Conformational Transitions in Adenylate Kinase

Abstract

Large conformational changes in the LID and NMP domains of adenylate kinase (AKE) are known to be key to ligand binding and catalysis, yet the order of binding events and domain motion is not well understood. Combining the multiple available structures for AKE with the energy landscape theory for protein folding, a theoretical model was developed for allostery, order of binding events, and efficient catalysis. Coarse-grained models and nonlinear normal mode analysis were used to infer that intrinsic structural fluctuations dominate LID motion, whereas ligand-protein interactions and cracking (local unfolding) are more important during NMP motion. In addition, LID-NMP domain interactions are indispensable for efficient catalysis. LID domain motion precedes NMP domain motion, during both opening and closing. These findings provide a mechanistic explanation for the observed 1:1:1 correspondence between LID domain closure, NMP domain closure, and substrate turnover. This catalytic cycle has likely evolved to reduce misligation, and thus inhibition, of AKE. The separation of allosteric motion into intrinsic structural fluctuations and ligand-induced contributions can be generalized to further our understanding of allosteric transitions in other proteins.Kinase-mediated phosphoryl transfer is a key component of many signaling pathways. Tight control of signaling requires regulation of kinase activity, which is influenced by allosteric transitions (1, 2). The classical description of allostery involves ligand-induced conformational rearrangements between static protein structures. It is now acknowledged that protein dynamics is statistical in nature and that allostery is often due to a change in the balance of pre-existing conformational substates upon ligand binding (3).This manuscript explores the subject of how protein structure determines allostery, order of ligand binding events, and ultimately, efficient catalysis, in the context of adenylate kinase (AKE).4 AKE is a three-domain (LID, NMP, and CORE) protein (Fig. 1) that undergoes large conformational changes as it catalyzes Reaction 1.Large motions of the LID and NMP domains are associated with nucleotide binding. It has been shown that substrate turnover and domain rearrangements ("open" state to "closed" state) occur at the same frequency (2). Despite several theoretical (6 -13) and experimental studies (2, 14 -17) on AKE, a catalytic mechanism that explains this high efficiency has not been proposed.Because it is known that conformational transitions can be well described as a superposition of normal modes (6, 7, 13, 18 -20), we use a simplified nonlinear elastic network model and a structure-based model with implicit ligand interactions to demonstrate a likely catalytic mechanism in AKE. We show that intrinsic structural fluctuations (21,22) in the LID domain account for the majority of the domain's motion and that ATP binding likely serves to lock the domain closed. The LID motion is an example of allosteric regulation that results from a shift in the popu...

Correlated Inter-Domain Motions in Adenylate Kinase

“…2b, the ATP and AMP domain movements are correlated, disfavouring conformations where the LID is open and the AMPbd is closed. Our findings agree with free energy calculations[34], molecular dynamics simulations[35] as well as with normal mode analysis [33], [36]. Although it is impossible to derive the closure pathway from equilibrium data, the conformational states observed favour a step-wise mechanism in which LID closure takes place before AMPbd closure.…”

“…5% at 37°C;[32] under the conditions used in this study (25°C) this state has a low population (<1%). Similarly, cracking of the AMPbd along the closing mechanism may play a role [26], [33]. However, cracking occurs at the transition state, and therefore has a very low population.…”

Computing free energies of protein conformations from explicit solvent simulations

“…In addition, both conformations A and B must correspond to finite segments of the ξ space, which means that conformations similar to A must have ξ close to ξ( A ) and vice versa, a region of ξ ≈ ξ( A ) must only contain conformations similar to A (the statement has to be true for conformation B as well). In some cases, the topology of the landscape or allosteric motions themselves can provide a good, physically meaningful collective coordinate [21,22]. The new path coordinate that we propose works for general case.…”

Interdomain communication revealed in the diabetes drug target mitoNEET

“…This led us to the hypothesis that functional regions in proteins may add roughness to the landscape because they are under separate evolutionary pressure than areas used for efficient folding. For example, structure-based simulations with adenylate kinase demonstrated that the introduction of frustration induced conformational transitions associated with enzymatic catalyisis through specific unfolding, or cracking (20,21). Folding simulations with Csk and IL-1β successfully captured long range communication to functional sites (22,23).…”