The Role of Protein-Ligand Contacts in Allosteric Regulation of the Escherichia coli Catabolite Activator Protein

Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring.     

Correlating allostery with rigidity

Allosteric proteins demonstrate the phenomenon of a ligand binding to a protein at a regulatory or effector site and thereby changing the chemical affinity of the catalytic site. As such, allostery is extremely important biologically as a regulatory mechanism for molecular concentrations in many cellular processes. One particularly interesting feature of allostery is that often the catalytic and effector sites are separated by a large distance. Structural comparisons of allosteric proteins resolved in both inactive and active states indicate that a variety of structural rearrangement and changes in motions may contribute to general allosteric behavior. In general it is expected that the coupling of catalytic and regulatory sites is responsible for allosteric behavior. We utilize a novel examination of allostery using rigidity analysis of the underlying graph of the protein structures. Our results indicate a general global change in rigidity associated with allosteric transitions where the R state is more rigid than the T state. A set of allosteric proteins with heterotropic interactions is used to test the hypothesis that catalytic and effector sites are structurally coupled. Observation of a rigid path connecting the effector and catalytic sites in 68.75% of the structures points to rigidity as a means by which the distal sites communicate with each other and so contribute to allosteric regulation. Thus structural rigidity is shown to be a fundamental underlying property that promotes cooperativity and non-locality seen in allostery.     

Ligand structure controlled allostery in cAMP-dependent protein kinase catalytic subunit

Protein kinase A (cAMP dependent protein kinase catalytic subunit, EC 2.7.11.11) binds simultaneously ATP and a phosphorylatable peptide. These structurally dissimilar allosteric ligands influence the binding effectiveness of each other. The same situation is observed with substrate congeners, which reversibly inhibit the enzyme. In this review these allosteric effects are quantified using the interaction factor, which compares binding effectiveness of ligands with the free enzyme and the pre-loaded enzyme complex containing another ligand. This analysis revealed that the allosteric effect depends upon structure of the interacting ligands, and the principle “better binding: stronger allostery” observed can be formalized in terms of linear free-energy relationships, which point to similar mechanism of the allosteric interaction between the enzyme-bound substrates and/or inhibitor molecules. On the other hand, the type of effect is governed by ligand binding effectiveness and can be inverted from positive allostery to negative allostery if we move from effectively binding ligands to badly binding compounds. Thus the outcome of the allostery in this monomeric enzyme is the same as defined by classical theories for multimeric enzymes: making the enzyme response more efficient if appropriate ligands bind.     

Coupling of global and local vibrational modes in dynamic allostery of proteins

It is now recognized that internal global protein dynamics play an important role in the allosteric function of many proteins. Alterations of protein flexibility on effector binding affect the entropic cost of binding at a distant site. We present a coarse-grained model for a potential amplification of such entropic allostery due to coupling of fast, localized modes to the slow, global modes. We show how such coupling can give rise to large compensating entropic and enthalpic terms. The model corresponds to the pattern of calorimetry and NMR data from experiments on the Met repressor.     

Protein dynamics and allostery: an NMR view

Allostery, the process by which distant sites within a protein system are energetically coupled, is an efficient and ubiquitous mechanism for activity regulation. A purely mechanical view of allostery invoking only structural changes has developed over the decades as the classical view of the phenomenon. However, a fast growing list of examples illustrate the intimate link between internal motions over a wide range of time scales and function in protein-ligand interactions. Proteins respond to perturbations by redistributing their motions and they use fluctuating conformational states for binding and conformational entropy as a carrier of allosteric energy to modulate association with ligands. In several cases allosteric interactions proceed with minimal or no structural changes. We discuss emerging paradigms for the central role of protein dynamics in allostery.     

Dynamic Allostery of the Catabolite Activator Protein Revealed by Interatomic Forces

The Catabolite Activator Protein (CAP) is a showcase example for entropic allostery. For full activation and DNA binding, the homodimeric protein requires the binding of two cyclic AMP (cAMP) molecules in an anti-cooperative manner, the source of which appears to be largely of entropic nature according to previous experimental studies. We here study at atomic detail the allosteric regulation of CAP with Molecular dynamics (MD) simulations. We recover the experimentally observed entropic penalty for the second cAMP binding event with our recently developed force covariance entropy estimator and reveal allosteric communication pathways with Force Distribution Analyses (FDA). Our observations show that CAP binding results in characteristic changes in the interaction pathways connecting the two cAMP allosteric binding sites with each other, as well as with the DNA binding domains. We identified crucial relays in the mostly symmetric allosteric activation network, and suggest point mutants to test this mechanism. Our study suggests inter-residue forces, as opposed to coordinates, as a highly sensitive measure for structural adaptations that, even though minute, can very effectively propagate allosteric signals.     

