NEGATIVE COOPERATIVITY EXHIBITED BY THE LYTIC AMINO-TERMINAL DOMAIN OF HUMAN PERFORIN - IMPLICATIONS FOR PERFORIN-MEDIATED CELL-LYSIS

Background: Cytolytic effector cells of the immune system recognize an d lyse cells that carry non-self epitopes. One mechanism of cell lysis involves release oi a 67-kDa pore-forming protein, perforin.;The amin o-terminal domain of perforin (greater than or equal to 19 residues) c an account for most of the lysis activity, by a mechanism that is simi lar to that of holoperforin. Detailed mechanistic studies of this doma in should yield useful insight Into the factors underlying perforin ac tivity in vivo. Results: The mechanism of pore formation by the 22-res idue amino-terminal domain of perforin was studied by kinetic and ther modynamic methods. Approximately 4 +/- 1 peptide monomers form an acti ve pore by a mechanism that displays negative cooperativity. Conclusio ns: A negatively-regulated aggregation mechanism is likely to be commo n for pore-forming peptides. The positively-charged domain B of perfor in (residues 7-15) may mediate cooperativity through electrostatic int eractions. Such a mechanism limits the number oi protein molecules tha t are committed to any particular channel. This data supports smaller pores as the physiologically relevant aggregate, rather than the large r ring sizes identified by electron microscopy at higher, non-biologic al concentrations.