (161) Google Scholar). While structures of secretion ATPases are available for T4SS and T2SS (Yeo et al., 2000Yeo H.J. Savvides S.N. Herr A.B. Lanka E. Waksman G. Mol. Cell. 2000; 6: 1461-1472Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, Robien et al., 2003Robien M.A. Krumm B.E. Sandkvist M. Hol W.G.J. J. Mol. Biol. 2003; 333: 657-674Crossref PubMed Scopus (100) Google Scholar, Hare et al., 2006Hare S. Bayliss R. Baron C. Waksman G. J. Mol. Biol. 2006; 360: 56-66Crossref PubMed Scopus (55) Google Scholar, Yamagata and Tainer, 2007Yamagata A. Tainer J.A. EMBO J. 2007; 26: 878-890Crossref PubMed Scopus (74) Google Scholar, Savvides et al., 2003Savvides S.N. Yeo H.J. Beck M.R. Blaesing F. Lurz R. Lanka E. Buhrdorf R. Fischer W. Haas R. Waksman G. EMBO J. 2003; 22: 1969-1980Crossref PubMed Scopus (140) Google Scholar), those of motor ATPases involved in type IV pilus assembly/retraction have remained elusive. In this issue of Structure, Forest and colleagues report the first crystallographic snapshots of the bacterial type IV pilus retraction motor, PilT. Additionally, they provide evidence for the way PilT can potentially undergo large-scale rigid-body motions per ATP binding event, thus making it possible to drag a pilus substrate protein over tens of Ångstroms (Satyshur et al., 2007Satyshur K.A. Worzalla G. Meyer L.S. Heiniger E.K. Aukema K.G. Misic A.M. Forest K.T. Structure. 2007; 15 (this issue): 363-376Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). This is highly anticipated work that complements ongoing studies on type IV pilus biogenesis (Craig et al., 2006Craig L. Volkmann N. Arvai A.S. Pique M.E. Yeager M. Egelman E.H. Tainer J.A. Mol. Cell. 2006; 23: 651-662Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Bacterial type IV pili are extraordinary macromolecular assemblies. They are thin filamentous structures (50–80 Å) with exceptional tensile strength (>100 pN), and are formed as a result of the ATP-dependent polymerization of pillin subunits. In their mature form, they emanate conspicuously from the surface of bacteria to mediate a multitude of cellular functions, such as surface motility, cell adhesion, biofilm formation, and cell signaling. In the case of some pathogenic bacteria, type IV pili constitute an important virulence factor (Craig et al., 2004Craig L. Pique M.E. Tainer J.A. Nat. Rev. Microbiol. 2004; 2: 363-378Crossref PubMed Scopus (525) Google Scholar). Like the other structurally characterized members of the secretion ATPase superfamily—the bacterial T4SS traffic ATPases HP0525 from Helicobacter pylori and VirB11 from Brucella suis, the bacterial T2SS motor EpsE from Vibrio Cholera, and the archaeal T2SS motor GspE from Arhaeoglobus fulgidus—nucleotide-bound forms of PilT feature a symmetric, toroidal hexameric structure constructed from intimately interacting subunits, which in turn are organized in N-terminal and C-terminal domains (NTD and CTD) separated by a linker (Figure 1). The NTD and CTD of secretion superfamily ATPases are, in general, structurally well conserved despite the sometimes marginal sequence identity among homologs. Differences in sequence length take the form of additional structural features. For instance, the NTD of PilT resembles Per/Arndt/Sim (PAS) domains due to additional secondary structure elements. Furthermore, the nucleotide binding fold of the CTD accommodates an extra motif of 7 short α helices, of which αJ contains the signature sequence for pilus retraction (AIRNLIRE) (Figure 1). Intriguingly, neither of the symmetric hexamers of PilT bound with ATP or the ADP corresponds to active forms due to their incomplete nucleotide binding sites, suggesting that other, functionally competent oligomeric forms of PilT must exist. Unexpectedly, Forest and colleagues managed to trap PilT in a grossly deformed, asymmetric hexameric assembly (Figure 2). In this squashed hexamer with pseudo 2-fold symmetry, subunits A, C, D, and F exhibit contracted conformations (the NTD-CTD grooves are closed by 16° compared to the 6-fold symmetric structure), while subunits B and E have moved away from the center of the assembly and have undergone a staggering 65° rotation about their linkers to yield wide-open conformations (Figure 2). In this structural context, the nucleotide binding site of subunit F becomes complete and reveals clusters of arginine residues contributed by adjacent subunits, leading to the formation of what the authors call an “arginine wire.” With the support of mutagenesis data and functional assays, the authors propose that PilT may utilize an arginine finger to drive pilus retraction and, by analogy to the arginine fingers