Crystal Structure of the Drug Discharge Outer Membrane Protein, OprM, of Pseudomonas aeruginosa

Abstract

The OprM lipoprotein of Pseudomonas aeruginosa is a member of the MexAB-OprM xenobiotic-antibiotic transporter subunits that is assumed to serve as the drug discharge duct across the outer membrane. The channel structure must differ from that of the porintype open pore because the protein facilitates the exit of antibiotics but not the entry. For better understanding of the structure-function linkage of this important pump subunit, we studied the x-ray crystallographic structure of OprM at the 2.56-Å resolution. The overall structure exhibited trimeric assembly of the OprM monomer that consisted mainly of two domains: the membrane-anchoring ␤-barrel and the cavity-forming ␣-barrel. OprM anchors the outer membrane by two modes of membrane insertions. One is via the covalently attached NH 2 -terminal fatty acids and the other is the ␤-barrel structure consensus on the outer membrane-spanning proteins. The ␤-barrel had a pore opening with a diameter of about 6 -8 Å, which is not large enough to accommodate the exit of any antibiotics. The periplasmic ␣-barrel was about 100 Å long formed mainly by a bundle of ␣-helices that formed a solvent-filled cavity of about 25,000 Å 3 . The proximal end of the cavity was tightly sealed, thereby not permitting the entry of any molecule. The result of this structure was that the resting state of OprM had a small outer membrane pore and a tightly closed periplasmic end, which sounds plausible because the protein should not allow free access of antibiotics. However, these observations raised another unsolved problem about the mechanism of opening of the OprM cavity ends. The crystal structure offers possible mechanisms of pore opening and pump assembly.

Diversity in the Oligomeric Channel Structure of the Multidrug Efflux Pumps in Pseudomonas aeruginosa

“…The faint bands below each main protein band may be produced as a result of the difference of the intra-protein crosslinking. This clearly indicates that OprM assumed a trimeric structure in the membrane and the result is consistent with the trimeric structure of OprM determined by X-ray crystallography (1).…”

Assembly and Channel Opening of Outer Membrane Protein in Tripartite Drug Efflux Pumps of Gram-negative Bacteria

“…When the interaction was disrupted, the TolC channel partially opened spontaneously (7). Similar interactions were also found in the crystal structure of OprM in the closed state, demonstrating 12 polar interactions (46). However, the number of intermolecular interactions associated with channel closing was less than the putative interactions for the association of OprM with MexA residues.…”

“…This open conformation is distinguished from the closed conformation observed in the crystal structure of the full-length OprM (Fig. 2C) (46). This chimeric protein containing the OprM ␣-barrel tip region (A. actinomycetemcomitans MacA-OprM␣ hybrid dimer) exhibited a strong affinity to another chimeric protein containing MexA ␣-hairpin on a size exclusion chromatographic column (Fig.…”

Lateral mobility of proteins in liquid membranes revisited

“…To further explore the dependence of D on the radius R, two ␤-barrel proteins, OmpA (20) and OprM (21), along with BR, were inserted in dodecane-free bilayers of C 12 E 5 . Compared with the diffusion of L 12 , the protein diffusion constant is significantly reduced.…”