NMP Domain Research Articles

Cooperativity and Folding Kinetics in a Multidomain Protein with Interwoven Chain Topology.

Although a large percentage of eukaryotic proteomes consist of proteins with multiple domains, not much is known about their assembly mechanism, especially those with intricate native state architectures. Some have a complex topology in which the structural elements along the sequence are interwoven in such a manner that the domains cannot be separated by cutting at any location along the sequence. Such proteins are multiply connected multidomain proteins (MMPs) with the three-domain (NMP, LID, and CORE) phosphotransferase enzyme adenylate kinase (ADK) being an example. We devised a coarse-grained model to simulate ADK folding initiated by changing either the temperature or guanidinium chloride (GdmCl) concentration. The simulations reproduce the experimentally measured melting temperatures (associated with two equilibrium transitions), FRET efficiency as a function of GdmCl concentration, and the folding times quantitatively. Although the NMP domain orders independently, cooperative interactions between the LID and the CORE domains are required for complete assembly of the enzyme. Kinetic simulations show that, on the collapse time scale, multiple interconnected metastable states are populated, attesting to the folding heterogeneity. The network of kinetically connected states reveals that the CORE domain folds only after the NMP and LID domains, reflecting the interwoven nature of the chain topology.

ADP-Induced Conformational Transition of Human Adenylate Kinase 1 Is Triggered by Suppressing Internal Motion of α3α4 and α7α8 Fragments on the ps-ns Timescale.

Human adenylate kinase 1 (hAK1) plays a vital role in the energetic and metabolic regulation of cell life, and impaired functions of hAK1 are closely associated with many diseases. In the presence of Mg2+ ions, hAK1 in vivo can catalyze two ADP molecules into one ATP and one AMP molecule, activating the downstream AMP signaling. The ADP-binding also initiates AK1 transition from an open conformation to a closed conformation. However, how substrate binding triggers the conformational transition of hAK1 is still unclear, and the underlying molecular mechanisms remain elusive. Herein, we determined the solution structure of apo-hAK1 and its key residues for catalyzing ADP, and characterized backbone dynamics characteristics of apo-hAK1 and hAK1-Mg2+-ADP complex (holo-hAK1) using NMR relaxation experiments. We found that ADP was primarily bound to a cavity surrounded by the LID, NMP, and CORE domains of hAK1, and identified several critical residues for hAK1 catalyzing ADP including G16, G18, G20, G22, T39, G40, R44, V67, D93, G94, D140, and D141. Furthermore, we found that apo-hAK1 adopts an open conformation with significant ps-ns internal mobility, and Mg2+-ADP binding triggered conformational transition of hAK1 by suppressing the ps-ns internal motions of α3α4 in the NMP domain and α7α8 in the LID domain. Both α3α4 and α7α8 fragments became more rigid so as to fix the substrate, while the catalyzing center of hAK1 experiences promoted µs-ms conformational exchange, potentially facilitating catalysis reaction and conformational transition. Our results provide the structural basis of hAK1 catalyzing ADP into ATP and AMP, and disclose the driving force that triggers the conformational transition of hAK1, which will deepen understanding of the molecular mechanisms of hAK1 functions.

Multiple Pathways and Time Scales for Conformational Transitions in apo-Adenylate Kinase.

The open/close transition in adenylate kinase (AK) is regarded as a representative example for large-scale conformational transition in proteins, yet its mechanism remains unclear despite numerous experimental and computational studies. Using extensive (∼50 μs) explicit solvent atomistic simulations and Markov state analysis, we shed new lights on the mechanism of this transition in the apo form of AK. The closed basin of apo AK features an open NMP domain while the LID domain closes and rotates toward it. Therefore, although the computed structural properties of the closed ensemble are consistent with previously reported FRET and PRE measurements, our simulations suggest that NMP closure is likely to follow AMP binding, in contrast to the previous interpretation of FRET and PRE data that the apo state was able to sample the fully closed conformation for "ligand selection". The closed state ensemble is found to be kinetically heterogeneous; multiple pathways and time scales are associated with the open/close transition, providing new clues to the disparate time scales observed in different experiments. Besides interdomain interactions, a novel mutual information analysis identifies specific intradomain interactions that correlate strongly to transition kinetics, supporting observations from previous chimera experiments. While our results underscore the role of internal domain properties in determining the kinetics of open/close transition in apo AK, no evidence is observed for any significant degree of local unfolding during the transition. These observations about AK have general implications to our view of conformational states, transition pathways, and time scales of conformational changes in proteins. The key features and time scales of observed transition pathways are robust and similar from simulations using two popular fixed charge force fields.

Ligand Docking to Intermediate and Close-To-Bound Conformers Generated by an Elastic Network Model Based Algorithm for Highly Flexible Proteins.

