Many Local Motions Cooperate to Produce the Adenylate Kinase Conformational Transition

Conformational transitions are functionally important in many proteins. In the enzyme adenylate kinase (AK), two small domains (LID and NMP) close over the larger CORE domain; the reverse (opening) motion limits the rate of catalytic turnover. Here, using double-well Gō simulations of Escherichia coli AK, we elaborate on previous investigations of the AK transition mechanism by characterizing the contributions of rigid-body (Cartesian), backbone dihedral, and contact motions to transition-state (TS) properties. In addition, we compare an apo simulation to a pseudo-ligand-bound simulation to reveal insights into allostery. In Cartesian space, LID closure precedes NMP closure in the bound simulation, consistent with prior coarse-grained models of the AK transition. However, NMP-first closure is preferred in the apo simulation. In backbone dihedral space, we find that, as expected, backbone fluctuations are reduced in the O/C transition in parts of all three domains. Among these “quenching” residues, most in the CORE, especially residues 11–13, are rigidified in the TS of the bound simulation, while residues 42–44 in the NMP are flexible in the TS. In contact space, in both apo and bound simulations, one nucleus of closed-state contacts includes parts of the NMP and CORE; CORE–LID contacts are absent in the TS of the apo simulation but formed in the TS of the bound simulation. From these results, we predict mutations that will perturb the opening and/or closing transition rates by changing the entropy of dihedrals and/or the enthalpy of contacts. Furthermore, regarding allostery, the fully closed structure is populated in the apo simulation, but our contact results imply that ligand binding shifts the preferred O/C transition pathway, thus precluding a simple conformational selection mechanism. Finally, the analytical approach and the insights derived from this work may inform the rational design of flexibility and allostery in proteins.     

Global Transitions of Proteins Explored by a Multiscale Hybrid Methodology: Application to Adenylate Kinase

Efficient and accurate mapping of transition pathways is a challenging problem in allosteric proteins. We propose here a to our knowledge new methodology called collective molecular dynamics (coMD). coMD takes advantage of the collective modes of motions encoded by the fold, simultaneously evaluating the interactions and energetics via a full-atomic MD simulation protocol. The basic approach is to deform the structure collectively along the modes predicted by the anisotropic network model, upon selecting them via a Monte Carlo/Metropolis algorithm from among the complete pool of all accessible modes. Application to adenylate kinase, an allosteric enzyme composed of three domains, CORE, LID, and NMP, shows that both open-to-closed and closed-to-open transitions are readily sampled by coMD, with large-scale motions of the LID dominating. An energy-barrier crossing occurs during the NMP movements. The energy barrier originates from a switch between the salt bridges K136-D118 at the LID-CORE interface and K57-E170 and D33-R156 at the CORE-NMP and LID-NMP interfaces, respectively. Despite its simplicity and computing efficiency, coMD yields ensembles of transition pathways in close accord with detailed full atomic simulations, lending support to its utility as a multiscale hybrid method for efficiently exploring the allosteric transitions of multidomain or multimeric proteins.     

Global Transitions of Proteins Explored by a Multiscale Hybrid Methodology: Application to Adenylate Kinase

Efficient and accurate mapping of transition pathways is a challenging problem in allosteric proteins. We propose here a to our knowledge new methodology called collective molecular dynamics (coMD). coMD takes advantage of the collective modes of motions encoded by the fold, simultaneously evaluating the interactions and energetics via a full-atomic MD simulation protocol. The basic approach is to deform the structure collectively along the modes predicted by the anisotropic network model, upon selecting them via a Monte Carlo/Metropolis algorithm from among the complete pool of all accessible modes. Application to adenylate kinase, an allosteric enzyme composed of three domains, CORE, LID, and NMP, shows that both open-to-closed and closed-to-open transitions are readily sampled by coMD, with large-scale motions of the LID dominating. An energy-barrier crossing occurs during the NMP movements. The energy barrier originates from a switch between the salt bridges K136-D118 at the LID-CORE interface and K57-E170 and D33-R156 at the CORE-NMP and LID-NMP interfaces, respectively. Despite its simplicity and computing efficiency, coMD yields ensembles of transition pathways in close accord with detailed full atomic simulations, lending support to its utility as a multiscale hybrid method for efficiently exploring the allosteric transitions of multidomain or multimeric proteins.     

