The Structural Basis of ATP as an Allosteric Modulator

Adenosine-5’-triphosphate (ATP) is generally regarded as a substrate for energy currency and protein modification. Recent findings uncovered the allosteric function of ATP in cellular signal transduction but little is understood about this critical behavior of ATP. Through extensive analysis of ATP in solution and proteins, we found that the free ATP can exist in the compact and extended conformations in solution, and the two different conformational characteristics may be responsible for ATP to exert distinct biological functions: ATP molecules adopt both compact and extended conformations in the allosteric binding sites but conserve extended conformations in the substrate binding sites. Nudged elastic band simulations unveiled the distinct dynamic processes of ATP binding to the corresponding allosteric and substrate binding sites of uridine monophosphate kinase, and suggested that in solution ATP preferentially binds to the substrate binding sites of proteins. When the ATP molecules occupy the allosteric binding sites, the allosteric trigger from ATP to fuel allosteric communication between allosteric and functional sites is stemmed mainly from the triphosphate part of ATP, with a small number from the adenine part of ATP. Taken together, our results provide overall understanding of ATP allosteric functions responsible for regulation in biological systems.     

The changing landscape of protein allostery

It is becoming increasingly clear that the fundamental capacity to undergo conformational change in response to ligand binding is intrinsic to proteins. This property confers on proteins the ability to be allosterically modulated in order to shift substrate binding affinities, alter enzymatic activity or regulate protein-protein interaction. How this allosteric modulation occurs--the pathways of communication, the shifting of conformational ensembles and the altered molecular dynamics--has received considerable attention during the past two years. Recent progress has helped outline the molecular origins of allostery in proteins as diverse as Hsp70 molecular chaperones and signal integrating proteins, such as WASP. In addition, allosteric properties have been successfully engineered into proteins for drug design or the development of novel biosensors. Methodological advances have provided exciting prospects for new insights and new biological roles of allosteric systems have been uncovered.     

Coupling between substrate binding and allosteric regulation in ribozyme catalysis

The contribution of substrate binding to allosteric regulation in the ribozyme catalysis has been investigated using allosteric ribozymes consisting of the hammerhead ribozyme and a flavin mononucleotide (FMN) aptamer. Kinetic parameters were measured for various lengths of the substrates with a wide range of binding energy. The maximum cleavage rate of each ribozyme was retained with the long substrates. However, the cleavage rates largely decreased by the truncation of the substrates according to loss in the free energy of substrate binding. The high sensitivity to the substrate lengths is attributed to the increase in the energetic requirement for the catalytic core folding, which is caused by the incorporation of the aptamer region. One role of FMN binding is assisting the promotion of the core folding through the stabilization of the aptamer domain. The allosteric effect is significantly expressed only when the substrate binding energy is insufficient for the core folding of the ribozyme-substrate complex. This type of allosteric interaction dominates the substrate dependency of another type of regulation. These results demonstrate that an adequate correlation between the type of regulation and the substrate binding is responsible for the effective allosteric interaction in the kinetic process.     

Contrasting catalytic and allosteric mechanisms for phosphoglycerate dehydrogenases

D-3-Phosphoglycerate dehydrogenases (PGDH) exist with at least three different structural motifs and the enzymes from different species display distinctly different mechanisms. In many species, particularly bacteria, the catalytic activity is regulated allosterically through binding of l-serine to a distinct structural domain, termed the ACT domain. Some species, such as Mycobacterium tuberculosis, contain an additional domain, called the "allosteric substrate binding" or ASB domain, that functions as a co-domain in the regulation of catalytic activity. That is, both substrate and effector function synergistically in the regulation of activity to give the enzyme some interesting properties that may have physiological relevance for the persistent state of tuberculosis. Both enzymes function through a V-type regulatory mechanism and, in the Escherichia coli enzyme, it has been demonstrated that this results from a dead-end complex that decreases the concentration of active species rather than a decrease in the velocity of the active species. This review compares and contrasts what we know about these enzymes and provides additional insight into their mechanism of allosteric regulation.     

Exploring the Structural Mechanism of Covalently Bound E3 Ubiquitin Ligase: Catalytic or Allosteric Inhibition?

