A novel view of domain flexibility in E. coli adenylate kinase based on structural mode-coupling 15N NMR relaxation

Abstract

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. In the ligand-free enzyme the domains AMPbd and LID execute large-amplitude movements controlling substrate binding and product release during catalysis. Domain flexibility is investigated herein with the slowly relaxing local structure (SRLS) model for 15N relaxation. SRLS accounts rigorously for coupling between the global and local N-H motions through a local ordering potential exerted by the protein structure at the N-H bond. The latter reorients with respect to its protein surroundings, which reorient on the slower time scale associated with the global protein tumbling. AKeco diffuses globally with correlation time τm=15.1 ns, while locally two different dynamic cases prevail. The domain CORE features ordering about the equilibrium N-H bond orientation with order parameters, S2, of 0.8-0.9 and local motional correlation times, τ, mainly between 5-130 ps. This represents a conventional rigid protein structure with rapid small-amplitude N-H fluctuations. The domains AMPbd and LID feature small parallel (ZM) ordering of S2=0.2-0.5 which can be reinterpreted as high perpendicular (YM) ordering. M denotes the local ordering/local diffusion frame. Local motion about ZM is given by τ∥≈5 ps and local motion of the effective ZM axis about YM by τ⊥=6-11 ns. ZM is tilted at approximately 20° from the N-H bond. The orientation of the YM axis may be considered parallel to the Ci−1α-Ciα axis. The τ⊥ mode reflects collective nanosecond peptide-plane motions, interpretable as domain motion. A powerful new model of protein flexibility/domain motion has been established. Conformational exchange (Rex) processes accompany the τ⊥ mode. The SRLS analysis is compared with the conventional model-free analysis.