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J. Biol. Chem., Vol. 279, Issue 25, 25939-25942, June 18, 2004

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Crystal Structure of the Membrane Fusion Protein, MexA, of the Multidrug Transporter in Pseudomonas aeruginosa^*

Hiroyuki Akama, Takanori Matsuura, Sachiko Kashiwagi, Hiroshi Yoneyama¶, Shin-ichiro Narita, Tomitake Tsukihara, 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 MexAB-OprM efflux pump of Pseudomonas aeruginosa is central to multidrug resistance of this organism, which infects immunocompromised hospital patients. The MexA, MexB, and OprM subunits were assumed to function as the membrane fusion protein, the body of the transporter, and the outer membrane channel protein, respectively. For better understanding of this important xenobiotic transporter, we show the x-ray crystallographic structure of MexA at a resolution of 2.40 Å. The global MexA structure showed unforeseen new features with a spiral assembly of six and seven protomers that were joined together at one end by a pseudo 2-fold image. The protomer showed a new protein structure with a tandem arrangement consisting of at least three domains and presumably one more. The rod domain had a long hairpin of twisted coiled-coil that extended to one end. The second domain adjacent to the rod -helical domain was globular and constructed by a cluster of eight short -sheets. The third domain located distal to the -helical rod was globular and composed of seven short -sheets and one short -helix. The 13-mer was shaped like a woven rattan cylinder with a large internal tubular space and widely opened flared ends. The 6-mer and 7-mer had a funnel-like structure consisting of a tubular rod at one side and a widely opened flared funnel top at the other side. Based on these results, we constructed a model of the MexAB-OprM pump assembly. The three pairs of MexA dimers interacted with the periplasmic -barrel domain of OprM via the -helical hairpin, the second domain interacted with both MexB and OprM at their contact site, and the third and disordered domains probably interacted with the distal domain of MexB. In this fashion, the MexA subunit connected MexB and OprM, indicating that MexA is the membrane bridge protein.

Received for publication, April 13, 2004 , and in revised form, April 26, 2004.

^* This work was supported in part by a grant-in-aid for the 21st Century Centers of Excellence Research and the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Culture, Sport, Science and Technology; a grant-in-aid from the New Energy and Industrial Technology Department Organization (NEDO); a Tokai University Project Research Grant; and by a Tokai University School of Medicine Research Project Grant. 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 Table I and supplemental material for "Results and Discussion."

The atomic coordinates and structure factors (code 1VF7) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^¶ Present address: Faculty of Agriculture, Tohoku University, Sendai 981-8555, Japan.

^|| To whom correspondence may be addressed: The Inst. for Protein Research, Osaka University, Suita 565-0871, Japan. Tel./Fax: 81-6-6879-4313; E-mail: atsushi{at}protein.osaka-u.ac.jp. ** To whom correspondence may be addressed: Dept. of Molecular Life Science, Tokai University School of Medicine, Isehara, 259-1193, Japan. Tel.: 81-463-93-5436; Fax: 81-463-93-5437; E-mail: nakae{at}is.icc.u-tokai.ac.jp.

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