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J. Biol. Chem., Vol. 279, Issue 51, 52816-52819, December 17, 2004

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

DUAL MODES OF MEMBRANE ANCHORING AND OCCLUDED CAVITY END^*

Hiroyuki Akama, Misa Kanemaki, Masato Yoshimura, Tomitake Tsukihara, Tomoe Kashiwagi, Hiroshi Yoneyama, Shin-ichiro Narita, Atsushi Nakagawa¶, and Taiji Nakae||

From the Department of Molecular Life Science, Tokai University School of Medicine, Isehara 259-1193, Japan and the Institute for Protein Research, Osaka University, Suita 565-0871, Japan

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 porin-type 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 NH₂-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 ų. 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.

Received for publication, September 22, 2004 , and in revised form, October 14, 2004.

^* This work was supported in part by grants from the Ministry of Education, Culture, Sport, Science and Technology; by the 21st Century Centers of Excellence Research; by Protein 3000 project; and by Tokai University (Project Research Grant) and Tokai University School of Medicine (Research Project Grant). This work was performed under the Cooperative Research Program of Institute for Protein Research, Osaka University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains the Experimental Procedures, supplemental Results, Table 1, and supplemental Figs. 1–4.

The atomic coordinates and structure factors (code 1WP1) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^¶ To whom correspondence may be addressed. Tel./Fax: 81-6-6879-4313; E-mail: atsushi{at}protein.osaka-u.ac.jp. || To whom correspondence may be addressed. Tel.: 81-463-93-5436; Fax: 81-463-93-5437; E-mail: nakae{at}is.icc.u-tokai.ac.jp.

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