The ATPase Motor Turns for Type IV Pilus Assembly
2016; Elsevier BV; Volume: 24; Issue: 11 Linguagem: Inglês
10.1016/j.str.2016.10.002
ISSN1878-4186
Autores Tópico(s)Enzyme Structure and Function
ResumoIn this issue of Structure, Mancl et al., 2016Mancl J.M. Black W.P. Robinson H. Yang Z. Schubot F.D. Structure. 2016; 24 (this issue): 1886-1897Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar elucidate the crystal structure of the PilB ATPase domain in complex with ATPγS and unveil how ATP binding and hydrolysis coordinates conformational change. Their results reveal a distinct symmetric rotary mechanism for ATP hydrolysis to power bacterial pilus assembly. In this issue of Structure, Mancl et al., 2016Mancl J.M. Black W.P. Robinson H. Yang Z. Schubot F.D. Structure. 2016; 24 (this issue): 1886-1897Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar elucidate the crystal structure of the PilB ATPase domain in complex with ATPγS and unveil how ATP binding and hydrolysis coordinates conformational change. Their results reveal a distinct symmetric rotary mechanism for ATP hydrolysis to power bacterial pilus assembly. Type IV pili (T4P) are bacterial filaments responsible for cell movement and signaling, DNA uptake, microcolony and biofilm formation, and mediating virulent invasion into a host. Many bacteria utilize T4P for twitching along surfaces by pilus extension, adhesion, and retraction (Craig et al., 2004Craig L. Pique M.E. Tainer J.A. Nat. Rev. Microbiol. 2004; 2: 363-378Crossref PubMed Scopus (570) Google Scholar). The remarkable ability of T4P to grow micrometers in seconds and generate >100 pN force during retraction make T4P among the most fascinating and powerful self-assembling molecular machines. The heart of this machine is its PilB assembly ATPase domain, whose structure is defined by Mancl et al., 2016Mancl J.M. Black W.P. Robinson H. Yang Z. Schubot F.D. Structure. 2016; 24 (this issue): 1886-1897Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar. The T4P machinery consists of >10 different proteins: its structural organization resembles the type II secretion system (T2SS) and the archaella (formerly archaeal flagella). The pilus is assembled by PilB ATPase and disassembled by antagonist ATPase PilT. Both ATPases are coordinated by the inner membrane platform protein PilC: PilB and PilT bind to the N-terminal and C-terminal cytoplasmic domains, respectively (Takhar et al., 2013Takhar H.K. Kemp K. Kim M. Howell P.L. Burrows L.L. J. Biol. Chem. 2013; 288: 9721-9728Crossref PubMed Scopus (80) Google Scholar). PilB ATPase belongs to the PilB/PilF/GspE family and is a hexamer by cryo-electron tomography (Chang et al., 2016Chang Y.W. Rettberg L.A. Treuner-Lange A. Iwasa J. Søgaard-Andersen L. Jensen G.J. Science. 2016; 351: aad2001Crossref PubMed Scopus (237) Google Scholar). A detailed crystal structure of the PilB core ATPase domain from Thermus thermophilus (TtPilBATP) in complex with ATPγS at 2.65 Å resolution now uncovers how ATP hydrolysis is coordinated by the TtPilBATP hexamer (Mancl et al., 2016Mancl J.M. Black W.P. Robinson H. Yang Z. Schubot F.D. Structure. 2016; 24 (this issue): 1886-1897Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). By determining the TtPilBATP crystal structure beyond 3 Å resolution, Mancl et al., 2016Mancl J.M. Black W.P. Robinson H. Yang Z. Schubot F.D. Structure. 2016; 24 (this issue): 1886-1897Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar gained impressive insights. The TtPilBATP shows a two-fold symmetric hexameric complex with ATPγS bound to the C-terminal domain (CTD) of each subunit. Full-length TtPilB forms a hexameric structure without ATP, but ATP-binding stabilizes the hexameric complex. Moreover, the conserved tetra-cysteine zinc-binding motif contributes to TtPilB hexameric complex stability by maintaining the correct subunit folding (Salzer et al., 2014Salzer R. Herzberg M. Nies D.H. Joos F. Rathmann B. Thielmann Y. Averhoff B. J. Biol. Chem. 