Intrinsically Disordered Protein Threads Through the Bacterial Outer-Membrane Porin OmpF

Housden, Nicholas G.; Hopper, Jonathan T. S.; Lukoyanova, Natalya; Rodríguez-Larrea, David; Wojdyla, Justyna Aleksandra; Klein, Alexander; Kaminska, Renata; Bayley, Hagan; Saibil, Helen R.; Robinson, Carol V.; Kleanthous, Colin

doi:10.1126/science.1237864

Cited by 113 publications

(176 citation statements)

References 51 publications

Supporting

Mentioning

167

Contrasting

Unclassified

Order By: Relevance

“…The T83 peptide is an intrinsically unstructured part of the N-terminal transport domain of E colicins, and it is the first domain to enter OmpF lumen (47). Indeed, recent studies show that the unstructured colicin E9 N-terminal domain threads through two adjacent OmpF trimer subunits in opposite orientation (48). Our results suggest the nature of the RcsF unstructured linker interaction with OmpC/F might be similar to that of unstructured colicin linker.…”

Section: Discussionmentioning

confidence: 65%

Transmembrane domain of surface-exposed outer membrane lipoprotein RcsF is threaded through the lumen of β-barrel proteins

Konovalova

Perlman

Cowles

et al. 2014

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

114

173

View full text Add to dashboard Cite

RcsF (regulator of capsule synthesis) is an outer membrane (OM) lipoprotein that functions to sense defects such as changes in LPS. However, LPS is found in the outer leaflet, and RcsF was thought to be tethered to the inner leaflet by its lipidated N terminus, raising the question of how it monitors LPS. We show that RcsF has a transmembrane topology with the lipidated N terminus on the cell surface and the C-terminal signaling domain in the periplasm. Strikingly, the short, unstructured, charged transmembrane domain is threaded through the lumen of β-barrel OM proteins where it is protected from the hydrophobic membrane interior. We present evidence that these unusual complexes, which contain one protein inside another, are formed by the Bam complex that assembles all β-barrel proteins in the OM. The ability of the Bam complex to expose lipoproteins at the cell surface underscores the mechanistic versatility of the β-barrel assembly machine.membrane biogenesis | protein folding | signal transduction | envelope stress response

show abstract

Section: Discussionmentioning

confidence: 65%

Transmembrane domain of surface-exposed outer membrane lipoprotein RcsF is threaded through the lumen of β-barrel proteins

Konovalova

Perlman

Cowles

et al. 2014

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

114

173

View full text Add to dashboard Cite

show abstract

“…1B) facilitates a search in two dimensions, via lateral diffusion and a "fishing pole" mechanism, by which the highly unstructured N-terminal T domain finds a copy of its OmpF translocator (8,13). For colicins E3 and E9, segments of their T domains were shown to be bound inside the pore of OmpF (14)(15)(16), and their T domains have also been shown to occlude OmpF channels in planar lipid bilayer membranes (16,17). For colicin Ia, a pore-forming colicin, a second copy of its Cir receptor serves as its translocator (10,18).…”

Section: Importancementioning

confidence: 99%

The Colicin E1 TolC Box: Identification of a Domain Required for Colicin E1 Cytotoxicity and TolC Binding

