Backbone dynamics of Escherichia coli adenylate kinase at the extreme stages of the catalytic cycle studied by N-15 NMR relaxation

Adenylate kinase from Escherichia coli (AKeco), consisting of a single 23.6 kDa polypeptide chain folded into domains CORE, AMPbd, and LID, catalyzes the reaction AMP + ATP --> 2ADP. Domains LID and AMPbd execute large-scale movements during catalysis. Backbone dynamics of ligand-free and AP(5)A-inh ibitor-bound AKeco were studied comparatively with N-15 NMR relaxation meth ods. Overall diffusion with correlation times of 15.05 (11.42) ns and aniso tropy D-parallel/D-perp = 1.25 (1.10), and fast internal motions with corre lation times up to 100 ps (50 ps), were determined for AKeco (AKeco*AP(5)A) . Fast internal motions affect 93% of the AKeco sites, with pronounced pref erence for domains AMPbd and LLD, and 47% of the AKeco*AP(5)A sites, with l imited variability along the chain. The mean squared generalized order para meters, [S-2], Of secondary structure elements and loops are affected by li gand binding differentially and in a domain-specific manner. Nanosecond mot ions predominate within AMPbd. Prominent exchange contributions, associated in particular with residue G10 of the nucleotide-binding P-loop motif, are interpreted to reflect hydrogen bond dynamics at the inhibitor-binding sit e. The hypothesis of energetic counter balancing of substrate binding based on crystallographic data is strongly supported by the solution NMR results . Correlations between backbone dynamics and domain displacement are establ ished.