Crystal structure of the membrane-fusion protein of the multidrug efflux pump


Takanori Matsuura1, Hiroaki Akama2, Tomitake Tsukihara1, Atsushi Nakagawa1, Taiji Nakae2

1Institute for Protein Research, Osaka University,
2Department of Molecular Life Science, Tokai University School of Medicine

BSR2004 (the 8th International Conference on Biology and Synchrotron Radiation), (Himeji, Japan 2004.9.7-11)

The MexAB-OprM efflux pump of Pseudomonas aeruginosa is central to multidrug resistance of this organism, which infects immunocompromised hospital patients. Emergence of infectious agents resistant to structurally and functionally dissimilar chemotherapeutic agents is increasingly problematic in human health.

The crystal structure of MexA was solved by the single isomorphous replacement with the anomalous scattering (SIRAS) method using native and lutetium derivative data set. SHELX-97 and SHARP was used for Lu site search and SIRAS phasing, respectively. The atomic model was built using program O. At the initial stage of refinement, a simulated-annealed torsion angle refinement using CNS was applied. Subsequent cycles of refinement was carried out by REFMAC5 in CCP4. The crystal structure was refined to the R-factor of 25.8 % for 99.8% of the data in the resolution range of 40-2.4 Å.

Only 68.3% of the MexA residues were visible. The best ordered monomer structure shows 252 residues (from 23 to 274) among 369 amino acid residues of Azu-MexA-(His)6. MexA monomer consisted mainly of three domains (α-domain, β-domain, and α+β-domain) and possibly one additional unseen domain due to a disorder as follows. β-domain shows a topology similar to the biotinyl/lipoyl carrier proteins and domains family was defined in the SCOP data base.

The crystal structure of MexA appeared as a spiral assembly of the 13 protomer by contiguous joining of the hexamer and heptamer forming a rod at the middle and a funnel-top structure at both ends. The hexamer and heptamer were spirally assembled with a side-by-side contiguity of each monomer so that the spiral structure continued until the first molecule touched the seventh molecule at the narrower end, but the wider part of the first and last molecules remained untouched exhibiting a large unsealed lateral side.

To construct an assembly model of the MexAB-OprM efflux pump, MexB is simulated by amino acid replacement of AcrB of E.coli. The first question to be answered was whether or not the MexA 13-mer is present. Although an approximate longitudinal size of the 13-mer agreed well with the distance between the inner and outer membranes, we assume that the 13-mer is most likely a crystallographic artifact by the reasons that MexA is mainly fractionated with the inner membrane and the α+β-domain is located at both ends of the 13-mer.

So we assume another two models. The first one is based on the sleeve model that can be seen the crystal structure. In this model, the contact region between MexB and OprM is surrounded by the contiguous joining of MexA. We calculated that a total 12 of MexA molecules might be needed to wind around a MexB-OprM trimer. Thus, we have not yet discarded this model. The second model is 3-fold MexA-dimer model. In this model, dimeric MexA is bound to the contact region between MexB and OprM. Presence of the dimeric MexA was supported by gel filtration experiments in which a small fraction of MexA was eluted in the position corresponding to the dimer, although the majority of MexA appeared to be monomeric in aqueous solution (data not shown). Fitting of α-helices of the MexA dimer with the lower part of OprM resulted in good helix-helix interaction of OprM and MexA with a contact angle of about 20°.



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MATSUURA Takanori
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