Multinuclear magnetic resonance studies of Escherichia coli adenylate kinase in free and bound forms -: Resonance assignment, secondary structure and ligand binding

The crystal structure of Escherichia coli adenylate kinase (AKe) revealed three main components: a CORE domain, composed of a five-stranded parallel beta-sheet surrounded by ol-helices, and two peripheral domains involved in covering the ATP in the active site (LID) and binding of the AMP (NMPbind) We initiated a long-term NMR study aiming to characterize the solution structure, binding mechanism and internal dynamics of the various domains. Using single (N-15) and double-labeled (C-13 and N-15) samples and double- and triple-resonance NMR experiments we assigned 97% of the H-1,C-13 and N-15 backbone resonances, and proton and C-13(beta) resonances for more than 40% of the side chains in the free protein. Analysis of a N-15-labeled enzyme in complex with the bi-substrate analogue [P-1,P-5-bis(S'-adenosine)-pentaphosphate] (Ap5A) resulted in the assignment of 90% of the backbone H-1 and N-15 resonances and 42% of the side chain resonances. Based on short-range NOEs and H-1 and C-13 secondary chemical shifts, we identified the elements of secondary structure and the topology of the beta-strands in the unliganded form. The alpha-helices and the beta-strands of the parallel beta-sheet in solution have the same limits (+/- 1 residue) as those observed in the crystal. The first helix (alpha 1) appears to have a frayed N-terminal side. Significant differences relative to the crystal were noticed in the LID domain, which in solution exhibits four antiparallel P-strands. The secondary structure of the nucleoside-bound form, as deduced from intramolecular NOEs and the H-1(alpha) chemical shifts, is similar to that of the free enzyme. The largest chemical shift differences allowed us to map the regions of protein-ligand contacts. H-1/H-2 exchange experiments performed on free and Ap5A-bound enzymes showed a general decrease of the structural flexibility in the complex which is accompanied by a local increased flexibility on the N-side of the parallel beta-sheet.