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A novel cholesterol-insensitive mode of membrane binding promotes cytolysin-mediated translocation by Streptolysin O.

ABSTRACT: Cytolysin-mediated translocation (CMT), performed by Streptococcus pyogenes, utilizes the cholesterol-dependent cytolysin Streptolysin O (SLO) to translocate the NAD(+) -glycohydrolase (SPN) into the host cell during infection. SLO is required for CMT and can accomplish this activity without pore formation, but the details of SLO's interaction with the membrane preceding SPN translocation are unknown. Analysis of binding domain mutants of SLO and binding domain swaps between SLO and homologous cholesterol-dependent cytolysins revealed that membrane binding by SLO is necessary but not sufficient for CMT, demonstrating a specific requirement for SLO in this process. Despite being the only known receptor for SLO, this membrane interaction does not require cholesterol. Depletion of cholesterol from host membranes and mutation of SLO's cholesterol recognition motif abolished pore formation but did not inhibit membrane binding or CMT. Surprisingly, SLO requires the coexpression and membrane localization of SPN to achieve cholesterol-insensitive membrane binding; in the absence of SPN, SLO's binding is characteristically cholesterol-dependent. SPN's membrane localization also requires SLO, suggesting a co-dependent, cholesterol-insensitive mechanism of membrane binding occurs, resulting in SPN translocation.

SUBMITTER: Mozola CC

PROVIDER: S-EPMC4213296 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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A novel cholesterol-insensitive mode of membrane binding promotes cytolysin-mediated translocation by Streptolysin O.

Mozola Cara C CC Magassa N'Goundo N Caparon Michael G MG

Molecular microbiology 20140923 3

Cytolysin-mediated translocation (CMT), performed by Streptococcus pyogenes, utilizes the cholesterol-dependent cytolysin Streptolysin O (SLO) to translocate the NAD(+) -glycohydrolase (SPN) into the host cell during infection. SLO is required for CMT and can accomplish this activity without pore formation, but the details of SLO's interaction with the membrane preceding SPN translocation are unknown. Analysis of binding domain mutants of SLO and binding domain swaps between SLO and homologous c ...[more]

PMID: 25196983

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Project description:P-glycoprotein (Pgp) is a biomedically important member of the ABC transporter superfamily that mediates multidrug resistance in various cancer types. Substrate binding and transport in Pgp are modulated by the presence of cholesterol in the membrane. Structural information on cholesterol binding sites and mechanistic details of its redistribution are, however, largely unknown. In this study, a set of 40 independent molecular dynamics (MD) simulations of Pgp embedded in cholesterol-rich lipid bilayers are reported, totaling 8 μs, enabling extensive sampling of cholesterol-protein interactions in Pgp. Clustering analyses of the ensemble of cholesterol molecules (∼5740) sampled around Pgp in these simulations reveal specific and asymmetric cholesterol-binding regions formed by the transmembrane (TM) helices TM1-6 and TM8. Notably, not all the putative cholesterol binding sites identified by MD can be predicted by the primary sequence based cholesterol-recognition amino acid consensus (CRAC) or inverted CRAC (CARC) motifs, an observation that we attribute to inadequacy of these motifs to account for binding sites formed by remote amino acids in the sequence that can still be spatially adjacent to each other. Binding of cholesterol to Pgp occurs more frequently through its rough β-face formed by the two protruding methyl groups, whereas the opposite smooth α-face prefers packing alongside the membrane lipids. One full and two partial cholesterol flipping events between the two leaflets of the bilayer mediated by the surface of Pgp are also captured in these simulations. All flipping events are observed in a region formed by helices TM1, TM2, and TM11, featuring two full and two partial CRAC/CARC motifs, with Tyr49 and Tyr126 identified as key residues interacting with cholesterol during this event. Our study is the first to report direct observation of unconventional cholesterol translocation on the surface of Pgp, providing a secondary transport model for the known flippase activity of ABC exporters of cholesterol. This article is part of a Special Issue entitled: Molecular biophysics of membranes and membrane proteins.

Dataset Information

A novel cholesterol-insensitive mode of membrane binding promotes cytolysin-mediated translocation by Streptolysin O.

Publications

A novel cholesterol-insensitive mode of membrane binding promotes cytolysin-mediated translocation by Streptolysin O.

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