Granulysin Crystal Structure and a Structure-derived Lytic Mechanism Daniel H. Anderson 1,2 *, Michael R. Sawaya 1,2 , Duilio Cascio 2 William Ernst 2 , Robert Modlin 2,3 , Alan Krensky 4 and David Eisenberg 1,2 1 Howard Hughes Medical Institute, 5-748 MacDonald Box 951662, Los Angeles, CA 90095-1662, USA 2 UCLA-DOE Institute of Genomics and Proteomics Molecular Biology Institute University of California, Los Angeles, Box 951570, Los Angeles, CA 90095-1570, USA 3 Division of Dermatology and Dept. of Microbiology, Immunology and Molecular Genetics David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA 4 Department of Paediatrics Stanford University, Stanford CA 94305-5164, USA 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 A ˚ resolution. We present the structure model refined using native diffraction data to 0.96 A ˚ 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 mem- brane could drive the subsequent lytic processes. The loosely packed core facilitates a hinge or scissors motion towards exposure of hydro- phobic surface that we propose tunnels the granulysin into the fracturing target membrane. q 2003 Elsevier Science Ltd. All rights reserved Keywords: granulysin; saposin fold; antimicrobial protein; crystal structure; lytic mechanism *Corresponding author 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 pro- truding 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 simul- taneous apoptosis. 5,6 Granulysin can also induce apoptosis of the host cell, in a mechanism involv- ing 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: sapo- sins 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 sub- strates 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 Leippe 13 overlaps that presented here. Qi and Grabowski 9 propose that differences in charge distributions confer specificity by steering the orientations of saposins 0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved E-mail address of the corresponding author: dha@mbi.ucla.edu doi:10.1016/S0022-2836(02)01234-2 J. Mol. Biol. (2003) 325, 355–365