Covalent inhibition has recently gained a resurgence of interest in several drug discovery areas. The expansion of this approach is based on evidence elucidating the selectivity and potency of covalent inhibitors when bound to particular amino acids of a biological target. The Nedd4-1, an E3 ubiquitin ligase, is characterized by two covalent binding sites, of which catalytic Cys^cat and allosteric Cys^allo are enclosed. This enzyme has demonstrated inhibition at both the above-mentioned binding sites; however, a detailed molecular understanding of the structural mechanism of inhibition upon Cys^cat and Cys^allo binding remains vague. This prompted us to provide the first account of investigating the preferential covalent binding mode and the underlying structural and molecular dynamic implications. Based on the molecular dynamic analyses, it was evident that although both catalytic and allosteric covalent binding led to greater stability of the enzyme, a preferential covalent mechanism of inhibition was seen in the allosteric-targeted system. This was supported by a more favorable binding energy in the allosteric site compared to the catalytic site, in addition to the larger number of residue interactions and stabilizing hydrogen bonds occurring in the allosteric covalent bound complex. The fundamental dynamic analysis presented in this report compliments, as well as adds to previous experimental findings, thus leading to a crucial understanding of the structural mechanism by which Nedd4-1 is inhibited. The findings from this study may assist in the design of more target-specific Nedd4-1 covalent inhibitors exploring the surface-exposed cysteine residues.     

Deviations from hyperbolic kinetics in slowly dissociating allosteric enzyme systems]

The feautres of kinetic behavior of dissociating enzyme systems for which the rate of equilibrium between the oligomeric forms is slow in comparison with the rate of the enzymatic process are discussed. It is shown that in slowly dissociating enzyme system of the type Np in equilibrium P (P is the enzyme oligomer, and p is the subunit: N greater than or equal to2) in which P and p forms differ by the character of allosteric interaction between the active and allosteric sites the plots of the initial reaction rate (v) versus substrate (S) or effector (F) concentration may be a very complicated shape. In similar systems the v versus [S]0 plots may have intermediate plateau, maximum and minimum simultaneously, sigmoidality followed by intermediate plateau and so on, and the v versus [F]0 plots may have intermediate plateau.     

A quantitative model for allosteric control of purine reduction by murine ribonucleotide reductase

The reduction of purine nucleoside diphosphates by murine ribonucleotide reductase requires catalytic (R1) and free radical-containing (R2) enzyme subunits and deoxynucleoside triphosphate allosteric effectors. A quantitative 16 species model is presented, in which all pertinent equilibrium constants are evaluated, that accounts for the effects of the purine substrates ADP and GDP, the deoxynucleoside triphosphate allosteric effectors dGTP and dTTP, and the dimeric murine R2 subunit on both the quaternary structure of murine R1 subunit and the dependence of holoenzyme (R1(2)R2(2)) activity on substrate and effector concentrations. R1, monomeric in the absence of ligands, dimerizes in the presence of substrate, effectors, or R2(2) because each of these ligands binds R1(2) with higher affinity than R1 monomer. This leads to apparent positive heterotropic cooperativity between substrate and allosteric effector binding that is not observed when binding to the dimeric protein itself is evaluated. Allosteric activation results from an increase in k(cat) for substrate reduction upon binding of the correct effector, rather than from heterotropic cooperativity between effector and substrate. Neither the allosteric site nor the active site displays nucleotide base specificity: dissociation constants for dGTP and dTTP are nearly equivalent and K(m) and k(cat) values for both ADP and GDP are similar. R2(2) binding to R1(2) shows negative heterotropic cooperativity vis-à-vis effectors but positive heterotropic cooperativity vis-à-vis substrates. Binding of allosteric effectors to the holoenzyme shows homotropic cooperativity, suggestive of a conformational change induced by activator binding. This is consistent with kinetic results indicating full dimer activation upon binding a single equivalent of effector per R1(2)R2(2).     

Allosteric sites can be identified based on the residue–residue interaction energy difference

Allosteric drugs act at a distance to regulate protein functions. They have several advantages over conventional orthosteric drugs, including diverse regulation types and fewer side effects. However, the rational design of allosteric ligands remains a challenge, especially when it comes to the identification allosteric binding sites. As the binding of allosteric ligands may induce changes in the pattern of residue–residue interactions, we calculated the residue–residue interaction energies within the allosteric site based on the molecular mechanics generalized Born surface area energy decomposition scheme. Using a dataset of 17 allosteric proteins with structural data for both the apo and the ligand‐bound state available, we used conformational ensembles generated by molecular dynamics simulations to compute the differences in the residue–residue interaction energies in known allosteric sites from both states. For all the known sites, distinct interaction energy differences (>25%) were observed. We then used CAVITY, a binding site detection program to identify novel putative allosteric sites in the same proteins. This yielded a total of 31 “druggable binding sites,” of which 21 exhibited >25% difference in residue interaction energies, and were hence predicted as novel allosteric sites. Three of the predicted allosteric sites were supported by recent experimental studies. All the predicted sites may serve as novel allosteric sites for allosteric ligand design. Our study provides a computational method for identifying novel allosteric sites for allosteric drug design. Proteins 2015; 83:1375–1384. © 2014 Wiley Periodicals, Inc.     