Incorporating receptor flexibility in small ligand-protein docking still poses a challenge for proteins undergoing large conformational changes. In the absence of bound structures, sampling conformers that are accessible by apo state may facilitate docking and drug design studies. For this aim, we developed an unbiased conformational search algorithm, by integrating global modes from elastic network model, clustering and energy minimization with implicit solvation. Our dataset consists of five diverse proteins with apo to complex RMSDs 4.7–15 Å. Applying this iterative algorithm on apo structures, conformers close to the bound-state (RMSD 1.4–3.8 Å), as well as the intermediate states were generated. Dockings to a sequence of conformers consisting of a closed structure and its “parents” up to the apo were performed to compare binding poses on different states of the receptor. For two periplasmic binding proteins and biotin carboxylase that exhibit hinge-type closure of two dynamics domains, the best pose was obtained for the conformer closest to the bound structure (ligand RMSDs 1.5–2 Å). In contrast, the best pose for adenylate kinase corresponded to an intermediate state with partially closed LID domain and open NMP domain, in line with recent studies (ligand RMSD 2.9 Å). The docking of a helical peptide to calmodulin was the most challenging case due to the complexity of its 15 Å transition, for which a two-stage procedure was necessary. The technique was first applied on the extended calmodulin to generate intermediate conformers; then peptide docking and a second generation stage on the complex were performed, which in turn yielded a final peptide RMSD of 2.9 Å. Our algorithm is effective in producing conformational states based on the apo state. This study underlines the importance of such intermediate states for ligand docking to proteins undergoing large transitions.

Molecular simulations of substrate release and coupled conformational motions in adenylate kinase

Conformational opening coupled substrate release is believed to be related to the rate limiting step in the catalysis cycle of the adenylate kinase. However, it is still unclear how the substrate dissociates from its active site and how the substrate release is coupled to conformational changes of the kinase. In this work, by using metadynamics simulations, we investigated the ADP release process and the coupled protein dynamics. We found that the ADP release involves overcoming a high free energy barrier, and protonation of the [Formula: see text]-phosphate of the ADP molecules can drastically reduce the barrier height, therefore, promote the ADP release. We identified several key residues contributing to the high free energy barrier. We also showed that the ADP attached to LID domain leaves the binding pocket earlier than the one attached to the NMP domain. We further observed that the ADP release is accompanied by almost fully opening of the LID domain and partially opening of the NMP domain. Our results provide insight into the molecular mechanism of the substrate release of adenylate kinase and the coupled conformational motions.

Molecular dynamics studies on the conformational transitions of adenylate kinase: a computational evidence for the conformational selection mechanism.

Escherichia coli adenylate kinase (ADK) is a monomeric phosphotransferase enzyme that catalyzes reversible transfer of phosphoryl group from ATP to AMP with a large-scale domain motion. The detailed mechanism for this conformational transition remains unknown. In the current study, we performed long time-scale molecular dynamics simulations on both open and closed states of ADK. Based on the structural analyses of the simulation trajectories, we detected over 20 times conformational transitions between the open and closed states of ADK and identified two novel conformations as intermediate states in the catalytic processes. With these findings, we proposed a possible mechanism for the large-scale domain motion of Escherichia coli ADK and its catalytic process: (1) the substrate free ADK adopted an open conformation; (2) ATP bound with LID domain closure; (3) AMP bound with NMP domain closure; (4) phosphoryl transfer occurred with ATP, and AMP converted into two ADPs, and no conformational transition was detected in the enzyme; (5) LID domain opened with one ADP released; (6) another ADP released with NMP domain open. As both open and closed states sampled a wide range of conformation transitions, our simulation strongly supported the conformational selection mechanism for Escherichia coli ADK.

Exploring the Dynamic Functional Landscape of Adenylate Kinase Modulated by Substrates.

Adenylate kinase (ADK) has been explored widely, through both experimental and theoretical studies. However, still less is known about how the functional dynamics of ADK is modulated explicitly by its natural substrates. Here, we report a quantitative study of the dynamic energy landscape for ADK responding to the substrate binding by integrating both experimental investigations and theoretical modeling. We make theoretical predictions which are in remarkable agreement with the single molecule experiments on the substrate-bound complex. With our combined models of ADK in its apo form, in the presence of AMP or ATP, and in complex with both substrates, we specifically address the following key questions: (1) Are there intermediate state(s) during their catalytic cycle and if so how many? (2) How many pathways are there along the open-to-closed transitions and what are their corresponding weights? (3) How do substrates influence the pathway weights and the stability of the intermediates? (4) Which lid's motion is rate-limiting along the turnover cycle, the NMP or the LID domain? Our models predict two major parallel stepwise pathways and two on-pathway intermediates which are denoted as IN (NMP domain open while LID domain closed) and IL (LID domain open and NMP domain closed), respectively. Further investigation of temperature effects suggests that the IN pathway is dominant at room temperature, but the IL pathway is dominant at the optimal temperature. This leads us to propose that the IL pathway is more dominant by entropy and IN pathway by enthalpy. Remarkably, our results show that even with maximum concentrations of natural substrates, ADK still fluctuates between multiple functional states, reflecting an intrinsic capability of large-scale conformational fluctuations which may be essential to its biological function. The results based on the dual-ligands model provide the theoretical validation of random bisubstrate biproducts (Bi-Bi) mechanism for the enzymatic reaction of ADK. Additionally, the pathway flux analysis strongly suggests that the motion of the NMP domain is the rate-determining step for the conformational cycle (opening and closing).

