Solution NMR analysis of the interaction between the actinoporin Sticholysin I and DHPC micelles—Correlation with backbone dynamics

López-Castilla, Aracelys; Pazos, Fabiola; Schreier, Shirley; Pires, Juliana Rico

doi:10.1002/prot.24475

Cited by 9 publications

(7 citation statements)

References 54 publications

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“…1). At least two of them (W110 and W114 in StnII) have been shown to be part of an exposed and conserved cluster of aromatic residues that are implied in the interaction with membranes (8,19,37,58,91,92). Our results (67).…”

Section: Discussionsupporting

confidence: 52%

Sticholysin, Sphingomyelin, and Cholesterol: A Closer Look at a Tripartite Interaction

Palacios-Ortega

García‐Linares

Rivera-de-Torre

et al. 2019

Biophysical Journal

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Actinoporins are a group of soluble toxic proteins that bind to membranes containing sphingomyelin (SM) and oligomerize to form pores. Sticholysin II (StnII) is a member of the actinoporin family produced by Stichodactyla helianthus. Cholesterol (Chol) is known to enhance the activity of StnII. However, the molecular mechanisms behind this activation have remained obscure, although the activation is not Chol specific but rather sterol specific. To further explore how bilayer lipids affect or are affected by StnII, we have used a multiprobe approach (fluorescent analogs of both Chol and SM) in combination with a series of StnII tryptophan (Trp) mutants to study StnII/bilayer interactions. First, we compared StnII bilayer permeabilization in the presence of Chol or oleoyl-ceramide (OCer). The comparison was done because both Chol and OCer have a 1-hydroxyl, which helps to orient the molecule in the bilayer (although OCer has additional polar functional groups). Both Chol and OCer also have increased affinity for SM, which StnII may recognize. However, our results show that only Chol was able to activate StnII-induced bilayer permeabilization; OCer failed to activate it. To further examine possible Chol/StnII interactions, we measured Fö rster resonance energy transfer between Trp in StnII and cholestatrienol, a fluorescent analog of Chol. We could show higher Fö rster resonance energy transfer efficiency between cholestatrienol and Trps in position 100 and 114 of StnII when compared to three other Trp positions further away from the bilayer binding region of StnII. Taken together, our results suggest that StnII was able to attract Chol to its vicinity, maybe by showing affinity for Chol. SM interactions are known to be important for StnII binding to bilayers, and Chol is known to facilitate subsequent permeabilization of the bilayers by StnII. Our results help to better understand the role of these important membrane lipids for the bilayer properties of StnII.

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Section: Discussionsupporting

confidence: 52%

Sticholysin, Sphingomyelin, and Cholesterol: A Closer Look at a Tripartite Interaction

Palacios-Ortega

García‐Linares

Rivera-de-Torre

et al. 2019

Biophysical Journal

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“…It forms a stable micelle that rotates freely in solution and mimics anisotropic environments such as the lipid membrane (15). Although DPC is one of the most commonly used detergents in solution NMR assays, many other lipids can be employed (16). Recently, many techniques have been developed to quantify motions that occur over a wide range of correlation times and to measure orientation constraints as indices to determine peptide structures (17).…”

