THE NMR SIDE-CHAIN ASSIGNMENTS AND SOLUTION STRUCTURE OF ENZYME IIBCELLOBIOSE OF THE PHOSPHOENOLPYRUVATE-DEPENDENT PHOSPHOTRANSFERASE SYSTEM OF ESCHERICHIA-COLI

The assignment of the side-chain Nh IR resonances and the determinatio n of the three-dimensional solution structure of the C10S mutant of en zyme IIBcellobiose (IIBcel) of the phosphoenolpyruvate-dependent phosp hotransferase system of Escherichia coli are presented. The side-chain resonances were assigned nearly completely using a variety of mostly heteronuclear NMR experiments, including HCCH-TOCSY, HCCH-COSY, and CO CCH-TOCSY experiments as well as CBCACOHA, CBCA(CO)NH, and HBHA(CBCA)( CO)NH experiments. In order to obtain the three-dimensional structure, NOE data were collected from N-15-NOESY-HSQC, C-13-HSQC-NOESY, and 2D NOE experiments. The distance restraints derived from these NOE data were used in distance geometry calculations followed by molecular dyna mics and simulated annealing protocols. In an iterative procedure, add itional NOE assignments were derived from the calculated structures an d new structures were calculated. The final set of structures, calcula ted with approximately 2000 unambiguous and ambiguous distance restrai nts, has an rms deviation of 1.1 Angstrom, on C alpha atoms. IIBcel co nsists of a four stranded parallel beta-sheet, in the order 2134. The sheet is flanked with two and three alpha-helices on either side. Resi due 10, a cysteine in the wild-type enzyme, which is phosphorylated du ring the catalytic cycle, is located at the end of the first beta-stra nd. A loop that is proposed to be involved in the binding of the phosp horyl-group follows the cysteine. The loop appears to be disordered in the unphosphorylated state.