The Crystal Structure of the Periplasmic Domain of the Type II Secretion System Protein EpsM From Vibrio cholerae: The Simplest Version of the Ferredoxin Fold

Abendroth, Jan; Rice, Adrian E.; McLuskey, Karen; Bagdasarian, Michael; Hól, Wim G. J.

doi:10.1016/j.jmb.2004.01.064

Cited by 61 publications

(75 citation statements)

References 40 publications

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“…In the crystal structure model of the dimer, subunit-subunit interactions occur primarily through contacts between residues 123 to 133 and 147 of the EpsM monomer (2). Removal of these residues should result in a form of EpsM that does not dimerize, consistent with our experimental finding that deletion of residues 100 to 165 to create GFP-EpsM⌬66 prevented interaction with full-length EpsM (Fig.…”

Section: Discussionsupporting

confidence: 82%

“…All T2S systems contain an EpsM homolog (26) that is essential for function, however. The X-ray crystal structure of a large portion of the periplasmic domain of EpsM has been determined and represents a novel version of the ferredoxin fold, but the extensive variety of functions that ferredoxin-like proteins perform lends few clues to the function of EpsM (2). In solution, this periplasmic EpsM construct, which consists of residues 65 to 165, forms dimers (2).…”

mentioning

confidence: 99%

“…The X-ray crystal structure of a large portion of the periplasmic domain of EpsM has been determined and represents a novel version of the ferredoxin fold, but the extensive variety of functions that ferredoxin-like proteins perform lends few clues to the function of EpsM (2). In solution, this periplasmic EpsM construct, which consists of residues 65 to 165, forms dimers (2). This suggests that neither the transmembrane domain nor the short N terminus of EpsM is needed for this process.…”

mentioning

confidence: 99%

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Mapping Critical Interactive Sites within the Periplasmic Domain of the Vibrio cholerae Type II Secretion Protein EpsM

2007

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The type II secretion (T2S) system is present in many gram-negative species, both pathogenic and nonpathogenic, where it supports the delivery of a variety of toxins, proteases, and lipases into the extracellular environment. In Vibrio cholerae, the T2S apparatus is composed of 12 Eps proteins that assemble into a multiprotein complex that spans the entire cell envelope. Two of these proteins, EpsM and EpsL, are key components of the secretion machinery present in the inner membrane. In addition to likely forming homodimers, EpsL and EpsM have been shown to form a stable complex in the inner membrane and to protect each other from proteolytic degradation. To identify and map the specific regions of EpsM involved in protein-protein interactions with both another molecule of EpsM and EpsL, we tested the interactions of deletion constructs of EpsM with full-length EpsM and EpsL by functional characterization and copurification as well as coimmunoprecipitation. Analysis of the truncated EpsM mutants revealed that the region of EpsM from amino acids 100 to 135 is necessary for EpsM to form homo-oligomers, while residues 84 to 99 appear to be critical for a stable interaction with EpsL.

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Section: Discussionsupporting

confidence: 82%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Mapping Critical Interactive Sites within the Periplasmic Domain of the Vibrio cholerae Type II Secretion Protein EpsM

2007

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“…Structural and functional similarities between the T2S and T4P systems have long been recognized (51). Despite minimal sequence identity between orthologous components of the alignment subcomplexes in the two systems, atomic resolution structures revealed a high degree of structural similarity, suggesting conservation of function (33,34,46,(52)(53)(54). Previous studies suggested that multiple segments of GspL and GspM, the T2S equivalents of PilMN and PilO, respectively, participate in their interaction (47,48).…”

Section: Discussionmentioning

confidence: 99%

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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“…Interestingly, the ␤-sheet face of ferredoxin-like proteins is so often involved in mediating interactions with other moieties that it has been described as a ''supersite'' for ligand binding (32). Some members of the ferredoxin-like family have been at least implicated in peptide binding and protein transport systems, for example, the EpsM protein of the bacterial type II secretion system (33), and others are chaperones involved in metalloprotein assembly processes, for example, CopZ (34), UreE (35), and HypF (36).…”

Section: Discussionmentioning

confidence: 99%

Structural diversity in twin-arginine signal peptide-binding proteins

Maillard

Spronk

Buchanan

et al. 2007

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

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The twin-arginine transport (Tat) system is dedicated to the translocation of folded proteins across the bacterial cytoplasmic membrane. Proteins are targeted to the Tat system by signal peptides containing a twin-arginine motif. In Escherichia coli, many Tat substrates bind redox-active cofactors in the cytoplasm before transport. Coordination of cofactor insertion with protein export involves a ''Tat proofreading'' process in which chaperones bind twin-arginine signal peptides, thus preventing premature export. The initial Tat signal-binding proteins described belonged to the TorD family, which are required for assembly of N-and S-oxide reductases. Here, we report that E. coli NapD is a Tat signal peptide-binding chaperone involved in biosynthesis of the Tatdependent nitrate reductase NapA. NapD binds tightly and specifically to the NapA twin-arginine signal peptide and suppresses signal peptide translocation activity such that transport via the Tat pathway is retarded. High-resolution, heteronuclear, multidimensional NMR spectroscopy reveals the 3D solution structure of NapD. The chaperone adopts a ferredoxin-type fold, which is completely distinct from the TorD family. Thus, NapD represents a new family of twin-arginine signal-peptide-binding proteins.metalloenzyme biosynthesis ͉ molecular chaperone ͉ NMR solution structure ͉ protein-protein interactions ͉ twin-arginine transport pathway

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The Crystal Structure of the Periplasmic Domain of the Type II Secretion System Protein EpsM From Vibrio cholerae: The Simplest Version of the Ferredoxin Fold

Cited by 61 publications

References 40 publications

Mapping Critical Interactive Sites within the Periplasmic Domain of the Vibrio cholerae Type II Secretion Protein EpsM

Mapping Critical Interactive Sites within the Periplasmic Domain of the Vibrio cholerae Type II Secretion Protein EpsM

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

Structural diversity in twin-arginine signal peptide-binding proteins

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