2014; 289: 30343-30354Crossref PubMed Scopus (22) Google Scholar). Here the TtPilBATP hexameric structure contains a total of six zinc metals within its C-terminal coordinated metal domain (CMD). Interestingly, this CMD domain is absent in all known PilT proteins, implying distinct functional interactions of the inner membrane platform complex with PilB through the CMD. In fact, mutations of cysteine residues in the zinc-binding motif impair twitching motility but promote hyperpiliation, which indicates an essential CMD role for pilus function (Salzer et al., 2014Salzer R. Herzberg M. Nies D.H. Joos F. Rathmann B. Thielmann Y. Averhoff B. J. Biol. Chem. 2014; 289: 30343-30354Crossref PubMed Scopus (22) Google Scholar). TtPilBATP AAA+ ATPase (ATPases associated with diverse cellular activities) contains conserved Walker A and B motifs for ATP binding and hydrolysis and forms a hexameric ring structures in the presence of nucleotide similarly to many AAA+ ATPases (Misic et al., 2010Misic A.M. Satyshur K.A. Forest K.T. J. Mol. Biol. 2010; 400: 1011-1021Crossref PubMed Scopus (69) Google Scholar, Reindl et al., 2013Reindl S. Ghosh A. Williams G.J. Lassak K. Neiner T. Henche A.L. Albers S.V. Tainer J.A. Mol. Cell. 2013; 49: 1069-1082Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, Salzer et al., 2014Salzer R. Herzberg M. Nies D.H. Joos F. Rathmann B. Thielmann Y. Averhoff B. J. Biol. Chem. 2014; 289: 30343-30354Crossref PubMed Scopus (22) Google Scholar, Yamagata and Tainer, 2007Yamagata A. Tainer J.A. EMBO J. 2007; 26: 878-890Crossref PubMed Scopus (77) Google Scholar). The TtPilBATP hexamer is ∼45Å maximum inner ring dimension, and ∼15Å at its narrowest. This asymmetry is created because the arginine fingers on the NTD (N-terminal domain) move in to interact with the ATP γ-phosphate, forming an "open conformation," and out for ATP hydrolysis, forming a "closed conformation" (Mancl et al., 2016Mancl J.M. Black W.P. Robinson H. Yang Z. Schubot F.D. Structure. 2016; 24 (this issue): 1886-1897Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The arginine fingers, generally conserved in the AAA+ ATPase superfamily, are essential for ATP-mediated hexamer stabilization. Mancl et al., 2016Mancl J.M. Black W.P. Robinson H. Yang Z. Schubot F.D. Structure. 2016; 24 (this issue): 1886-1897Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar overlay the open and closed conformations and reveal a shift of the 17-residue flexible linker region connecting NTD and CTD, resulting in ∼55° rotation of NTD with respect to CTD. The "open conformation" was further divided into two subgroups due to differences in solvent accessibility of the active sites. These distinct TtPilBATP conformations in the hexameric complex structure form three structural states during ATP hydrolysis that occur simultaneously on three pairs of TtPilBATP subunits (Figure 1) (Mancl et al., 2016Mancl J.M. Black W.P. Robinson H. Yang Z. Schubot F.D. Structure. 2016; 24 (this issue): 1886-1897Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Spectacularly, rigid-body analysis of these conformations from two adjacent subunits suggests a distinct symmetric rotary motor mechanism for TtPilBATP. How the ATPase motor coordinates ATP hydrolysis for bacterial T4P and T2SS and for archaellum has been under debate. Perhaps no unified mechanism exists. Yet, T4P/T2SS ATPase studies suggest the possibility of a few general models for ATP catalysis within the hexamer. For example, the PilT structures from Aquifex aeolicus and Pseudomonas aeruginosa suggested a concerted (Satyshur et al., 2007Satyshur K.A. Worzalla G.A. Meyer L.S. Heiniger E.K. Aukema K.G. Misic A.M. Forest K.T. Structure. 