Biochemistry zyxwvu 1992, 31, zyxwvu 5017-5021 5017 Channel-Forming Activity of the Perforin N-Terminus and a Putative a-Helical Region Homologous with Complement C9t Pedro M. Persechini,*J David M. Ojcius,fJ Sandro C. Adeodato,: Paulo C. Notaroberto,: Carlos B. Daniel,$ and John Ding-E Young8 The Rockefeller University, I230 York Avenue, New York. New York 10021, and Instituto Bioflsica, Universidade Federal do Rio de Janeiro, 21 941 Rio de Janeiro, Brazil Received August 28, 1991; Revised Manuscript Received February 26, I992 zyxwvutsrqponmlkjihgfedcbaZYXW ABSTRACT: Cytolytic lymphocytes are endowed with a pore-forming protein called perforin. Recently, a cytolytic domain was located in the first 34 residues of the perforin N-terminus. It has been proposed that the first 19 residues are composed of a 3-domain structure including a putative amphipathic @-sheet and that the 19 residues are sufficient for cytolytic activity. This model has now been tested by synthesizing peptides covering different portions of the N-terminus, and testing their ability to lyse lipid vesicles or increase the conductance of lipid bilayers or plasma membranes. It was found that the putative @-sheet is indispensable for lytic activity and that the first 19 residues of the N-terminus are required for optimal lytic activity but that shorter peptides, containing only 16 residues, can form pores in lipid bilayers and cell membranes. A putative amphipathic a-helix from the central portion of perforin, homologous to complement C9, is nonlytic to lipid vesicles, but it can form pores in lipid bilayers. Taken together, these results support the model that the perforin N-terminus is important in initial pore formation and that the putative a-helical domain may be involved in subsequent perforin polymerization into large pores. cytotoxic T lymphocytes (CTL)' and natural killer (NK) cells lyse cancerous and virally infected cells through a con- tact-dependent mechanism. According to a widely held view, recognition of the target cell triggers exocytosis of the killer cell, which results in vectorial release of a number of putative cytotoxins from the killer cell granules. Prominent among these toxins is a 70-kDa pore-forming protein called perforin or cytolysin (Tschopp 8c Nabholz, 1990; Podack et al., 1991; Young, 1989; Henkart, 1985). In its monomeric form, perforin is thought to bind to the target cell surface, where it inserts into the plasma membrane and polymerizes into nonspecific pores of up to 20 nm in diameter. The target cell thus swells and subsequently bursts due to the colloid osmotic pressure that ensues. On the basis of electron microscopy and immunoblot analysis, it was known that the tubular lesions produced by perforin are ultrastructurally and immunologically related to the pores formed by the membrane attack complex (MAC) of complement (Young et al., 1986). Sequence analysis of the cDNA clones for perforin and the MAC components subse- quently confirmed that there is significant homology between these 2 groups of pore-forming proteins, which is largely re- stricted to a region of about 270 amino acids (Lichtenheld et al., 1988; Ishikawa et al., 1989; Shinkai et al., 1988; Kwon et al., 1989; Lowrey et al., 1989); 40 amino acids from the N-terminus and 100 from the C-terminus of perforin are unique to perforin. The central one-third of the perforin This work was financed by grants from the Leukemia Society of America, the American Cancer Society, the National Institutes of Health, the Conselho Nacional de Desenvolvimento Cientifico e Tecnoldgicodo Brasil, and the FundapHo de Amparo a Pesquisa do Rio de Janeiro. *Address correspondence to this author at the Instituto de Biofkica da UFRJ, Blow G do CCS, Ilha do FundHo, 21941, Rio de Janeiro, RJ, Brazil. 8 Universidade Federal do Rio de Janeiro. zyxwvutsrq (I Present address: Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris The Rockefeller University. Cdex 15, France. protein is zyxwv conserved in the complement components C6 through C9, and contains four candidate membrane-spanning domains that could potentially form amphipathic a-helices or 8-sheets (Kwon et al., 1989). However, recent attempts to locate a lytic domain in this region have failed, as none of the synthetic peptides based on the amino acid sequences of the four can- didate domains are able to lyse cells or lipid vesicles (Ojcius et al., 1991). Nevertheless, a search outside of the homologous region did yield lytic peptides comprising the first 34 amino acids of the N-terminus of human and murine perforin. These peptides were also shown to form pores in lipid bilayers, strongly suggesting that the N-terminus is involved in perforin insertion and pore formation (Ojcius et al., 1991). As these peptides lack hydrophobic segments long enough to span the membrane, they would have to assume an amphipathic con- figuration and to polymerize in order to form pores. It was therefore proposed that the lytic activity could be due to the first 19 amino acids, which can be broken down into 3 separate domains, consisting of amino acids 1-5 (domain A), 6-16 (domain B), and 17-19 (domain C). Figure 1 shows the proposed structure of the perforin N-terminus in the presence of a lipid bilayer. Both flanking regions of the N-terminus (domains A and C) are clearly hydrophobic, while the central region (domain B), drawn as a potential 8-sheet, is predom- inantly hydrophilic, having several charged (mostly basic) residues. This finding, however, did not lead us to the complete structure of the perforin pore. Although peptides have been used as models to study specific ion channels and pore-forming proteins, the complete structure of such channels/pores is usually formed by multiple domains organized in a complex form (Montal, 1990; Lear et al., 1988). In the case of zy perforin, part of the focus has also been concentrated on the putative amphipathic a-helix comprising amino acids 189-218 (a1) ' Abbreviations: CF, carboxyfluorescein; CTL, cytotoxic T lym- phocyte(s); MAC, membrane attack complex; PC, phosphatidylcholine, SUV, small unilamellar vesicle(s). 0006-2960/92/0431-5017$03.00/0 zyxwvut 0 1992 American Chemical Society