THE CRYSTAL STRUCTURE OF THE IIA DOMAIN OF THE MANNOSE TRANSPORTER FROM ESCHERICHIA COLI AT 1.7 Å RESOLUTION. R. S. Nunn1, Z. Markovic-Housley1,2, J.C. Genovesio-Taverne1, K. Flukiger2, P.J. Rizkallah3, J.N. Jansoniusl, B. Erni2 and T. Schirmer1, 1Biozentrum, Abteilung für Strukturbiologie, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland, 2Institut fur Biochemie, Universitat Bern, Freiestrasse 3, CH-3012 Bern, Switzerland, 3Daresbury Laboratory,Daresbury, Warrington,WA4 4AD, UK
The mannose uansporter from Escherichia coli is a member of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The multi-subunit complex couples translocation across the bacterial inner membrane with solute phosphorylation. A functional fragment, 14.8 kDa (IIAMan, residues 2-133) of the membrane associated IIABMan subunit of the mannose transporter was expressed as a selenomethionine protein and the unphosphorylated structure was solved by X-ray crystallography. The protein consists of a central 5-stranded ß-sheet flanked by helices. The order of the secondary structure elements is (ßa)4,(aß) with strand order 21345. Four parallel ß-strands are linked by helices forming right-handed cross-over connections. The fifth strand is antiparallel to the others and is swapped between the subunits of the dimeric structure. Helices D and E form a helical hairpin. The active site consists of a buried aspartyl group and a histidine residue which is reminiscent of the family of serine proteases. His10, which is known to be transiently phosphorylated during catalysis, is located at the topologial switch-point of the structure and close to the subunit interface. Nd1 of Hisl0 is hydrogen bonded to the side-chain of Asp67. It is likely that Asp67 acts as a general base and thus increases the nucleophilicity of the histidine. Data were collected to 1.6 Å at station 9.5 in Daresbury and the crystal structure was solved in a hexagonal spacegroup P6(1)22 (Imol/a.u.) with cell constants a=b=76.4 Å, c=88.7 Å, a=ß=90deg., g=120deg. using MIRAS. The initial Se-SIRAS map calculated to 2.4 Å showed part of a helix (later identified as helix D) which improved upon addition of the platinum phases (calculated to 3.2 Å) and density modification. The atomic model was built and refined to 1.7 Å resolution with a crystallographic Rfactor of 18.9% and Rfree of 22.1% for 989 protein atoms and 70 water molecules.