Structure of yeast hexokinase: IV. Low-resolution structure of enzyme—Substrate complexes revealing negative co-operativity and allosteric interactions

We conclude from X-ray diffraction studies at low resolution (7 Å) that the binding of sugar and nucleotide substrates to dimeric yeast hexokinase BII crystals exhibits both negative co-operativity and positive allosteric co-operativity. Difference electron density maps show the positions of sugar and nucleotide binding sites and extensive substrate-induced structural changes in the protein. Sugar substrates and inhibitors bind in the deep cleft that divides each subunit into two lobes and nucleotide substrates bind nearby to one site per dimer, which lies between the subunits and on the molecular symmetry axis. Although the inhibitors o- and p-iodobenzoylglucosamine and o-toluoylglucosamine bind equally to both subunits, the degree of substitution of glucose or xylose is very different for the two subunits. The substrate analog β, γ-imido ATP shows only one strong binding site per dimer. This negative co-operativity in substrate binding may result from the heterologous or non-equivalent association of the two subunits (Anderson et al., 1974), which provides non-equivalent environments for the two chemically identical subunits.Further, there is a positive allosteric interaction between the sugar and nucleotide binding sites. Sugar binding is required for nucleotide binding at the intersubunit site and the binding of nucleotide modifies the binding of sugars. These positive heterotropic interactions appear to be mediated by extensive substrate-induced structural changes in the enzyme.     

Exploring Mechanisms of Allosteric Regulation and Communication Switching in the Multiprotein Regulatory Complexes of the Hsp90 Chaperone with Cochaperones and Client Proteins: Atomistic Insights from Integrative Biophysical Modeling and Network Analysis of Conformational Landscapes

Understanding molecular principles underlying Hsp90 chaperone functions and modulation of client activity is fundamental to dissect activation mechanisms of many proteins. In this work, we performed a computational investigation of the Hsp90-Hsp70-Hop-CR client complex to examine allosteric regulatory mechanisms underlying dynamic chaperone interactions and principles of chaperone-dependent client recognition and remodeling. Conformational dynamics analysis using high-resolution coarse-grained simulations and ensemble-based local frustration analysis suggest that the Hsp90 chaperone could recognize and recruit the GR client by invoking reciprocal dynamic exchanges near the intermolecular interfaces with the client. Using mutational scanning of the intermolecular residues in the Hsp90-Hsp70-Hop-GR complex, we identified binding energy hotspots in the regulatory complex. Perturbation-based network analysis and dynamic fluctuations-based modeling of allosteric residue potentials are employed for a detailed analysis of allosteric interaction networks and identification of conformational communication switches. We found that allosteric interactions between the Hsp90, the client-bound Hsp70 and Hop cochaperone can define two allosteric residue clusters that control client recruitment in which the intrinsic Hsp70 allostery is exploited to mediate integration of the Hsp70-bound client into the Hsp90 chaperone system. The results suggest a model of dynamics-driven allostery that enables efficient client recruitment and loading through allosteric couplings between intermolecular interfaces and communication switch centers. This study showed that the Hsp90 interactions with client proteins may operate under dynamic-based allostery in which ensembles of preexisting conformational states and intrinsic allosteric pathways present in the Hsp90 and Hsp70 chaperones can be exploited for recognition and integration of substrate proteins.     

Thrombin

Thrombin is a Na+-activated, allosteric serine protease that plays opposing functional roles in blood coagulation. Binding of Na+ is the major driving force behind the procoagulant, prothrombotic and signaling functions of the enzyme, but is dispensable for cleavage of the anticoagulant protein C. The anticoagulant function of thrombin is under the allosteric control of the cofactor thrombomodulin. Much has been learned on the mechanism of Na+ binding and recognition of natural substrates by thrombin. Recent structural advances have shed light on the remarkable molecular plasticity of this enzyme and the molecular underpinnings of thrombin allostery mediated by binding to exosite I and the Na+ site. This review summarizes our current understanding of the molecular basis of thrombin function and allosteric regulation. The basic information emerging from recent structural, mutagenesis and kinetic investigation of this important enzyme is that thrombin exists in three forms, E*, E and E:Na+, that interconvert under the influence of ligand binding to distinct domains. The transition between the Na+ -free slow from E and the Na+ -bound fast form E:Na+ involves the structure of the enzyme as a whole, and so does the interconversion between the two Na+ -free forms E* and E. E* is most likely an inactive form of thrombin, unable to interact with Na + and substrate. The complexity of thrombin function and regulation has gained this enzyme pre-eminence as the prototypic allosteric serine protease. Thrombin is now looked upon as a model system for the quantitative analysis of biologically important enzymes.     

