Structure and assembly of an inner membrane platform for initiation of type IV pilus biogenesis

Karuppiah, V.; Collins, Richard F.; Thistlethwaite, Angela; Ya, Gao; Derrick, Jeremy P.

doi:10.1073/pnas.1312313110

Cited by 58 publications

(73 citation statements)

References 73 publications

(94 reference statements)

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“…This stoichiometry is consistent with our previous data, where PilNOP were shown by gel filtration to elute as either a trimer or a dimer of trimers (20). A dimeric complex of PilMNO (2:2:2) was reported for T. thermophilus (29), fitting with the idea that P. aeruginosa PilN and PilO form a dimer of dimers (17,19,20,29).…”

Section: Piln and Pilo May Form Functional Dimers Of Dimers Insupporting

confidence: 92%

“…2B), with masses corresponding to PilNO heterodimers and PilN homodimers. Although a PilN homodimer (bound to PilM) from the T4P system of T. thermophilus was observed in vitro (29) (Fig. 3, A and B), suggesting that there are (at least) two possible interfaces for each protein.…”

Section: Piln and Pilo May Form Functional Dimers Of Dimers Inmentioning

confidence: 99%

“…The structure of a periplasmic portion of PilN from T. thermophilus is available (29); however, due to the low primary sequence identity with PilN from P. aeruginosa, as well as a different arrangement of secondary structure elements (Thermus PilN has an extra ␣-helix inserted in the first ␣␤␤ motif), we were unable to generate a high confidence model using that structural template. P. aeruginosa PilN is predicted to have a structural organization similar to PilO, and thus we used the truncated P. aeruginosa PilO homodimer structure (18) to generate a high confidence Phyre 2 (45) model of a PilN ⌬57 monomer, and we used the PilO homodimer interface to model a PilNO heterodimer using the same core-core interface (Fig.…”

Section: Selection Of Residues For Cys Substitution-ourmentioning

confidence: 99%

“…2D), suggesting that the homo-and heterodimer interfaces might be angled differently. Models of the core interaction interface between PilN and PilO in T. thermophilus (29) and M. xanthus (49) show them as tilted, possibly because of longer predicted coiled coils in PilO (18). The persistence of PilN and PilO monomers (Figs.…”

Section: Piln and Pilo May Form Functional Dimers Of Dimers Inmentioning

confidence: 99%

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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

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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...

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Section: Piln and Pilo May Form Functional Dimers Of Dimers Insupporting

confidence: 92%

Section: Piln and Pilo May Form Functional Dimers Of Dimers Inmentioning

confidence: 99%

Section: Selection Of Residues For Cys Substitution-ourmentioning

confidence: 99%

Section: Piln and Pilo May Form Functional Dimers Of Dimers Inmentioning

confidence: 99%

See 2 more Smart Citations

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

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“…2). The proteins encoded in the pilMNOPQ cluster initiate the formation of the biogenesis apparatus across the outer and inner membranes (Karuppiah et al ., 2013). In Myxococcus xanthus, another delta‐proteobacterium, assembly is initiated with the formation of the PilQ secretin ring on the outer membrane and the recruitment of the inner membrane PilP lipoprotein and the PilNO subcomplex (Friedrich et al ., 2014).…”

Section: A Nanowire Pathway For Metal Reductionmentioning

confidence: 99%

Harnessing the power of microbial nanowires

Reguera

2018

Microbial Biotechnology

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SummaryThe reduction of iron oxide minerals and uranium in model metal reducers in the genus Geobacter is mediated by conductive pili composed primarily of a structurally divergent pilin peptide that is otherwise recognized, processed and assembled in the inner membrane by a conserved Type IVa pilus apparatus. Electronic coupling among the peptides is promoted upon assembly, allowing the discharge of respiratory electrons at rates that greatly exceed the rates of cellular respiration. Harnessing the unique properties of these conductive appendages and their peptide building blocks in metal bioremediation will require understanding of how the pilins assemble to form a protein nanowire with specialized sites for metal immobilization. Also important are insights into how cells assemble the pili to make an electroactive matrix and grow on electrodes as biofilms that harvest electrical currents from the oxidation of waste organic substrates. Genetic engineering shows promise to modulate the properties of the peptide building blocks, protein nanowires and current‐harvesting biofilms for various applications. This minireview discusses what is known about the pilus material properties and reactions they catalyse and how this information can be harnessed in nanotechnology, bioremediation and bioenergy applications.

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The traffic ATPase PilF interacts with the inner membrane platform of the DNA translocator and type IV pili from Thermus thermophilus

2018

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A major driving force for the adaptation of bacteria to changing environments is the uptake of naked DNA from the environment by natural transformation, which allows the acquisition of new capabilities. Uptake of the high molecular weight DNA is mediated by a complex transport machinery that spans the entire cell periphery. This DNA translocator catalyzes the binding and splitting of double‐stranded DNA and translocation of single‐stranded DNA into the cytoplasm, where it is recombined with the chromosome. The thermophilic bacterium Thermus thermophilus exhibits the highest transformation frequencies reported and is a model system to analyze the structure and function of this macromolecular transport machinery. Transport activity is powered by the traffic ATPase PilF, a soluble protein that forms hexameric complexes. Here, we demonstrate that PilF physically binds to an inner membrane assembly platform of the DNA translocator, comprising PilMNO, via the ATP‐binding protein PilM. Binding to PilMNO or PilMN stimulates the ATPase activity of PilF ~ 2‐fold, whereas there is no stimulation when binding to PilM or PilN alone. A PilM K26A variant defective in ATP binding still binds PilF and, together with PilN, stimulates PilF‐mediated ATPase activity. PilF is unique in having three conserved GSPII (general secretory pathway II) domains (A–C) at its N terminus. Deletion analyses revealed that none of the GSPII domains is essential for binding PilMN, but GSPIIC is essential for PilMN‐mediated stimulation of ATP hydrolysis by PilF. Our data suggest that PilM is a coupling protein that physically and functionally connects the soluble motor ATPase PilF to the DNA translocator via the PilMNO assembly platform.

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Structure and assembly of an inner membrane platform for initiation of type IV pilus biogenesis

Cited by 58 publications

References 73 publications

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

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

Harnessing the power of microbial nanowires

The traffic ATPase PilF interacts with the inner membrane platform of the DNA translocator and type IV pili from Thermus thermophilus

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