Sequence of events underlying the allosteric transition of rod cyclic nucleotide-gated channels

Activation of cyclic nucleotide-gated (CNG) ion channels involves a conformational change in the channel protein referred to as the allosteric transition. The amino terminal region and the carboxyl terminal cyclic nucleotide-binding domain of CNG channels have been shown to be involved in the allosteric transition, but the sequence of molecular events occurring during the allosteric transition is unknown. We recorded single-channel currents from bovine rod CNG channels in which mutations had been introduced in the binding domain at position 604 and/or the rat olfactory CNG channel amino terminal region had been substituted for the bovine rod amino terminal region. Using a hidden Markov modeling approach, we analyzed the kinetics of these channels activated by saturating concentrations of cGMP, cIMP, and cAMP. We used thermodynamic mutant cycles to reveal an interaction during the allosteric transition between the purine ring of the cyclic nucleotides and the amino acid at position 604 in the binding site. We found that mutations at position 604 in the binding domain alter both the opening and closing rate constants for the allosteric transition, indicating that the interactions between the cyclic nucleotide and this amino acid are partially formed at the time of the transition state. In contrast, the amino terminal region affects primarily the closing rate constant for the allosteric transition, suggesting that the state-dependent stabilizing interactions between amino and carboxyl terminal regions are not formed at the time of the transition state for the allosteric transition. We propose that the sequence of events that occurs during the allosteric transition involves the formation of stabilizing interactions between the purine ring of the cyclic nucleotide and the amino acid at position 604 in the binding domain followed by the formation of stabilizing interdomain interactions.     

Minimum free energy path of ligand-induced transition in adenylate kinase

Large-scale conformational changes in proteins involve barrier-crossing transitions on the complex free energy surfaces of high-dimensional space. Such rare events cannot be efficiently captured by conventional molecular dynamics simulations. Here we show that, by combining the on-the-fly string method and the multi-state Bennett acceptance ratio (MBAR) method, the free energy profile of a conformational transition pathway in Escherichia coli adenylate kinase can be characterized in a high-dimensional space. The minimum free energy paths of the conformational transitions in adenylate kinase were explored by the on-the-fly string method in 20-dimensional space spanned by the 20 largest-amplitude principal modes, and the free energy and various kinds of average physical quantities along the pathways were successfully evaluated by the MBAR method. The influence of ligand binding on the pathways was characterized in terms of rigid-body motions of the lid-shaped ATP-binding domain (LID) and the AMP-binding (AMPbd) domains. It was found that the LID domain was able to partially close without the ligand, while the closure of the AMPbd domain required the ligand binding. The transition state ensemble of the ligand bound form was identified as those structures characterized by highly specific binding of the ligand to the AMPbd domain, and was validated by unrestrained MD simulations. It was also found that complete closure of the LID domain required the dehydration of solvents around the P-loop. These findings suggest that the interplay of the two different types of domain motion is an essential feature in the conformational transition of the enzyme.     

A novel view of domain flexibility in E. coli adenylate kinase based on structural mode-coupling (15)N 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. 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 (15)N 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 tau(m)=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, S(2), of 0.8-0.9 and local motional correlation times, tau, 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 (Z(M)) ordering of S(2)=0.2-0.5 which can be reinterpreted as high perpendicular (Y(M)) ordering. M denotes the local ordering/local diffusion frame. Local motion about Z(M) is given by tau( parallel) approximately 5 ps and local motion of the effective Z(M) axis about Y(M) by tau( perpendicular)=6-11 ns. Z(M) is tilted at approximately 20 degrees from the N-H bond. The orientation of the Y(M) axis may be considered parallel to the C(alpha)(i-1)-C(alpha)(i) axis. The tau( perpendicular) 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 (R(ex)) processes accompany the tau( perpendicular) mode. The SRLS analysis is compared with the conventional model-free analysis.     

Event detection and sub‐state discovery from biomolecular simulations using higher‐order statistics: Application to enzyme adenylate kinase

