The amino terminus of Pseudomonas aeruginosa outer membrane protein OprF forms channels in lipid bilayer membranes: correlation with a three-dimensional model

Abstract

Pseudomonas aeruginosa OprF forms 0.36-nS channels and, rarely, 2- to 5-nS channels in lipid bilayer membranes. We show that a protein comprising only the N-terminal 162-amino-acid domain of OprF formed the smaller, but not the larger, channels in lipid bilayers. Circular dichroism spectroscopy indicated that this protein folds into a beta-sheet-rich structure, and three-dimensional comparative modeling revealed that it shares significant structural similarity with the amino terminus of the orthologous protein Escherichia coli OmpA, which has been shown to form a beta-barrel. OprF and OmpA share only 15% identity in this domain, yet these results support the utility of modeling such widely divergent beta-barrel domains in three dimensions in order to reveal similarities not readily apparent through primary sequence comparisons. The model is used to further hypothesize why porin activity differs for the N-terminal domains of OprF and OmpA.

FIG. 1

Histograms of single-channel conductance measurements showing channel size distributions for OprF (A) and OprF_1–162 (B).

FIG. 2

CD spectral analysis of OprF_1–162 in 0.1% sodium dodecyl sulfate.

FIG. 3

Alignment of the sequences of OprF_1–162 and OmpA_1–171, according to sequence hydrophobicity and location of charged residues (see the text). Predicted transmembrane regions are boxed, and stars mark identical residues. The two underlined tryptophans are examples of residues conserved in location in three-dimensional space (according to our modeling) but not conserved in location along the sequence.

FIG. 4

Three-dimensional model of OprF_1–162, constructed by threading the sequence of OprF_1–162 on a crystal structure of OmpA_1–171. (A) Overview of the molecule, highlighting all aromatic residues on the outside of the protein (yellow). Note the striking rings of aromatic residues at the proposed water-lipid interfaces. (B) A slice horizontally through the barrel of the proposed model of OprF (blue), overlaid on the structure of OmpA (yellow), illustrating how residues previously proposed to form a barrier to pore formation in OmpA (12) are not conserved in OprF and allow for a larger channel with no salt bridge in that region.