Journal of Molecular Biology
Volume 325, Issue 2, 10 January 2003, Pages 355-365
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Granulysin Crystal Structure and a Structure-derived Lytic Mechanism

https://doi.org/10.1016/S0022-2836(02)01234-2Get rights and content

Abstract

Our crystal structure of granulysin suggests a mechanism for lysis of bacterial membranes by granulysin, a 74-residue basic protein from human cytolytic T lymphocyte and natural killer cells. We determined the initial crystal structure of selenomethionyl granulysin by MAD phasing at 2 Å resolution. We present the structure model refined using native diffraction data to 0.96 Å resolution. The five-helical bundle of granulysin resembles other “saposin folds” (such as NK-lysin). Positive charges distribute in a ring around the granulysin molecule, and one face has net positive charge. Sulfate ions bind near the segment of the molecule identified as most membrane-lytic and of highest hydrophobic moment. The ion locations may indicate granulysin's orientation of initial approach towards the membrane. The crystal packing reveals one way to pack a sheet of granulysin molecules at the cell surface for a concerted lysis effort. The energy of binding granulysin charges to the bacterial membrane could drive the subsequent lytic processes. The loosely packed core facilitates a hinge or scissors motion towards exposure of hydrophobic surface that we propose tunnels the granulysin into the fracturing target membrane.

Introduction

Granulysin, a 74-residue basic protein from human cytolytic T lymphocyte and natural killer cells, directly lyses a variety of bacterial, tumor, and fungal cells, including Mycobacterium tuberculosis and Mycobacterium leprae.1 Patients expressing large amounts of granulysin can contain the spread of leprosy infection.2., 3. One visible result of granulysin action on M. tuberculosis is the formation of protruding lesions on the target cell surface.1 Increase in membrane permeability of the M. tuberculosis and Escherichia coli substrates results in osmotic lysis.4 In collaboration with perforin, granulysin kills intracellular M. tuberculosis without simultaneous apoptosis.5., 6. Granulysin can also induce apoptosis of the host cell, in a mechanism involving caspase and other pathways.7., 8.

Granulysin is one member of the “saposin fold” family of membrane-interacting proteins of various functions. Other examples of the family are: saposins A and C;9 porcine NK-lysin;10 the cyclic peptide bacteriocin AS-48;11 one domain of prophytepsin;12 and amoebapores.13 Saposins A and C appear to alter membranes to become substrates for other enzymes, but do not lyse the membranes they bind. The saposin-like domain of prophytepsin appears to anchor the protein for transport to vacuoles. Bacteriocin protects Enterococcus faecalis from bacterial infection by opening pores in the target membrane. NK-lysin and granulysin directly lyse membranes.

Evidence and speculations on the actions of saposin-like proteins have been published. For reviews of antimicrobial peptides and their modes of action, see Zasloff,14 Shai,15 and Bechinger.16 Also, the thinking of Bruhn and Leippe13 overlaps that presented here. Qi and Grabowski9 propose that differences in charge distributions confer specificity by steering the orientations of saposins A and C relative to the membrane surface. The N and C termini were found by them to be buried in the membrane, with a conformational change observed for saposin C. NK-lysin lyses membranes possibly by “molecular electroporation” followed by pore formation.17 NK-lysin buries its one tryptophan (Trp58) in the membrane but does not lose its secondary structure.18 Several peptides derived from the NK-lysin and granulysin sequences lyse membranes.19., 20. Experiments identifying the most lytic peptides do not address the function of the entire molecule, as the protein charge distributions and shape and adjacency effects are not retained in such experiments.

We present our crystal structure of granulysin. Based on interpretation of this structure, and on previous work with membrane-lytic peptides and with other saposin-like proteins, we propose a schematic model of action. The locations of charged solvent species identified in the crystal structure indicate the orientation of granulysin relative to the membrane. The packing of the granulysin in the crystal is a plausible “carpet” arrangement as implicated in the function of other membrane-lytic peptides.15

Section snippets

Granulysin structure

The granulysin molecule is a five-helix bundle (Figure 1), resembling other “saposin fold” proteins. Our granulysin model includes all 74 residues, of which ten side-chains are split into two conformations each. The granulysin amino acid residues are numbered 1–74, and other modeled species have non-sequential “residue” numbers 81–1107. The model is refined with 0.96 Å resolution diffraction data (R-factor is 0.138; Rfree for the penultimate model was 0.190). For details and statistics, see

Discussion

From the structural detail observed via crystallography, we extrapolate to the function of granulysin on the target cell membrane, assisted by the sparse matrix of previous experiments and thoughts on related membrane-lytic proteins. The granulysin structure implies its function, but not as explicitly as would a ligand-bound enzyme structure. We narrow discussion to lysis of bacterial membranes in culture. On an exposed substrate, the action of granulysin is not complicated by partners in

Crystallization

Granulysin was produced and purified as described,4., 33. but scaled up to 5 mg injections into a larger reverse-phase column (Varian Dynamax C18; 21.4 mm×25 cm, plus guard cartridge). The flow rate was 10 ml min−1, and detection was by absorbance at 280 nm. The granulysin peak fractions were dried in a vacuum centrifuge (Speed Vac from Savant) for storage. The dried granulysin was dissolved to about 10 mg ml−1 by addition of water. Crystals grew by the hanging drop vapor diffusion method. The

Acknowledgements

We thank Peter Müller for assistance with handling of diffraction data, and Annaliza Legaspi for production of granulysin. This work was supported by: NIH grants AI07118 and AR40312 to R.M. and AI43348 to A.K.; HHMI award to D.E. This material is based upon work supported by the National Science Foundation under grant number 9904671 to D.E.

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