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Negative cooperativity exhibited by the lytic amino-terminal domain of human perforin: implications for
1Department of Chemistry, The Ohio State University, 120 West 18th Avenue, Columbus, OH 43210, USA.
Background:
Cytolytic effector cells of the immune system recognize and lyse cells that carry non-self epitopes. One mechanism of cell lysis involves release of a 67-kDa pore-forming protein, perforin. The amino-terminal domain of perforin (>/= 19 residues) can account for most of the lysis activity, by a mechanism that is similar to that of holoperforin. Detailed mechanistic studies of this domain should yield useful insight into the factors underlying perforin activity in vivo.
Results:
The mechanism of pore formation by the 22-residue amino-terminal domain of perforin was studied by kinetic and thermodynamic methods. Approximately 4 +/- 1 peptide monomers form an active pore by a mechanism that displays negative cooperativity.
Conclusions:
A negatively-regulated aggregation mechanism is likely to be common for pore-forming peptides. The positively-charged domain B of perforin (residues 7- 15) may mediate cooperativity through electrostatic interactions. Such a mechanism limits the number of protein molecules that are committed to any particular channel. This data supports smaller pores as the physiologically relevant aggregate, rather than the larger ring sizes identified by electron microscopy at higher, non-biological concentrations.