Lipid binding attenuates channel closure of the outer membrane protein OmpF

Liko, Idlir; Degiacomi, Matteo T.; Lee, Sejeong; Newport, Thomas D.; Gault, Joseph; Reading, Eamonn; Hopper, Jonathan T. S.; Housden, Nicholas G.; White, Paul; Colledge, Matthew; Sula, Altin; Wallace, B.A.; Kleanthous, Colin; Stansfeld, Phillip J.; Bayley, Hagan; Benesch, Justin L. P.; Allison, Timothy M.; Robinson, Carol V.

doi:10.1073/pnas.1721152115

Cited by 41 publications

(44 citation statements)

References 48 publications

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“… 16 , 27 , 32 , 33 The lipid membrane environment may also play a role in facilitating the release of the colicin immunity protein 34 and in altering the binding properties of porins. 35 More elaborate in vitro reconstitution of the ColE9 translocation machinery may be required for a detailed elucidation of this intriguing process.…”

Section: Discussionmentioning

confidence: 99%

Transmembrane Epitope Delivery by Passive Protein Threading through the Pores of the OmpF Porin Trimer

et al. 2020

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Trimeric porins in the outer membrane (OM) of Gram-negative bacteria are the conduits by which nutrients and antibiotics diffuse passively into cells. The narrow gateways that porins form in the OM are also exploited by bacteriocins to translocate into cells by a poorly understood process. Here, using single-channel electrical recording in planar lipid bilayers in conjunction with protein engineering, we explicate the mechanism by which the intrinsically unstructured N-terminal translocation domain (IUTD) of the endonuclease bacteriocin ColE9 is imported passively across the Escherichia coli OM through OmpF. We show that the import is dominated by weak interactions of OmpF pores with binding epitopes within the IUTD that are orientationally biased and result in the threading of over 60 amino acids through 2 subunits of OmpF. Single-molecule kinetic analysis demonstrates that the IUTD enters from the extracellular side of OmpF and translocates to the periplasm where the polypeptide chain does an about turn in order to enter a neighboring subunit, only for some of these molecules to pop out of this second subunit before finally re-entering to form a stable complex. These intimately linked transport/binding processes generate an essentially irreversible, hook-like assembly that constrains an import activating peptide epitope between two subunits of the OmpF trimer.

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Section: Discussionmentioning

confidence: 99%

Transmembrane Epitope Delivery by Passive Protein Threading through the Pores of the OmpF Porin Trimer

et al. 2020

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“…Because the outer membrane is composed almost entirely of anionic lipopolysaccharide, with similar headgroup properties to POPG, such anionic lipid binding could prevent closure of OmpF channels, thereby increasing access of antibiotics that use porin-mediated pathways. That these lipids can maintain open channels was discovered almost entirely by accident, through our attempts to rationalize changes in lipid binding in response to changes in electrospray polarity (38).…”

Section: Uncovering Lipids That Fine-tune Membrane Protein Functionmentioning

confidence: 99%

Mass spectrometry: From plasma proteins to mitochondrial membranes

Robinson

2019

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

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In this Inaugural Article, I trace some key steps that have enabled the development of mass spectrometry for the study of intact protein complexes from a variety of cellular environments. Beginning with the preservation of the first soluble complexes from plasma, I describe our early experiments that capitalize on the heterogeneity of subunit composition during assembly and exchange reactions. During these investigations, we observed many assemblies and intermediates with different subunit stoichiometries, and were keen to ascertain whether or not their overall topology was preserved in the mass spectrometer. Adapting ion mobility and soft-landing methodologies, we showed how ring-shaped complexes could survive the phase transition. The next logical progression from soluble complexes was to membrane protein assemblies but this was not straightforward. We encountered many pitfalls along the way, largely due to the use of detergent micelles to protect and stabilize complexes. Further obstacles presented when we attempted to distinguish lipids that copurify from those that are important for function. Developing new experimental protocols, we have subsequently defined lipids that change protein conformation, mediate oligomeric states, and facilitate downstream coupling of G protein-coupled receptors. Very recently, using a radical method—ejecting protein complexes directly from native membranes into mass spectrometers—we provided insights into associations within membranes and mitochondria. Together, these developments suggest the beginnings of mass spectrometry meeting with cell biology.

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“…Cholesterol photoaffinity probes have also characterized lipid-protein interactions with ion channels (27,28). The advent of mass spectrometry (MS) methodologies offers the opportunity to examine membrane protein structure (29) to more sensitively and accurately detect and map the interaction of lipids with membrane proteins (30)(31)(32). In this study, we conduct studies analogous to those used in examining cholesterol interactions with the glycine receptor (33) to directly probe the lipid accessible regions of hSERT in its apo-state.…”

Section: Introductionmentioning

confidence: 99%

Characterizing the lipid-protein interface of the human serotonin transporter by crosslinking mass spectrometry

DeMarco

Ferraro

Sweigard

et al. 2020

Preprint

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Altered serotonin (5-HT) levels contribute to disease states such as depression and anxiety. Synaptic serotonin concentration is partially regulated by the serotonin transporter (SERT), making this transporter an important therapeutic target. This study seeks to examine the lipid accessible domains of hSERT to provide critical information regarding the apo-state of this transporter in a lipid environment. Recombinant hSERT was inducibly expressed in a human cell line. Solubilized SERT was purified by affinity chromatography using a FLAG Tag and reconstituted into mixed lipid vesicles containing our photoactivatable lipid probe. The lipidaccessible domains of the reconstituted transporter in membranes in its apo-state were probed via photocrosslinking to azi-cholesterol followed by quadrupole time of flight liquid chromatography-mass spectrometry (QTOF-LC-MS). MS studies identified crosslinks in three transmembrane loops consistent with the known topology of SERT. Surprisingly, the amino-and carboxy-terminal domains were similarly crosslinked by cholesterol, suggesting that these regions may also be intimately associated with the lipid bilayer. The data presented herein assist in further refining our understanding of the topography of the apo-state of hSERT via analysis of lipid accessibility. INTRODUCTION:

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Lipid binding attenuates channel closure of the outer membrane protein OmpF

Cited by 41 publications

References 48 publications

Transmembrane Epitope Delivery by Passive Protein Threading through the Pores of the OmpF Porin Trimer

Transmembrane Epitope Delivery by Passive Protein Threading through the Pores of the OmpF Porin Trimer

Mass spectrometry: From plasma proteins to mitochondrial membranes

Characterizing the lipid-protein interface of the human serotonin transporter by crosslinking mass spectrometry

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