PilN Binding Modulates the Structure and Binding Partners of the Pseudomonas aeruginosa Type IVa Pilus Protein PilM

McCallum, Matthew; Tammam, Stephanie; Little, Dustin J.; Robinson, Howard; Koo, Jarley; Shah, Megha; Calmettes, Charles; Moraes, Trevor F.; Burrows, Lori L.; Howell, P. Lynne

doi:10.1074/jbc.m116.718353

Cited by 57 publications

(62 citation statements)

References 71 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…PilG is likely to become a stable part of the machinery once PilM has been recruited to the PilNOP complex (PilG does not form a stable complex with PilNOP), which is consistent with Tfp subtomograms showing that the PilG "dome" structure requires the presence of the PilM ring (34). Similarly, the ATPase PilF is likely to be recruited to the complex via interactions with PilM (PilF does not copurify with PilNOP) (33,43) and/or PilG (32), which is consistent with Tfp subtomograms where the PilF "disc" requires both the PilM ring and PilG dome (34). Finally, PilE from a pool of processed subunits awaiting in the membrane would diffuse to the PilFGMNOP complex (28), which would scoop them out of the lipid bilayer and into the base of a growing filament (Fig.…”

Section: Discussionsupporting

confidence: 52%

“…but there is ample evidence that they establish multiple binary/ ternary interactions at the cytoplasmic membrane (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33). Moreover, in a recent study in Myxococcus xanthus, in which the entire Tfp machinery was visualized by cryoelectron tomography (34), it was shown that these five proteins form a series of interconnected layers spanning the cytoplasmic membrane, which is a priori compatible with a role in filament assembly.…”

Section: Significancementioning

confidence: 99%

See 1 more Smart Citation

Reconstitution of a minimal machinery capable of assembling periplasmic type IV pili

Goosens

Büsch

Georgiadou

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Type IV pili (Tfp), which are key virulence factors in many bacterial pathogens, define a large group of multipurpose filamentous nanomachines widespread in Bacteria and Archaea. Tfp biogenesis is a complex multistep process, which relies on macromolecular assemblies composed of 15 conserved proteins in model gramnegative species. To improve our limited understanding of the molecular mechanisms of filament assembly, we have used a synthetic biology approach to reconstitute, in a nonnative heterologous host, a minimal machinery capable of building Tfp. Here we show that eight synthetic genes are sufficient to promote filament assembly and that the corresponding proteins form a macromolecular complex at the cytoplasmic membrane, which we have purified and characterized biochemically. Our results contribute to a better mechanistic understanding of the assembly of remarkable dynamic filaments nearly ubiquitous in prokaryotes.type IV pili | type IV filamentous nanomachines | filament assembly | synthetic biology E volution has provided prokaryotes with sophisticated surface nanomachines that endow them with many functions instrumental to their ability to colonize most niches on Earth. Among these engineering marvels, type IV filamentous (Tff) nanomachines (1), of which type IV pili (Tfp) are the paradigm, are unique for two reasons. They are exceptionally (i) widespread, with genes encoding distinctive proteins found in virtually every prokaryotic genome, and (ii) multipurpose, associated with functions as diverse as adhesion, motility, protein secretion, DNA uptake, electric conductance, and so forth (1). Much of this broad distribution and multifunctionality is due to Tfp (1).All Tff nanomachines share multiple components and are thought to use common basic operating principles. They have at their core a filament, which can be long or short and is a polymeric assembly of a protein named pilin, PilE in our model Tfp-expressing species Neisseria meningitidis (meningococcal nomenclature will be used here). Type IV pilins are produced as prepilins with a distinctive N-terminal class III signal peptide (2), consisting of a short hydrophilic leader peptide followed by a stretch of 21 hydrophobic residues, always forming an extended α-helix (3). This signal peptide is first recognized by the Sec machinery (4, 5), which translocates prepilins across the cytoplasmic membrane, where they remain embedded as bitopic proteins. The leader peptide is then cleaved by an integral membrane aspartic protease (6, 7), the prepilin peptidase PilD. This processing, which does not require other Pil proteins (8), is a prerequisite for polymerization of pilins into filaments. Filaments are helical polymers in which the pilins' extended N-terminal α-helices are buried within the filament core, almost parallel to its long axis (9). Finally, in gram-negative Tfp-expressing bacteria, filaments cross the outer membrane through a pore formed by the secretin PilQ (10).The molecular mechanisms of filament assembly remain poorly understood. However,...

