Crystal structure of the full-length ATPase GspE from the Vibrio vulnificus type II secretion system in complex with the cytoplasmic domain of GspL

Lü, Chan; Korotkov, Konstantin V.; Hól, Wim G. J.

doi:10.1016/j.jsb.2014.07.006

Cited by 36 publications

(40 citation statements)

References 79 publications

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“…Formation of "rods" of the cytoplasmic domain of GspL were recently observed in several crystal structures (68), suggesting that similar to GspL a higher oligomeric form of PilM is assembled in the presence of nucleotides and may play a role in the assembly or the function of the T4P system. In archaea, this role might be fulfilled by either the FlaH ATPase that interacts with the FlaI secretion ATPase (69) or FlaX (38), a membrane protein with a cytosolic domain that forms a ring-like structure around the FlaI secretion ATPase.…”

Section: Discussionmentioning

confidence: 84%

The Type IV Pilus Assembly ATPase PilB of Myxococcus xanthus Interacts with the Inner Membrane Platform Protein PilC and the Nucleotide-binding Protein PilM

Bischof

Friedrich

Harms

et al. 2016

Journal of Biological Chemistry

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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) 3 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 (1-3). 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, 5), bacterial competence systems that are involved in the uptake of DNA (6), and systems that are involved in the assembly of archaeal surface structures (7).T4P are highly dynamic structures that undergo cycles of extension and retraction (8, 9). 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 ϳ6 nm and can extend up to several micrometers in length (10). 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, 11). T4P retraction generates forces up to 150 piconewtons (12, 13), pulling a cell forward, and making T4P systems the strongest molecular motors known.The rod-shaped cells of the ␦-proteobacterium Myxococcus xanthus, assemble 5-10 T4P at the leading cell pole that extend and retract to generate cell movemen...

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Section: Discussionmentioning

confidence: 84%

The Type IV Pilus Assembly ATPase PilB of Myxococcus xanthus Interacts with the Inner Membrane Platform Protein PilC and the Nucleotide-binding Protein PilM

Bischof

Friedrich

Harms

et al. 2016

Journal of Biological Chemistry

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“…This model could explain why the PilN chimera was more detrimental to the function of the system than the PilO chimera, as the TMS swap in PilN might alter relay of conformational changes from PilM. In the T2S and the type IVb pilus (T4bP) systems, PilB-like ATPases have been cocrystallized with their PilM homologs, confirming interactions between these components (61)(62)(63). Alternatively, communication between the PilMNOP subcomplex and PilQ could be altered such that the secretin is not appropriately gated to allow for normal pilus assembly/disassembly dynamics (37,38).…”

Section: Discussionmentioning

confidence: 93%

Novel Role for PilNO in Type IV Pilus Retraction Revealed by Alignment Subcomplex Mutations

et al. 2015

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Type IV pili (T4P) are dynamic protein filaments that mediate bacterial adhesion, biofilm formation, and twitching motility. The highly conserved PilMNOP proteins form an inner membrane alignment subcomplex required for function of the T4P system, though their exact roles are unclear. Three potential interaction interfaces for PilNO were identified: core-core, coiled coils (CC), and the transmembrane segments (TMSs). A high-confidence PilNO heterodimer model was used to select key residues for mutation, and the resulting effects on protein-protein interactions were examined both in a bacterial two-hybrid (BTH) system and in their native Pseudomonas aeruginosa context. Mutations in the oppositely charged CC regions or the TMS disrupted PilNO heterodimer formation in the BTH assay, while up to six combined mutations in the core failed to disrupt the interaction. When the mutations were introduced into the P. aeruginosa chromosome at the pilN or pilO locus, specific changes at each of the three interfaces-including core mutations that failed to disrupt interactions in the BTH system-abrogated surface piliation and/or impaired twitching motility. Unexpectedly, specific CC mutants were hyperpiliated but nonmotile, a hallmark of pilus retraction defects. These data suggest that PilNO participate in both the extension and retraction of T4P. Our findings support a model of multiple, precise interaction interfaces between PilNO; emphasize the importance of studying protein function in a minimally perturbed context and stoichiometry; and highlight potential target sites for development of small-molecule inhibitors of the T4P system. IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen that uses type IV pili (T4P) for host attachment. The T4P machinery is composed of four cell envelope-spanning subcomplexes. PilN and PilO heterodimers are part of the alignment subcomplex and essential for T4P function. Three potential PilNO interaction interfaces (the core-core, coiled-coil, and transmembrane segment interfaces) were probed using site-directed mutagenesis followed by functional assays in an Escherichia coli two-hybrid system and in P. aeruginosa. Several mutations blocked T4P assembly and/or motility, including two that revealed a novel role for PilNO in pilus retraction, while other mutations affected extension dynamics. These critical PilNO interaction interfaces represent novel targets for small-molecule inhibitors with the potential to disrupt T4P function.A larming increases in antibiotic resistance have prompted the search for alternative strategies to combat bacterial infection (1). Genomic strategies designed to identify essential bacterial targets have been informative but less successful for drug development than originally predicted due to high mutation rates and resistance (2). Alternative strategies-such as phage therapy and the use of "antivirulence" drugs that target bacterial factors involved in pathogenesis-show promise since they can effectively disarm bacteria, which are then more easily...

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“…This interface corresponds to the PilN binding pocket of PilM and may be a conserved binding site because it is located at a hinge point that can rotate upon ATP binding (62). Hence, where it has been evaluated, peptide binding to this interface can be modulated by ATP binding and/or catalysis (53,63 (28) suggests that GspL and BfpC do not need to regulate association to their PilN-like portions, consistent with fusion of these domains.…”

Section: Discussionmentioning

confidence: 93%

“…However, GspL and BfpC deviate substantially from PilM. These proteins are more than 100 residues shorter and consequently lack the ability to bind ATP (28), thereby limiting the extent to which we can generate testable hypotheses for PilM based on the structures of GspL and BfpC.…”

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confidence: 99%

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

McCallum

Tammam

Little

et al. 2016

Journal of Biological Chemistry

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Pseudomonas aeruginosa is an opportunistic bacterial pathogen that expresses type IVa pili. The pilus assembly system, which promotes surface-associated twitching motility and virulence, is composed of inner and outer membrane subcomplexes, connected by an alignment subcomplex composed of PilMNOP. PilM binds to the N terminus of PilN, and we hypothesize that this interaction causes functionally significant structural changes in PilM. To characterize this interaction, we determined the crystal structures of PilM and a PilM chimera where PilM was fused to the first 12 residues of PilN (PilM⅐PilN(1-12)).

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Crystal structure of the full-length ATPase GspE from the Vibrio vulnificus type II secretion system in complex with the cytoplasmic domain of GspL

Cited by 36 publications

References 79 publications

The Type IV Pilus Assembly ATPase PilB of Myxococcus xanthus Interacts with the Inner Membrane Platform Protein PilC and the Nucleotide-binding Protein PilM

The Type IV Pilus Assembly ATPase PilB of Myxococcus xanthus Interacts with the Inner Membrane Platform Protein PilC and the Nucleotide-binding Protein PilM

Novel Role for PilNO in Type IV Pilus Retraction Revealed by Alignment Subcomplex Mutations

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

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