Saposin fold revealed by the NMR structure of NK-lysin

Лиепиньш, Э. Э.; Andersson, Mats; Ruysschaert, Jean‐Marie; Otting, Gottfried

doi:10.1038/nsb1097-793

Cited by 231 publications

(221 citation statements)

References 28 publications

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“…A model of granulysin was constructed based on the nuclear magnetic resonance (NMR) structure of NK-lysin (PDB accession code 1NKL) (14). Briefly, using the fold recognition server (http://fold.doe-mbi.ucla.edu/), the sequence of granulysin was threaded onto the NMR backbone of NKlysin (15).…”

Section: Circular Dichroic Analysis Of Granulysinmentioning

confidence: 99%

Granulysin, a T Cell Product, Kills Bacteria by Altering Membrane Permeability

Ernst

Thoma-Uszynski

Teitelbaum

et al. 2000

The Journal of Immunology

198

172

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Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has antimicrobial activity against a broad spectrum of microbial pathogens. A predicted model generated from the nuclear magnetic resonance structure of a related protein, NK lysin, suggested that granulysin contains a four α helical bundle motif, with the α helices enriched for positively charged amino acids, including arginine and lysine residues. Denaturation of the polypeptide reduced the α helical content from 49 to 18% resulted in complete inhibition of antimicrobial activity. Chemical modification of the arginine, but not the lysine, residues also blocked the antimicrobial activity and interfered with the ability of granulysin to adhere to Escherichia coli and Mycobacterium tuberculosis. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic β-galactosidase to its impermeant substrate. By electron microscopy, granulysin triggered fluid accumulation in the periplasm of M. tuberculosis, consistent with osmotic perturbation. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on direct interaction with the microbial cell wall and/or membrane, leading to increased permeability and lysis.

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Section: Circular Dichroic Analysis Of Granulysinmentioning

confidence: 99%

Granulysin, a T Cell Product, Kills Bacteria by Altering Membrane Permeability

Ernst

Thoma-Uszynski

Teitelbaum

et al. 2000

The Journal of Immunology

198

172

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“…Six conserved cysteines form three intrapeptide disulfide bonds and define a fold that has been conserved for an estimated 300 million years (27). The experimentally derived structures of three members of the superfamily, including saposin B [PDB entry 1N69 (28)], saposin C [PDB entry 1M12 (29)], and NK-lysin [PDB entry 1NKL (30)], all display this fold, although the helical splay of each of the proteins may differ.…”

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confidence: 99%

NMR Structures of the C-Terminal Segment of Surfactant Protein B in Detergent Micelles and Hexafluoro-2-propanol

et al. 2004

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Although the membrane-associated surfactant protein B (SP-B) is an essential component of lung surfactant, which is itself essential for life, the molecular basis for its activity is not understood. SP-B's biophysical functions can be partially mimicked by subfragments of the protein, including the C-terminus. We have used NMR to determine the structure of a C-terminal fragment of human SP-B that includes residues 63-78. Structure determination was performed both in the fluorinated alcohol hexafluoro-2-propanol (HFIP) and in sodium dodecyl sulfate (SDS) micelles. In both solvents, residues 68-78 take on an amphipathic helical structure, in agreement with predictions made by comparison to homologous saposin family proteins. In HFIP, the five N-terminal residues of the peptide are largely unstructured, while in SDS micelles, these residues take on a well-defined compact conformation. Differences in helical residue side chain positioning between the two solvents were also found, with better agreement between the structures for the hydrophobic face than the hydrophilic face. A paramagnetic probe was used to investigate the position of the peptide within the SDS micelles and indicated that the peptide is located at the water interface with the hydrophobic face of the helix oriented inward, the hydrophilic face of the helix oriented outward, and the N-terminal residues even farther from the micelle center than those on the hydrophilic face of the R-helix. Interactions of basic residues of SP-B with anionic lipid headgroups are known to have an impact on function, and these studies demonstrate structural ramifications of such interactions via the differences observed between the peptide structures determined in HFIP and SDS.

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“…NK-lysin has a high ␣-helical content and is folded tightly through three intramolecular disulfide bonds formed by six conserved cysteine residues (7,8). By analogy to NK-lysin, granulysin is predicted to adopt a similar structure (1), although the 9-kDa form of granulysin contains only four cysteine residues that form two rather than three predicted intramolecular disulfide bonds.…”

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confidence: 99%

Bactericidal and Tumoricidal Activities of Synthetic Peptides Derived from Granulysin

Wang¹,

Choice

Kaspar

et al. 2000

The Journal of Immunology

110

105

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Granulysin, a 9-kDa protein localized to human CTL and NK cell granules, is cytolytic against tumor cells and microbes. Molecular modeling predicts that granulysin is composed of five α-helices separated by short loop regions. In this report, synthetic peptides corresponding to the linear granulysin sequence were characterized for lytic activity. Peptides corresponding to the central region of granulysin lyse bacteria, human cells, and synthetic liposomes, while peptides corresponding to the amino or carboxyl regions are not lytic. Peptides corresponding to either helix 2 or helix 3 lyse bacteria, while lysis of human cells and liposomes is dependent on the helix 3 sequence. Peptides in which positively charged arginine residues are substituted with neutral glutamine exhibit reduced lysis of all three targets. While reduction of recombinant 9-kDa granulysin increases lysis of Jurkat cells, reduction of cysteine-containing granulysin peptides decreases lysis of Jurkat cells. In contrast, lysis of bacteria by recombinant granulysin or by cysteine-containing granulysin peptides is unaffected by reducing conditions. Jurkat cells transfected with either CrmA or Bcl-2 are protected from lysis by recombinant granulysin or the peptides. Differential activity of granulysin peptides against tumor cells and bacteria may be exploited to develop specific antibiotics without toxicity for mammalian cells.

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Saposin fold revealed by the NMR structure of NK-lysin

Abstract: NK-lysin is the first representative of a family of sequence related proteins--saposins, surfactant-associated protein B, pore forming amoeba proteins, and domains of acid sphingomyelinase, acyloxyacylhydrolase and plant aspartic proteinases--for which a structure has been determined.

Cited by 231 publications

References 28 publications

Granulysin, a T Cell Product, Kills Bacteria by Altering Membrane Permeability

Granulysin, a T Cell Product, Kills Bacteria by Altering Membrane Permeability

NMR Structures of the C-Terminal Segment of Surfactant Protein B in Detergent Micelles and Hexafluoro-2-propanol

Bactericidal and Tumoricidal Activities of Synthetic Peptides Derived from Granulysin

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