Section: Introductionmentioning

confidence: 99%

NMR Structural Studies of Antimicrobial Peptides: LPcin Analogs

Jeong

Kim

Choi

et al. 2016

Biophysical Journal

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Lactophoricin (LPcin), a component of proteose peptone (113-135) isolated from bovine milk, is a cationic amphipathic antimicrobial peptide consisting of 23 amino acids. We designed a series of N- or C-terminal truncated variants, mutated analogs, and truncated mutated analogs using peptide-engineering techniques. Then, we selected three LPcin analogs of LPcin-C8 (LPcin-YK1), LPcin-T2WT6W (LPcin-YK2), and LPcin-T2WT6W-C8 (LPcin-YK3), which may have better antimicrobial activities than LPcin, and successfully expressed them in E. coli with high yield. We elucidated the 3D structures and topologies of the three LPcin analogs in membrane environments by conducting NMR structural studies. We investigated the purity of the LPcin analogs and the α-helical secondary structures by performing (1)H-(15)N 2D HSQC and HMQC-NOESY liquid-state NMR spectroscopy using protein-containing micelle samples. We measured the 3D structures and tilt angles in membranes by conducting (15)N 1D and 2D (1)H-(15)N SAMMY type solid-state NMR spectroscopy with an 800 MHz in-house-built (1)H-(15)N double-resonance solid-state NMR probe with a strip-shield coil, using protein-containing large bicelle samples aligned and confirmed by molecular-dynamics simulations. The three LPcin analogs were found to be curved α-helical structures, with tilt angles of 55-75° for normal membrane bilayers, and their enhanced activities may be correlated with these topologies.

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“…Early studies of actinoporins aiming at elucidating their mechanism of action have proposed that these sea anemone toxins bind to membranes, promoting lysis via formation of toroidal pores, and implied their N-terminal domain in pore formation [ 44 ]. High resolution conformational studies of sticholysins, Eqt II, and FragC [ 4 , 5 , 10 , 11 , 23 , 51 ] show structural similarities between their folds, including the N-terminal region. All of them present a more, or less, hydrophobic stretch (residues 1–11 in St I and 1–10 in St II) followed by a region with a high propensity to acquire an amphipathic α-helical conformation.…”

Section: Discussionmentioning

confidence: 99%

Dissecting the mechanism of action of actinoporins. Role of the N-terminal amphipathic α-helix in membrane binding and pore activity of sticholysins I and II

et al. 2018

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Actinoporins sticholysin I and sticholysin II (St I, St II) are proposed to lyse model and biomembranes via toroidal pore formation by their N-terminal domain. Based on the hypothesis that peptide fragments can reproduce the structure and function of this domain, the behavior of peptides containing St I residues 12–31 (StI12-31), St II residues 11–30 (StII11-30), and its TOAC-labeled analogue (N-TOAC-StII11-30) was examined. Molecular modeling showed a good match with experimental structures, indicating amphipathic α-helices in the same regions as in the toxins. CD spectra revealed that the peptides were essentially unstructured in aqueous solution, acquiring α-helical conformation upon interaction with micelles and large unilamellar vesicles (LUV) of variable lipid composition. Fluorescence quenching studies with NBD-containing lipids indicated that N-TOAC-StII11-30’s nitroxide moiety is located in the membranes polar head group region. Pyrene-labeled phospholipid inter-leaflet redistribution suggested that the peptides form toroidal pores, according to the mechanism of action proposed for the toxins. Binding occurred only to negatively charged LUV, indicating the importance of electrostatic interactions; in contrast the peptides bound to both negatively charged and zwitterionic micelles, pointing to a lesser influence of these interactions. In addition, differences between bilayers and micelles in head group packing and in curvature led to differences in peptide-membrane interaction. We propose that the peptides topography in micelles resembles that of the toxins in the toroidal pore. The peptides mimicked the toxins permeabilizing activity, St II peptides being more effective than StI12-31. To our knowledge, this is the first demonstration that differences in the toxins N-terminal amphipathic α-helix play a role in the difference between St I and St II activities.

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Solution NMR analysis of the interaction between the actinoporin Sticholysin I and DHPC micelles—Correlation with backbone dynamics

Cited by 9 publications

References 54 publications

Sticholysin, Sphingomyelin, and Cholesterol: A Closer Look at a Tripartite Interaction

Sticholysin, Sphingomyelin, and Cholesterol: A Closer Look at a Tripartite Interaction

NMR Structural Studies of Antimicrobial Peptides: LPcin Analogs

Dissecting the mechanism of action of actinoporins. Role of the N-terminal amphipathic α-helix in membrane binding and pore activity of sticholysins I and II

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