Molecular basis for the polyamine-OmpF porin interactions: Inhibitor and mutant studies

By testing the sensitivity of Escherichia coli OmpF porin to various natura l and synthetic polyamines of different lengths, charge and other molecular characteristics, we were able to identify the molecular properties require d for compounds to act as inhibitors of OmpF in the nanomolar range. Inhibi tors require at least two amine groups to be effective. For diamines, the o ptimum length of the hydrocarbon spacer was found to be of eight to ten met hylene groups. Triamine molecules based on a 12-carbon motif were found to be more effective that spermidine, an eight-carbon trivalent derivative. Bu t differences in inhibition efficiencies were also found for trivalent comp ounds depending on the relative position of the internal secondary amine gr oup with respect to the terminal groups. Finally, quaternary ammonium deriv atives had no effect, suggesting that the nature of the terminal amine is i mportant for the interaction. From these observations, we deduce that inhib ition efficiency in the nanomolar range requires a 12-carbon chain triamine with terminal primary amine groups and replacement of the eighth methylene by a secondary amine. The need for this type of molecular architecture sug gests that inhibition is governed by interactions between specific amine gr oups and protein residues, and that this is not simply due to the accumulat ion of charges into the pore. Together with previous observations from site -directed mutagenesis studies and inspection of the crystal structure of Om pF, these results allowed us to propose three residues (D113, D121 and Y294 ) as putative sites of interaction between the channel and spermine. Alanin e substitution at each of these three residues resulted in a loss of inhibi tion by spermine, while mutations of only D113 and D121 affected inhibition by spermidine. Based on these observations, we suggest a model for the mol ecular determinants involved in the porin-polyamine interaction. (C) 2000 A cademic Press.