show abstract

Section: Discussionsupporting

confidence: 52%

Section: Significancementioning

confidence: 99%

Reconstitution of a minimal machinery capable of assembling periplasmic type IV pili

Goosens

Büsch

Georgiadou

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…In our work, only PilM or PilP could modulate the PilNO core interface. We speculate that subtle reorientation of PilN and PilO may occur during the transition between resting and active or "extension" versus "retraction" states of the T4P system, consistent with the hypothesis that PilM may disengage from PilN (41). Although a ratcheting-type mechanism, in which PilNO conformational dynamics might contribute to the stepwise addition or removal of pilin subunits cannot yet be ruled out, our data do not support this idea.…”

Section: Piln and Pilo Homodimers May Represent A Functional Rather Tcontrasting

confidence: 52%

“…6, B and C), suggesting that the PilNO interface no longer adopts an orientation permissive for disulfide bond formation. New crystal structures of a P. aeruginosa PilM homodimer and of monomeric PilM fused to part of its binding partner, PilN (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), revealed that in the absence of PilN, the seven N-terminal residues of PilM (PilM (1-7)) bind in the PilN binding cleft of a second PilM monomer (41). Thus, PilM dimerizes in the absence of PilN.…”

Section: Piln and Pilo Homodimers May Represent A Functional Rather Tmentioning

confidence: 99%

Type IV Pilus Alignment Subcomplex Proteins PilN and PilO Form Homo- and Heterodimers in Vivo

Leighton

Yong

Howell

et al. 2016

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and is resistant to many antibiotics. Type IV pili (T4P) are among the key virulence factors used by P. aeruginosa for host cell attachment, biofilm formation, and twitching motility, making this system a promising target for novel therapeutics. Point mutations in the conserved PilMNOP alignment subcomplex were previously shown to have distinct effects on assembly and disassembly of T4P, suggesting that it may function in a dynamic manner. We introduced mutations encoding Cys substitutions into pilN and/or pilO on the chromosome to maintain normal stoichiometry and expression levels and captured covalent PilNO heterodimers, as well as PilN and PilO homodimers, in vivo. Most covalent PilN or PilO homodimers had minimal functional impact in P. aeruginosa, suggesting that homodimers are a physiologically relevant state. However, certain covalent homo-or heterodimers eliminated twitching motility, suggesting that specific PilNO configurations are essential for T4P function. These data were verified using soluble N-terminal truncated fragments of PilN and PilO Cys mutants, which purified as a mixture of homo-and heterodimers at volumes consistent with a tetramer. Deletion of genes encoding alignment subcomplex components, PilM or PilP, but not other T4P components, including the motor ATPases PilB or PilT, blocked in vivo formation of disulfide-bonded PilNO heterodimers, suggesting that both PilM and PilP influence the heterodimer interface. Combined, our data suggest that T4P function depends on rearrangements at PilN and PilO interfaces.Many bacteria, including Pseudomonas aeruginosa, use type IV pili (T4P) 3 for surface attachment/adhesion, biofilm formation, and twitching motility (1-6). T4P are divided into T4aP and T4bP subgroups, based on differences in pilin size and organization of the assembly machinery (6, 7). Here we focus on T4aP, henceforth referred to as T4P. T4P are long, thin, fibrous appendages, which extend from the bacteria, attach to a surface, and are retracted back into the cell, winching the cell forward. This flagellum-independent form of locomotion is called twitching motility. Four subcomplexes make up a T4P machine in P. aeruginosa as follows: the outer membrane (OM) secretin (PilQ) and its pilotin (PilF) (8 -12); the inner membrane (IM) platform protein (PilC) and cytoplasmic motor proteins (ATPases PilBTU) (13,14); the helical pilus fiber, composed mainly of the major pilin subunit (PilA) plus minor pilins (PilVWXE and FimU) and adhesin PilY1 (5, 15, 16); and the alignment subcomplex proteins (PilMNOP) that span from the cytoplasm to the OM secretin (17-20). PilN and PilO are bitopic IM proteins with similar predicted secondary structures. These proteins are essential for T4P function and connect the cytoplasmic alignment subcomplex component, PilM, with the IM-associated lipoprotein PilP that interacts with the N0 domain of the secretin monomer PilQ (19,21). Proposed functions of the PilMNOP subcomplex include ali...