of GTPase-activating proteins and AAA+ ATPases, to facilitate communication between subunits. However, the true impact of observing an asymmetric PilT is that it provides the opportunity to propose models for its mode of action. Satyshur et al. postulate that PilT may oscillate between a structurally heterogeneous, unliganded form and the active ellipsoidal hexamer they have observed. ATP binding to one of the open subunits would cause a dramatic closure of the CTD domain. The immediate implications of this large-scale motion would be that a pillin subunit interacting with one of the swinging CTD domains of PilT would be dragged along over several Ångstroms, thus resulting in pilus retraction. The proposed mode of action also implies that only a fraction of the PilT subunits in the hexamer can be active at any given moment and that the rest would play a supporting role. It also raises the possibility that biologically relevant forms of PilT may never achieve hexameric symmetry, but instead cycle between distinct asymmetric assemblies. That mechanism would be in contrast to other secretion superfamily ATPases characterized to date. Nonetheless, one may be able to rationalize this scenario in terms of a need for a conformationally more diverse motor ATPase driving pilus retraction given the relatively large forces needed to translate pillin subunits by tens of Ångstroms. The coupling of ATP binding and hydrolysis to the generation of mechanical leverage is indeed the essence of secretion ATPases, which by definition should be able to cycle between structurally distinct nucleotide-bound and nucleotide-free forms. Earlier studies on the traffic ATPase from the T4SS of the human pathogen H. pylori (Savvides et al., 2003Savvides S.N. Yeo H.J. Beck M.R. Blaesing F. Lurz R. Lanka E. Buhrdorf R. Fischer W. Haas R. Waksman G. EMBO J. 2003; 22: 1969-1980Crossref PubMed Scopus (140) Google Scholar) and recent elegant work on GspE from an archaeal T2SS (Yamagata and Tainer, 2007Yamagata A. Tainer J.A. EMBO J. 2007; 26: 878-890Crossref PubMed Scopus (74) Google Scholar), have combined crystallographic studies with solution methods, such as analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS), to show that secretion superfamily ATPases are dynamic, modular hexameric assemblies that can undergo large nucleotide-dependent domain rearrangements. PilT is now the third such ATPase for which this dynamic behavior has been shown, albeit exclusively via crystallographic methods. Although very revealing, crystallographic studies of secretion superfamily ATPases are bound to be limited by what can be crystallized. In fact, the range of biologically relevant conformational ensembles is likely to be much greater than what has been documented so far. Nonetheless, the available studies already point to a universal paradigm that calls for a nucleotide-dependent “push-n-pull” mode of action. This is a testable proposal for which methods, such as fluorescence resonance energy transfer (FRET), AUC, SAXS, and NMR, and in silico approaches, such as molecular dynamics, could provide invaluable insights. PilT would be well suited for FRET studies because its subunits can swing extensively, and because the platform interacting with target pillins is the solvent exposed helix αJ in the CTD (Figure 1). Furthermore, a dissection of the operating principles of secretion ATPases will be needed in light of recently proposed models for ATP binding and hydrolysis (Martin et al., 2005Martin A. Baker T.A. Sauer R.T. Nature. 2005; 437: 1115-1120Crossref PubMed Scopus (289) Google Scholar). In this regard, detailed (pre-)steady state kinetic experiments complemented by site-directed mutagenesis and crystallographic titrations using substrate and substrate-analogs could provide a wealth of new information. The stage has been set! Crystal Structures of the Pilus Retraction Motor PilT Suggest Large Domain Movements and Subunit Cooperation Drive MotilitySatyshur et al.StructureMarch, 2007In BriefPilT is a hexameric ATPase required for bacterial type IV pilus retraction and surface motility. Crystal structures of ADP- and ATP-bound Aquifex aeolicus PilT at 2.8 and 3.2 Å resolution show N-terminal PAS-like and C-terminal RecA-like ATPase domains followed by a set of short C-terminal helices. The hexamer is formed by extensive polar subunit interactions between the ATPase core of one monomer and the N-terminal domain of the next. An additional structure captures a nonsymmetric PilT hexamer in which approach of invariant arginines from two subunits to the bound nucleotide forms an enzymatically competent active site. Full-Text PDF Open Archive