Dataset Information

Mechanistic Insights into the Cholesterol-dependent Binding of Perfringolysin O-based Probes and Cell Membranes.

ABSTRACT: Cholesterol distribution in the cell is maintained by both vesicular and non-vesicular sterol transport. Non-vesicular transport is mediated by the interaction of membrane-embedded cholesterol and water-soluble proteins. Small changes to the lipid composition of the membrane that do not change the total cholesterol content, can significantly affect how cholesterol interacts with other molecules at the surface of the membrane. The cholesterol-dependent cytolysin Perfringolysin O (PFO) constitutes a powerful tool to detect cholesterol in membranes, and the use of PFO-based probes has flourished in recent years. By using a non-lytic PFO derivative, we showed that the sensitivity of the probes for cholesterol can be tuned by modifications introduced directly in the membrane-interacting loops and/or by modifying residues away from the membrane-interacting domain. Through the use of these biosensors on live RAW 264.7 cells, we found that changes in the overall cholesterol content have a limited effect on the average cholesterol accessibility at the surface of the membrane. We showed that these exquisite biosensors report on changes in cholesterol reactivity at the membrane surface independently of the overall cholesterol content in the membrane.

SUBMITTER: Johnson BB

PROVIDER: S-EPMC5653841 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Mechanistic Insights into the Cholesterol-dependent Binding of Perfringolysin O-based Probes and Cell Membranes.

Johnson Benjamin B BB Breña Mariana M Anguita Juan J Heuck Alejandro P AP

Scientific reports 20171023 1

Cholesterol distribution in the cell is maintained by both vesicular and non-vesicular sterol transport. Non-vesicular transport is mediated by the interaction of membrane-embedded cholesterol and water-soluble proteins. Small changes to the lipid composition of the membrane that do not change the total cholesterol content, can significantly affect how cholesterol interacts with other molecules at the surface of the membrane. The cholesterol-dependent cytolysin Perfringolysin O (PFO) constitutes ...[more]

PMID: 29061991

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Project description:[Structure: see text]. FHA domains are protein modules that switch signals in diverse biological pathways by monitoring the phosphorylation of threonine residues of target proteins. As part of the effort to gain insight into cellular avoidance of cancer, FHA domains involved in the cellular response to DNA damage have been especially well-characterized. The complete protein where the FHA domain resides and the interaction partners determine the nature of the signaling. Thus, a key biochemical question is how do FHA domains pick out their partners from among thousands of alternatives in the cell? This Account discusses the structure, affinity, and specificity of FHA domains and the formation of their functional structure. Although FHA domains share sequence identity at only five loop residues, they all fold into a beta-sandwich of two beta-sheets. The conserved arginine and serine of the recognition loops recognize the phosphorylation of the threonine targeted. Side chains emanating from loops that join beta-strand 4 with 5, 6 with 7, or 10 with 11 make specific contacts with amino acids of the ligand that tailor sequence preferences. Many FHA domains choose a partner in extended conformation, somewhat according to the residue three after the phosphothreonine in sequence (pT + 3 position). One group of FHA domains chooses a short carboxylate-containing side chain at pT + 3. Another group chooses a long, branched aliphatic side chain. A third group prefers other hydrophobic or uncharged polar side chains at pT + 3. However, another FHA domain instead chooses on the basis of pT - 2, pT - 3, and pT + 1 positions. An FHA domain from a marker of human cancer instead chooses a much longer protein fragment that adds a beta-strand to its beta-sheet and that presents hydrophobic residues from a novel helix to the usual recognition surface. This novel recognition site and more remote sites for the binding of other types of protein partners were predicted for the entire family of FHA domains by a bioinformatics approach. The phosphopeptide-dependent dynamics of an FHA domain, SH2 domain, and PTB domain suggest a common theme: rigid, preformed binding surfaces support van der Waals contacts that provide favorable binding enthalpy. Despite the lack of pronounced conformational changes in FHA domains linked to binding events, more subtle adjustments may be possible. In the one FHA domain tested, phosphothreonine peptide binding is accompanied by increased flexibility just outside the binding site and increased rigidity across the beta-sandwich. The folding of the same FHA domain progresses through near-native intermediates that stabilize the recognition loops in the center of the phosphoprotein-binding surface; this may promote rigidity in the interface and affinity for targets phosphorylated on threonine.

Dataset Information

Mechanistic Insights into the Cholesterol-dependent Binding of Perfringolysin O-based Probes and Cell Membranes.

Publications

Mechanistic Insights into the Cholesterol-dependent Binding of Perfringolysin O-based Probes and Cell Membranes.

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