2007; 15: 363-376Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) and stochastic (Misic et al., 2010Misic A.M. Satyshur K.A. Forest K.T. J. Mol. Biol. 2010; 400: 1011-1021Crossref PubMed Scopus (69) Google Scholar) model, respectively, for pilus retraction. Whereas crystallography and X-ray scattering of archaeal FlaI supports the concerted mechanism for archaellum assembly (Reindl et al., 2013Reindl S. Ghosh A. Williams G.J. Lassak K. Neiner T. Henche A.L. Albers S.V. Tainer J.A. Mol. Cell. 2013; 49: 1069-1082Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), similar studies on archaeal AfGspE T2SS suggest a piston-like, push–pull mechanism consistent with sequential or stochastic mechanisms (Yamagata and Tainer, 2007Yamagata A. Tainer J.A. EMBO J. 2007; 26: 878-890Crossref PubMed Scopus (77) Google Scholar). The structure of the TtPilBATP hexamer reported by Mancl et al., 2016Mancl J.M. Black W.P. Robinson H. Yang Z. Schubot F.D. Structure. 2016; 24 (this issue): 1886-1897Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar supports a distinct symmetric rotary mechanism for T4P assembly in which ATP hydrolysis steps proceed sequentially from one subunit to its adjacent subunit in a two-fold symmetric way (Figure 1). The ATP binding and competitive displacement experiments further support the exchange of ADP with ATP (Mancl et al., 2016Mancl J.M. Black W.P. Robinson H. Yang Z. Schubot F.D. Structure. 2016; 24 (this issue): 1886-1897Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The conserved inner membrane protein PilC forms a dimer with cytoplasmic domains of ∼45Å and the N-terminal cytoplasmic domain directly interacting with PilB (Chang et al., 2016Chang Y.W. Rettberg L.A. Treuner-Lange A. Iwasa J. Søgaard-Andersen L. Jensen G.J. Science. 2016; 351: aad2001Crossref PubMed Scopus (237) Google Scholar, Takhar et al., 2013Takhar H.K. Kemp K. Kim M. Howell P.L. Burrows L.L. J. Biol. Chem. 2013; 288: 9721-9728Crossref PubMed Scopus (80) Google Scholar). Therefore, the asymmetric hexameric ring can probably lock onto the PilC dimer platform. During this symmetric rotary process, the coordination of ATP hydrolysis on TtPilBATP hexamer drives the PilC dimer to rotate to scoop up a pilus subunit to promote pilus assembly (Figures 1 and 2). Overall, the atomic resolution structure of TtPilBATP determined by Mancl et al., 2016Mancl J.M. Black W.P. Robinson H. Yang Z. Schubot F.D. Structure. 2016; 24 (this issue): 1886-1897Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar provides wonderful insights into a distinct rotary motor mechanism for ATP hydrolysis and T4P assembly. This mechanism unveils a testable model for the PilB/GspE family and its protein partners. Because PilB directly interacts with PilC and the cytosolic nucleotide binding protein PilM (Bischof et al., 2016Bischof L.F. Friedrich C. Harms A. Søgaard-Andersen L. van der Does C. J. Biol. Chem. 2016; 291: 6946-6957Crossref PubMed Scopus (39) Google Scholar), it will be exciting to see how this symmetric rotary mechanism is applied in the PilM-PilB-PilC complex. As more structures are determined for T4P systems, better mechanisms revealing how bacteria use their molecular motors and pili for movement and virulence will enhance our ability to predict and control these amazing molecular machines for biology, biotechnology, and medicine. The authors apologize that many important related papers were not cited or discussed due to space limitations. We thank Ching-Wen Wu for assistance with the figure. The authors' efforts on self-assembling molecular machines are supported by a Robert A. Welch Chemistry Chair and the United States Department of Energy program Integrated Diffraction Analysis Technologies (IDAT). Crystal Structure of a Type IV Pilus Assembly ATPase: Insights into the Molecular Mechanism of PilB from Thermus thermophilusMancl et al.StructureSeptember 22, 2016In BriefHexameric ATPases power numerous cellular activities critical for bacterial virulence. Mancl et al. have solved the structure of a type IV assembly ATPase to produce insights into the mechanism of force generation and broadly into the mechanism of catalysis of PilB/GspE family enzymes. Full-Text PDF Open Archive
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