Biomolecular simulations at millisecond and longer time‐scales can provide vital insights into functional mechanisms. Because post‐simulation analyses of such large trajectory datasets can be a limiting factor in obtaining biological insights, there is an emerging need to identify key dynamical events and relating these events to the biological function online, that is, as simulations are progressing. Recently, we have introduced a novel computational technique, quasi‐anharmonic analysis (QAA) (Ramanathan et al., PLoS One 2011;6:e15827), for partitioning the conformational landscape into a hierarchy of functionally relevant sub‐states. The unique capabilities of QAA are enabled by exploiting anharmonicity in the form of fourth‐order statistics for characterizing atomic fluctuations. In this article, we extend QAA for analyzing long time‐scale simulations online. In particular, we present HOST4MD—a higher‐order statistical toolbox for molecular dynamics simulations, which (1) identifies key dynamical events as simulations are in progress, (2) explores potential sub‐states, and (3) identifies conformational transitions that enable the protein to access those sub‐states. We demonstrate HOST4MD on microsecond timescale simulations of the enzyme adenylate kinase in its apo state. HOST4MD identifies several conformational events in these simulations, revealing how the intrinsic coupling between the three subdomains (LID, CORE, and NMP) changes during the simulations. Further, it also identifies an inherent asymmetry in the opening/closing of the two binding sites. We anticipate that HOST4MD will provide a powerful and extensible framework for detecting biophysically relevant conformational coordinates from long time‐scale simulations. Proteins 2012. © 2012 Wiley Periodicals, Inc.     

In the Multi-domain Protein Adenylate Kinase,Domain Insertion Facilitates Cooperative Folding while Accommodating Function at Domain Interfaces

Having multiple domains in proteins can lead to partial folding and increased aggregation. Folding cooperativity, the all or nothing folding of a protein, can reduce this aggregation propensity. In agreement with bulk experiments, a coarse-grained structure-based model of the three-domain protein, E. coli Adenylate kinase (AKE), folds cooperatively. Domain interfaces have previously been implicated in the cooperative folding of multi-domain proteins. To understand their role in AKE folding, we computationally create mutants with deleted inter-domain interfaces and simulate their folding. We find that inter-domain interfaces play a minor role in the folding cooperativity of AKE. On further analysis, we find that unlike other multi-domain proteins whose folding has been studied, the domains of AKE are not singly-linked. Two of its domains have two linkers to the third one, i.e., they are inserted into the third one. We use circular permutation to modify AKE chain-connectivity and convert inserted-domains into singly-linked domains. We find that domain insertion in AKE achieves the following: (1) It facilitates folding cooperativity even when domains have different stabilities. Insertion constrains the N- and C-termini of inserted domains and stabilizes their folded states. Therefore, domains that perform conformational transitions can be smaller with fewer stabilizing interactions. (2) Inter-domain interactions are not needed to promote folding cooperativity and can be tuned for function. In AKE, these interactions help promote conformational dynamics limited catalysis. Finally, using structural bioinformatics, we suggest that domain insertion may also facilitate the cooperative folding of other multi-domain proteins.     

Probing the catalytic allosteric mechanism of rabbit muscle pyruvate kinase by tryptophan fluorescence quenching

Pyruvate kinase acts as an allosteric enzyme, playing a crucial role in the catalysis of the final step of the glycolytic pathway. In this study, site-specific mutagenesis and tryptophan fluorescence quenching were used to probe the catalytic allosteric mechanism of rabbit muscle pyruvate kinase. Movement of the B domain was found to be essential for the catalytic reaction. Rotation of the B domain in the opening of the cleft between domains B and A induced by the binding of activating cations allows substrates to bind, whereas substrate binding shifts the rotation of the B domain in the closure of the cleft. Trp-157 accounts for the differences in tryptophan fluorescence signal with and without activating cations and substrates. Trp-481 and Trp-514 are brought into an aqueous environment after phenylalanine binding.     

Conformational changes during the catalytic cycle of gluconate kinase as revealed by X-ray crystallography

The crystal structure of gluconate kinase from Escherichia coli has been determined to 2.0 A resolution by X-ray crystallography. The three-dimensional structure was solved by multi-wavelength anomalous dispersion, using a crystal of selenomethionine-substituted enzyme. Gluconate kinase is an alpha/beta structure consisting of a twisted parallel beta-sheet surrounded by alpha-helices with overall topology similar to nucleoside monophosphate (NMP) kinases, such as adenylate kinase. In order to identify residues involved in substrate binding and catalysis, structures of binary complexes with ATP, the ATP analogue adenosine 5'-(beta,gamma-methylene) triphosphate and the product, gluconate-6-phosphate have been determined. Significant conformational changes are induced upon binding of ATP to the enzyme. The largest changes involve a hinge-bending motion of the NMP(bind) part and a motion of the LID with adjacent helices, which opens the cavity to the second substrate, gluconate. Opening of the active site cleft upon ATP binding is the opposite of what has been observed in the NMP kinase family so far, which usually close their active site to prevent fortuitous hydrolysis of ATP. The conformational change positions the side-chain of Arg120 to stack with the purine ring of ATP and the side-chain of Arg124 is shifted to interact with the alpha-phosphate in ATP, at the same time protecting ATP from solvent water. The beta and gamma-phosphate groups of ATP bind in the predicted P-loop. A conserved lysine side-chain interacts with the gamma-phosphate group, and might promote phosphoryl transfer. Gluconate-6-phosphate binds with its phosphate group in a similar position as the gamma-phosphate of ATP, consistent with inline phosphoryl transfer. The gluconate binding-pocket in GntK is located in a different position than the nucleoside binding-site usually found in NMP kinases.     