Interconversion of Functional Motions between Mesophilic and Thermophilic Adenylate Kinases

Dynamic properties are functionally important in many proteins, including the enzyme adenylate kinase (AK), for which the open/closed transition limits the rate of catalytic turnover. Here, we compare our previously published coarse-grained (double-well Gō) simulation of mesophilic AK from E. coli (AKmeso) to simulations of thermophilic AK from Aquifex aeolicus (AKthermo). In AKthermo, as with AKmeso, the LID domain prefers to close before the NMP domain in the presence of ligand, but LID rigid-body flexibility in the open (O) ensemble decreases significantly. Backbone foldedness in O and/or transition state (TS) ensembles increases significantly relative to AKmeso in some interdomain backbone hinges and within LID. In contact space, the TS of AKthermo has fewer contacts at the CORE-LID interface but a stronger contact network surrounding the CORE-NMP interface than the TS of AKmeso. A “heated” simulation of AKthermo at 375K slightly increases LID rigid-body flexibility in accordance with the “corresponding states” hypothesis. Furthermore, while computational mutation of 7 prolines in AKthermo to their AKmeso counterparts produces similar small perturbations, mutation of these sites, especially positions 8 and 155, to glycine is required to achieve LID rigid-body flexibility and hinge flexibilities comparable to AKmeso. Mutating the 7 sites to proline in AKmeso reduces some hinges' flexibilities, especially hinge 2, but does not reduce LID rigid-body flexibility, suggesting that these two types of motion are decoupled in AKmeso. In conclusion, our results suggest that hinge flexibility and global functional motions alike are correlated with but not exclusively determined by the hinge residues. This mutational framework can inform the rational design of functionally important flexibility and allostery in other proteins toward engineering novel biochemical pathways.

Dynamic coupling between the LID and NMP domain motions in the catalytic conversion of ATP and AMP to ADP by adenylate kinase

The catalytic conversion of adenosine triphosphate (ATP) and adenosine monophosphate (AMP) to adenosine diphosphate (ADP) by adenylate kinase (ADK) involves large amplitude, ligand induced domain motions, involving the opening and the closing of ATP binding domain (LID) and AMP binding domain (NMP) domains, during the repeated catalytic cycle. We discover and analyze an interesting dynamical coupling between the motion of the two domains during the opening, using large scale atomistic molecular dynamics trajectory analysis, covariance analysis, and multidimensional free energy calculations with explicit water. Initially, the LID domain must open by a certain amount before the NMP domain can begin to open. Dynamical correlation map shows interesting cross-peak between LID and NMP domain which suggests the presence of correlated motion between them. This is also reflected in our calculated two-dimensional free energy surface contour diagram which has an interesting elliptic shape, revealing a strong correlation between the opening of the LID domain and that of the NMP domain. Our free energy surface of the LID domain motion is rugged due to interaction with water and the signature of ruggedness is evident in the observed root mean square deviation variation and its fluctuation time correlation functions. We develop a correlated dynamical disorder-type theoretical model to explain the observed dynamic coupling between the motion of the two domains in ADK. Our model correctly reproduces several features of the cross-correlation observed in simulations.

Role of Water in the Enzymatic Catalysis: Study of ATP + AMP → 2ADP Conversion by Adenylate Kinase

The catalytic conversion ATP + AMP → 2ADP by the enzyme adenylate kinase (ADK) involves the binding of one ATP molecule to the LID domain and one AMP molecule to the NMP domain. The latter is followed by a phosphate transfer and then the release of two ADP molecules. We have computed a novel two-dimensional configurational free energy surface (2DCFES), with one reaction coordinate each for the LID and the NMP domain motions, while considering explicit water interactions. Our computed 2DCFES clearly reveals the existence of a stable half-open half-closed (HOHC) intermediate state of the enzyme. Cycling of the enzyme through the HOHC state reduces the conformational free energy barrier for the reaction by about 20 kJ/mol. We find that the stability of the HOHC state (missed in all earlier studies with implicit solvent model) is largely because of the increase of specific interactions of the polar amino acid side chains with water, particularly with the arginine and the histidine residues. Free energy surface of the LID domain is rather rugged, which can conveniently slow down LID's conformational motion, thus facilitating a new substrate capture after the product release in the catalytic cycle.

Conformational Transitions in Adenylate Kinase: ALLOSTERIC COMMUNICATION REDUCES MISLIGATION

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.