show abstract

“…In the model bacterium Pseudomonas aeruginosa, an inner membrane motor subcomplex consisting of the platform protein PilC and three hexameric ATPases-PilB, PilT, and PilU-provide energy for T4aP extension and retraction (7)(8)(9). A second inner membrane alignment subcomplex composed of PilMNOP connects the motor subcomplex with an outer membrane secretin composed of multimeric PilQ (2,(10)(11)(12)(13). Finally, the pilus fiber is composed of the major pilus subunit PilA and a set of minor pilins that prime pilus assembly and export the adhesin PilY1 to the cell surface (14).…”

mentioning

confidence: 99%

The Conserved Tetratricopeptide Repeat-Containing C-Terminal Domain of Pseudomonas aeruginosa FimV Is Required for Its Cyclic AMP-Dependent and -Independent Functions

et al. 2016

Self Cite

View full text Add to dashboard Cite

FimV is a Pseudomonas aeruginosa inner membrane protein that regulates intracellular cyclic AMP (cAMP) levels-and thus type IV pilus (T4P)-mediated twitching motility and type II secretion (T2S)-by activating the adenylate cyclase CyaB. Its cytoplasmic domain contains three predicted tetratricopeptide repeat (TPR) motifs separated by an unstructured region: two proximal to the inner membrane and one within the "FimV C-terminal domain," which is highly conserved across diverse homologs. Here, we present the crystal structure of the FimV C terminus, FimV 861-919 , containing a TPR motif decorated with solvent-exposed, charged side chains, plus a C-terminal capping helix. FimV 689 , a truncated form lacking this C-terminal motif, did not restore wild-type levels of twitching or surface piliation compared to the full-length protein. FimV 689 failed to restore wild-type levels of the T4P motor ATPase PilU or T2S, suggesting that it was unable to activate cAMP synthesis. Bacterial two-hybrid analysis showed that TPR3 interacts directly with the CyaB activator, FimL. However, FimV 689 failed to restore wild-type motility in a fimV mutant expressing a constitutively active CyaB (fimV cyaB-R456L), suggesting that the C-terminal motif is also involved in cAMP-independent functions of FimV. The data show that the highly conserved TPR-containing C-terminal domain of FimV is critical for its cAMP-dependent and -independent functions. IMPORTANCEFimV is important for twitching motility and cAMP-dependent virulence gene expression in P. aeruginosa. FimV homologs have been identified in several human pathogens, and their functions are not limited to T4P expression. The C terminus of FimV is remarkably conserved among otherwise very diverse family members, but its role is unknown. We provide here biological evidence for the importance of the C-terminal domain in both cAMP-dependent (through FimL) and -independent functions of FimV. We present X-ray crystal structures of the conserved C-terminal domain and identify a consensus sequence for the C-terminal TPR within the conserved domain. Our data extend our knowledge of FimV's functionally important domains, and the structures and consensus sequences provide a foundation for studies of FimV and its homologs. T ype IV pili (T4P) are filamentous surface appendages produced by a wide range of bacteria and archaea (1, 2), where they assist in DNA uptake, surface attachment, and twitching motility (3-5). There are two major subfamilies of T4P: T4aP and T4bP. T4aP are typically associated with twitching (2), a process in which pili undergo repeated cycles of extension, adhesion, and retraction, thus acting as molecular grappling hooks to pull cells along a surface.The T4aP machinery is composed of four structural subcomplexes (6). In the model bacterium Pseudomonas aeruginosa, an inner membrane motor subcomplex consisting of the platform protein PilC and three hexameric ATPases-PilB, PilT, and PilU-provide energy for T4aP extension and retraction (7-9). A second inner membrane al...

show abstract

PilN Binding Modulates the Structure and Binding Partners of the Pseudomonas aeruginosa Type IVa Pilus Protein PilM

Cited by 57 publications

References 71 publications

Reconstitution of a minimal machinery capable of assembling periplasmic type IV pili

Reconstitution of a minimal machinery capable of assembling periplasmic type IV pili

Type IV Pilus Alignment Subcomplex Proteins PilN and PilO Form Homo- and Heterodimers in Vivo

The Conserved Tetratricopeptide Repeat-Containing C-Terminal Domain of Pseudomonas aeruginosa FimV Is Required for Its Cyclic AMP-Dependent and -Independent Functions

Contact Info

Product

Resources

About