Crystal structure of human adenylate kinase 4 (L171P) suggests the role of hinge region in protein domain motion

It is well known that motion of LID and NMP-binding (NMP_bind) domains in adenylate kinase (AK) is important in ligand binding and catalysis. However, the nature of such domain motions is poorly characterized. One of the critical hinge regions is hinge IV, which connects the CORE and LID domains. In addition, the hinge IV contains a strictly conserved residue, L171, in the AK family. To investigate the role of hinge IV, crystal structure of human adenylate kinase 4 (AK4) L171P mutant was determined. This mutation dramatically changes the orientation of the LID domain, which could be described as a novel twisted-and-closed conformation in contrast to the open and closed conformations in other AKs. This mutant provides a new example of domain motions in AK family.     

A spring-loaded release mechanism regulates domain movement and catalysis in phosphoglycerate kinase

Phosphoglycerate kinase (PGK) is the enzyme responsible for the first ATP-generating step of glycolysis and has been implicated extensively in oncogenesis and its development. Solution small angle x-ray scattering (SAXS) data, in combination with crystal structures of the enzyme in complex with substrate and product analogues, reveal a new conformation for the resting state of the enzyme and demonstrate the role of substrate binding in the preparation of the enzyme for domain closure. Comparison of the x-ray scattering curves of the enzyme in different states with crystal structures has allowed the complete reaction cycle to be resolved both structurally and temporally. The enzyme appears to spend most of its time in a fully open conformation with short periods of closure and catalysis, thereby allowing the rapid diffusion of substrates and products in and out of the binding sites. Analysis of the open apoenzyme structure, defined through deformable elastic network refinement against the SAXS data, suggests that interactions in a mostly buried hydrophobic region may favor the open conformation. This patch is exposed on domain closure, making the open conformation more thermodynamically stable. Ionic interactions act to maintain the closed conformation to allow catalysis. The short time PGK spends in the closed conformation and its strong tendency to rest in an open conformation imply a spring-loaded release mechanism to regulate domain movement, catalysis, and efficient product release.     

Solution structure and function of an essential CMP kinase of Streptococcus pneumoniae

Streptococcus pneumoniae is a major human pathogen that causes high mortality and morbidity and has developed resistance to many antibiotics. We show that the gene product from SP1603, identified from S. pneumoniae TIGR4, is a CMP kinase that is essential for bacterial growth. It represents an attractive drug target for the development of a novel antibiotic to overcome the problems of drug resistance development for this organism. Here we describe the three-dimensional solution structure of the S. pneumoniae CMP kinase as determined by NMR spectroscopy. The structure consists of eight alpha-helices and two beta-sheets that fold into the classical core domain, the substrate-binding domain, and the LID domain. The three domains of the protein pack together to form a central cavity for substrate-binding and enzymatic catalysis. The S. pneumoniae CMP kinase resembles the fold of the Escherichia coli homolog. An insertion of one residue is observed at the beta-turn in the substrate-binding domain of the S. pneumoniae CMP kinase when compared with the E. coli homolog. Chemical shift perturbations caused by the binding of CMP, CDP, and ATP revealed that CMP or CDP binds to the junction between the core and substrate-binding domains, whereas ATP binds to the junction between the core and LID domains. From NMR relaxation studies, we determined that the loops in the LID domain are highly mobile. These mobile loops could aid in the closing/opening of the LID domain during enzyme catalysis.     