H/D Exchange Characterization of Silent Coupling: Entropy-Enthalpy Compensation in Allostery

The allosteric coupling constant in K-type allosteric systems is defined as a ratio of the binding of substrate in the absence of effector to the binding of the substrate in the presence of a saturating concentration of effector. As a result, the coupling constant is itself an equilibrium value comprised of a ΔH and a TΔS component. In the scenario in which TΔS completely compensates ΔH, no allosteric influence of effector binding on substrate affinity is observed. However, in this “silent coupling” scenario, the presence of effector causes a change in the ΔH associated with substrate binding. A suggestion has now been made that “silent modulators” are ideal drug leads because they can be modified to act as either allosteric activators or inhibitors. Any attempt to rationally design the effector to be an allosteric activator or inhibitor is likely to be benefitted by knowledge of the mechanism that gives rise to coupling. Hydrogen/deuterium exchange with mass spectrometry detection has now been used to identify regions of proteins that experience conformational and/or dynamic changes in the allosteric regulation. Here, we demonstrate the expected temperature dependence of the allosteric regulation of rabbit muscle pyruvate kinase by Ala to demonstrate that this effector reduces substrate (phosphoenolpyruvate) affinity at 35°C and at 10°C but is silent at intermediate temperatures. We then explore the use of hydrogen/deuterium exchange with mass spectrometry to evaluate the areas of the protein that are modified in the mechanism that gives rise to the silent coupling between Ala and phosphoenolpyruvate. Many of the peptide regions of the protein identified as changing in this silent system (Ala as the effector) were included in changes previously identified for allosteric inhibition by Phe.     

Delineation of the allosteric mechanism of a cytidylyltransferase exhibiting negative cooperativity.

The dimeric enzyme CTP:glycerol-3-phosphate cytidylyltransferase (GCT) displays strong negative cooperativity between the first and second binding of its substrate, CTP. Using NMR to study the allosteric mechanism of this enzyme, we observe widespread chemical shift changes for the individual CTP binding steps. Mapping these changes onto the molecular structure allowed the formulation of a detailed model of allosteric conformational change. Upon the second step of ligand binding, NMR experiments indicate an extensive loss of conformational exchange broadening of the backbone resonances of GCT. This suggests that a fraction of the free energy of negative cooperativity is entropic in origin.     

Structural basis for allosteric regulation of the monomeric allosteric enzyme human glucokinase

Glucokinase is a monomeric enzyme that displays a low affinity for glucose and a sigmoidal saturation curve for its substrate, two properties that are important for its playing the role of a glucose sensor in pancreas and liver. The molecular basis for these two properties is not well understood. Herein we report the crystal structures of glucokinase in its active and inactive forms, which demonstrate that global conformational change, including domain reorganization, is induced by glucose binding. This suggests that the positive cooperativity of monomeric glucokinase obeys the "mnemonical mechanism" rather than the well-known concerted model. These structures also revealed an allosteric site through which small molecules may modulate the kinetic properties of the enzyme. This finding provided the mechanistic basis for activation of glucokinase as a potential therapeutic approach for treating type 2 diabetes mellitus.     

Networks for the allosteric control of protein kinases

The allosteric regulation of protein kinases serves as an efficient strategy for molecular communication, event coupling and interconversion between catalytic states. Recent co-crystal structures have revealed novel ways in which kinases control activity and substrate specificity following phosphorylation, dimerization, or binding to regulatory proteins, substrates and scaffolds. In addition, hydrogen exchange coupled with mass spectrometry is emerging as a complementary strategy to probe the solution behavior of kinases; recent results have shown that allosteric regulation may involve transitions in protein motions as well as structural rearrangements.     

Inversion of the allosteric response of Escherichia coli glucosamine-6-P deaminase to N-acetylglucosamine 6-P, by single amino acid replacements

Amino acid replacements in the active site of glucosamine-6-P deaminase from Escherichia coli (GlcN6P deaminase, EC 3.5.99.6) involving the residues D141 and E148 produce atypical allosteric kinetics. These residues are located in the chain segment 139-156 which is part of the active site and which also forms several intersubunit contacts close to the allosteric site. In the D141N and E148Q mutant forms of this deaminase, there is an inversion of the effect of its physiological allosteric effector, N-acetylglucosamine 6-P, which becomes an inhibitor at substrate concentrations above a critical value. For both mutants, this particular point appears at low substrate concentration and the inhibition by the allosteric activator is the dominant effect in velocity versus substrate curves. These effects are analyzed as a particular case of the concerted allosteric model, assuming that the R state, the conformer displaying the higher affinity for the substrate, is the less catalytic state, thus producing an inverted allosteric response.