Jakes

2017

J Bacteriol

View full text Add to dashboard Cite

Colicins are protein toxins made by Escherichia coli to kill related bacteria that compete for scarce resources. All colicins must cross the target cell outer membrane in order to reach their intracellular targets. Normally, the first step in the intoxication process is the tight binding of the colicin to an outer membrane receptor protein via its central receptor-binding domain. It is shown here that for one colicin, E1, that step, although it greatly increases the efficiency of killing, is not absolutely necessary. For colicin E1, the second step, translocation, relies on the outer membrane/transperiplasmic protein TolC. The normal role of TolC in bacteria is as an essential component of a family of tripartite drug and toxin exporters, but for colicin E1, it is essential for its import. Colicin E1 and some N-terminal translocation domain peptides had been shown previously to bind in vitro to TolC and occlude channels made by TolC in planar lipid bilayer membranes. Here, a set of increasingly shorter colicin E1 translocation domain peptides was shown to bind to Escherichia coli in vivo and protect them from subsequent challenge by colicin E1. A segment of only 21 residues, the "TolC box," was thereby defined; that segment is essential for colicin E1 cytotoxicity and for binding of translocation domain peptides to TolC. IMPORTANCEThe Escherichia coli outer membrane/transperiplasmic protein TolC is normally an essential component of the bacterium's tripartite drug and toxin export machinery. The protein toxin colicin E1 instead uses TolC for its import into the cells that it kills, thereby subverting its normal role. Increasingly shorter constructs of the colicin's N-terminal translocation domain were used to define an essential 21-residue segment that is required for both colicin cytotoxicity and for binding of the colicin's translocation domain to bacteria, in order to protect them from subsequent challenge by active colicin E1. Thus, an essential TolC binding sequence of colicin E1 was identified and may ultimately lead to the development of drugs to block the bacterial drug export pathway.KEYWORDS TolC, colicins, drug efflux, membrane translocation, toxins E scherichia coli competes for scarce resources by making plasmid-encoded protein toxins called colicins, which efficiently kill closely related bacteria. All of the few dozen or so colicins that have been identified kill their targets by one of a few basic mechanisms: (i) making an ion-permeable channel in the inner membrane of the target cell, which depolarizes and kills the cell (1); (ii) enzymatically cleaving its rRNA, tRNA, or DNA in the cytoplasm (2, 3); or (iii) degrading peptidoglycan precursors in the periplasm (4, 5). Regardless of their ultimate killing mechanism, all colicins must cross at least the outer membrane in order to reach their targets. The way one particular colicin, E1, makes that transit is the subject of this study.Colicins have a well-defined domain structure, with the killing (catalytic or channelforming) domain at the ...

show abstract

“…Exploring further the quantitative aspects of this native DESI platform we selected OBS1 a 17‐residue peptide known to bind within the pores of OmpF with binding constants determined previously by both ITC and nanoES MS 24, 25. We deposited OmpF on the stage in OG detergent and incubated OmpF with increasing concentrations of OBS1 (0–75 μ m ) and deposited these protein‐peptide complexes onto the native DESI stage.…”

mentioning

confidence: 99%

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces

et al. 2017

Self Cite

View full text Add to dashboard Cite

Mass spectrometry (MS) applications for intact protein complexes typically require electrospray (ES) ionization and have not been achieved via direct desorption from surfaces. Desorption ES ionization (DESI) MS has however transformed the study of tissue surfaces through release and characterisation of small molecules. Motivated by the desire to screen for ligand binding to intact protein complexes we report the development of a native DESI platform. By establishing conditions that preserve non‐covalent interactions we exploit the surface to capture a rapid turnover enzyme–substrate complex and to optimise detergents for membrane protein study. We demonstrate binding of lipids and drugs to membrane proteins deposited on surfaces and selectivity from a mix of related agonists for specific binding to a GPCR. Overall therefore we introduce this native DESI platform with the potential for high‐throughput ligand screening of some of the most challenging drug targets including GPCRs.

show abstract

Intrinsically Disordered Protein Threads Through the Bacterial Outer-Membrane Porin OmpF

Cited by 113 publications

References 51 publications

Transmembrane domain of surface-exposed outer membrane lipoprotein RcsF is threaded through the lumen of β-barrel proteins

Transmembrane domain of surface-exposed outer membrane lipoprotein RcsF is threaded through the lumen of β-barrel proteins

The Colicin E1 TolC Box: Identification of a Domain Required for Colicin E1 Cytotoxicity and TolC Binding

Native Desorption Electrospray Ionization Liberates Soluble and Membrane Protein Complexes from Surfaces

Contact Info

Product

Resources

About