Communication between the nucleotide site and the main molecular hinge of 3-phosphoglycerate kinase

3-Phosphoglycerate kinase is a hinge-bending enzyme with substrate-assisted domain closure. However, the closure mechanism has not been described in terms of structural details. Here we present experimental evidence of the participation of individual substrate binding side chains in the operation of the main hinge which is distant from the substrate binding sites. The combined mutational, kinetic, and structural (DSC and SAXS) data for human 3-phosphoglycerate kinase have shown that catalytic residue R38, which also binds the substrate 3-phosphoglycerate, is essential in inducing domain closure. Similarly, residues K219, N336, and E343 which interact with the nucleotide substrates are involved in the process of domain closure. The other catalytic residue, K215, covers a large distance during catalysis but has no direct role in domain closure. The transmission path of the nucleotide effect toward the main hinge of PGK is described for the first time at the level of interactions existing in the tertiary structure.     

Domain flexibility in ligand-free and inhibitor-bound Escherichia coli adenylate kinase based on a mode-coupling analysis of 15N spin relaxation

Adenylate kinase from Escherichia coli (AKeco), consisting of a 23.6-kDa polypeptide chain folded into domains CORE, AMPbd, and LID catalyzes the reaction AMP + ATP <--> 2ADP. The domains AMPbd and LID execute large-amplitude movements during catalysis. Backbone dynamics of ligand-free and AP(5)A-inhibitor-bound AKeco is studied with slowly relaxing local structure (SRLS) (15)N relaxation, an approach particularly suited when the global (tau(m)) and the local (tau) motions are likely to be coupled. For AKeco tau(m) = 15.1 ns, whereas for AKeco*AP(5)A tau(m) = 11.6 ns. The CORE domain of AKeco features an average squared order parameter, , of 0.84 and correlation times tau(f) = 5-130 ps. Most of the AKeco*AP(5)A backbone features = 0.90 and tau(f) = 33-193 ps. These data are indicative of relative rigidity. Domains AMPbd and LID of AKeco, and loops beta(1)/alpha(1), alpha(2)/alpha(3), alpha(4)/beta(3), alpha(5)/beta(4), and beta(8)/alpha(7) of AKeco*AP(5)A, feature a novel type of protein flexibility consisting of nanosecond peptide plane reorientation about the C(i-1)(alpha)-C(i)(alpha) axis, with correlation time tau(perpendicular) = 5.6-11.3 ns. The other microdynamic parameters underlying this dynamic model include S(2) = 0.13-0.5, tau(parallel) on the ps time scale, and a diffusion tilt beta(MD) ranging from 12 to 21 degrees. For the ligand-free enzyme the tau(perpendicular) mode was shown to represent segmental domain motion, accompanied by conformational exchange contributions R(ex) < or = 4.4 s(-1). Loop alpha(4)/beta(3) and alpha(5)/beta(4) dynamics in AKeco*AP(5)A is related to the "energetic counter-balancing of substrate binding" effect apparently driving kinase catalysis. The other flexible AKeco*AP(5)A loops may relate to domain motion toward product release.     

Allosteric regulation of the biotin-dependent enzyme pyruvate carboxylase by acetyl-CoA

The activity of the biotin-dependent enzyme pyruvate carboxylase from many organisms is highly regulated by the allosteric activator acetyl-CoA. A number of X-ray crystallographic structures of the native pyruvate carboxylase tetramer are now available for the enzyme from Rhizobium etli and Staphylococcus aureus. Although all of these structures show that intersubunit catalysis occurs, in the case of the R. etli enzyme, only two of the four subunits have the allosteric activator bound to them and are optimally configured for catalysis of the overall reaction. However, it is apparent that acetyl-CoA binding does not induce the observed asymmetrical tetramer conformation and it is likely that, under normal reaction conditions, all of the subunits have acetyl-CoA bound to them. Thus the activation of the enzyme by acetyl-CoA involves more subtle structural effects, one of which may be to facilitate the correct positioning of Arg353 and biotin in the biotin carboxylase domain active site, thereby promoting biotin carboxylation and, at the same time, preventing abortive decarboxylation of carboxybiotin. It is also apparent from the crystal structures that there are allosteric interactions induced by acetyl-CoA binding in the pair of subunits not optimally configured for catalysis of the overall reaction.     

Crystal structure of bacteriophage T4 deoxynucleotide kinase with its substrates dGMP and ATP.

NMP kinases catalyse the phosphorylation of the canonical nucleotides to the corresponding diphosphates using ATP as a phosphate donor. Bacteriophage T4 deoxynucleotide kinase (DNK) is the only member of this family of enzymes that recognizes three structurally dissimilar nucleotides: dGMP, dTMP and 5-hydroxymethyl-dCMP while excluding dCMP and dAMP. The crystal structure of DNK with its substrate dGMP has been determined at 2.0 A resolution by single isomorphous replacement. The structure of the ternary complex with dGMP and ATP has been determined at 2.2 A resolution. The polypeptide chain of DNK is folded into two domains of equal size, one of which resembles the mononucleotide binding motif with the glycine-rich P-loop. The second domain, consisting of five alpha-helices, forms the NMP binding pocket. A hinge connection between the domains allows for large movements upon substrate binding which are not restricted by dimerization of the enzyme. The mechanism of active centre formation via domain closure is described. Comparison with other P-loop-containing proteins indicates an induced-fit mode of NTP binding. Protein-substrate interactions observed at the NMP and NTP sites provide the basis for understanding the principles of nucleotide discrimination.     

Mechanisms for activation and antagonism of an AMPA-sensitive glutamate receptor: crystal structures of the GluR2 ligand binding core

Crystal structures of the GluR2 ligand binding core (S1S2) have been determined in the apo state and in the presence of the antagonist DNQX, the partial agonist kainate, and the full agonists AMPA and glutamate. The domains of the S1S2 ligand binding core are expanded in the apo state and contract upon ligand binding with the extent of domain separation decreasing in the order of apo > DNQX > kainate > glutamate approximately equal to AMPA. These results suggest that agonist-induced domain closure gates the transmembrane channel and the extent of receptor activation depends upon the degree of domain closure. AMPA and glutamate also promote a 180 degrees flip of a trans peptide bond in the ligand binding site. The crystal packing of the ligand binding cores suggests modes for subunit-subunit contact in the intact receptor and mechanisms by which allosteric effectors modulate receptor activity.     

Crystal structure of unligated guanylate kinase from yeast reveals GMP-induced conformational changes

The crystal structure of guanylate kinase (GK) from yeast (Saccharomyces cerevisiae) with a non-acetylated N terminus has been determined in its unligated form (apo-GK) as well as in complex with GMP (GK.GMP). The structure of apo-GK was solved with multiwavelength anomalous diffraction data and refined to an R-factor of 0.164 (R(free)=0.199) at 2.3 A resolution. The structure of GK.GMP was determined using the crystal structure of GK with an acetylated N terminus as the search model and refined to an R-factor of 0.156 (R(free)=0.245) at 1.9 A. GK belongs to the family of nucleoside monophosphate (NMP) kinases and catalyzes the reversible phosphoryl transfer from ATP to GMP. Like other NMP kinases, GK consists of three dynamic domains: the CORE, LID, and NMP-binding domains. Dramatic movements of the GMP-binding domain and smaller but significant movements of the LID domain have been revealed by comparing the structures of apo-GK and GK.GMP. apo-GK has a much more open conformation than the GK.GMP complex. Systematic analysis of the domain movements using the program DynDom shows that the large movements of the GMP-binding domain involve a rotation around an effective hinge axis approximately parallel with helix 3, which connects the GMP-binding and CORE domains. The C-terminal portion of helix 3, which connects to the CORE domain, has strikingly higher temperature factors in GK.GMP than in apo-GK, indicating that these residues become more mobile upon GMP binding. The results suggest that helix 3 plays an important role in domain movement. Unlike the GMP-binding domain, which moves toward the active center of the enzyme upon GMP binding, the LID domain moves away from the active center and makes the presumed ATP-binding site more open. Therefore, the LID domain movement may facilitate the binding of MgATP. The structure of the recombinant GK.GMP complex superimposes very well with that of the native GK.GMP complex, indicating that N-terminal acetylation does not have significant impact on the three-dimensional structure of GK.     

Binding leverage as a molecular basis for allosteric regulation

Allosteric regulation involves conformational transitions or fluctuations between a few closely related states, caused by the binding of effector molecules. We introduce a quantity called binding leverage that measures the ability of a binding site to couple to the intrinsic motions of a protein. We use Monte Carlo simulations to generate potential binding sites and either normal modes or pairs of crystal structures to describe relevant motions. We analyze single catalytic domains and multimeric allosteric enzymes with complex regulation. For the majority of the analyzed proteins, we find that both catalytic and allosteric sites have high binding leverage. Furthermore, our analysis of the catabolite activator protein, which is allosteric without conformational change, shows that its regulation involves other types of motion than those modulated at sites with high binding leverage. Our results point to the importance of incorporating dynamic information when predicting functional sites. Because it is possible to calculate binding leverage from a single crystal structure it can be used for characterizing proteins of unknown function and predicting latent allosteric sites in any protein, with implications for drug design.