Review: allostery in chaperonins

Chaperonins mediate protein folding in an ATP-dependent manner. ATP binding and hydrolysis by chaperonins are subject to both homotropic and heterotropic allosteric regulation. In the case of GroEL and CCT, homotropic regulation by ATP is manifested in nested cooperativity, which involves positive intra-ring cooperativity and negative inter-ring cooperativity in ATP binding. Both types of cooperativity are modulated by various heterotropic allosteric effectors, which include nonfolded proteins, ADP, Mg2+, monovalent ions such as K+, and cochaperonins in the case of type I chaperonins such as GroEL. Here, the allosteric properties of chaperonins are reviewed and new results of ours are presented with regard to allosteric effects of ADP. The role of allostery in the reaction cycle and folding function of chaperonins is discussed.     

IgG cooperativity – Is there allostery? Implications for antibody functions and therapeutic antibody development

A central dogma in immunology is that an antibody's in vivo functionality is mediated by 2 independent events: antigen binding by the variable (V) region, followed by effector activation by the constant (C) region. However, this view has recently been challenged by reports suggesting allostery exists between the 2 regions, triggered by conformational changes or configurational differences. The possibility of allosteric signals propagating through the IgG domains complicates our understanding of the antibody structure-function relationship, and challenges the current subclass selection process in therapeutic antibody design. Here we review the types of cooperativity in IgG molecules by examining evidence for and against allosteric cooperativity in both Fab and Fc domains and the characteristics of associative cooperativity in effector system activation. We investigate the origin and the mechanism of allostery with an emphasis on the C-region-mediated effects on both V and C region interactions, and discuss its implications in biological functions. While available research does not support the existence of antigen-induced conformational allosteric cooperativity in IgGs, there is substantial evidence for configurational allostery due to glycosylation and sequence variations.     

Global conformational changes in allosteric proteins. A study of Escherichia coli cAMP receptor protein and muscle pyruvate kinase.

One of the basic features in allosteric regulation involves long range transduction of information. Based on crystallographic data on protein systems that are regulated by allosteric mechanisms, a global conformational change has always been observed. It is, therefore, important and useful to correlate the cooperativity of global structural change with the mode of binding of the regulatory ligand. Two systems were chosen for study, namely Escherichia coli cAMP receptor protein and muscle pyruvate kinase, which show negative and positive cooperativity in the binding of allosteric ligands, respectively. Quantitative titration of the global structural change, monitored by a high precision analytical gel chromatography technique, was conducted as a function of allosteric effector concentration. The results obtained for cAMP receptor protein show that the protein undergoes contraction upon binding of cAMP. The decreases in Stokes radius associated with complex formation are 0.1 +/- 0.1 and 0.7 +/- 0.1 A when one and two cAMP-binding sites are filled, respectively. The results for the pyruvate kinase system show a concerted structural change that quantitatively match the predicted behavior based on equilibrium constants derived from the analysis of steady state kinetic data by a two-state model. Hence, for these two systems, these results show that negative and positive cooperativity are correlated with sequential and concerted modes of structural change, respectively.     

Allosteric modulation of kinases and GPCRs: design principles and structural diversity

Allosteric binding sites, as opposed to traditional orthosteric binding sites, offer unparalleled opportunities for drug discovery by providing high levels of selectivity, mimicking physiological conditions, affording fewer side effects because of desensitization/downregulation, and engendering ligands with chemotypes divergent from orthosteric ligands. For kinases, allosteric mechanisms described to date include alteration of protein kinase conformation blocking productive ATP binding which appear 'ATP competitive' or blocking kinase activation by conformational changes that are 'ATP non-competitive'. For GPCRs, allosteric mechanisms impart multiple modes of target modulation (positive allosteric modulation (PAM), negative allosteric modulation (NAM), neutral cooperativity, partial antagonism (PA), allosteric agonism and allosteric antagonism). Here, we review recent developments in the design principles and structural diversity of allosteric ligands for kinases and GPCRs.     

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).     

Elucidation of direct competition and allosteric modulation of small‐molecular‐weight protein ligands using surface plasmon resonance methods

The present work introduces a surface plasmon resonance‐based method for the discrimination of direct competition and allosteric effects that occur in ternary systems comprising a receptor protein and two small‐molecular‐weight ligands that bind to it. Fatty acid binding protein 4, fructose‐1,6‐bisphosphatase and human serum albumin were used as model receptor molecules to demonstrate the performance of the method. For each of the receptor molecules, pairs of ligand molecules were selected for which either direct competition or an allosteric effect had already been determined by other methods. The method of discrimination introduced here is based on the surface plasmon resonance responses observed at equilibrium when an immobilized receptor protein is brought into contact with binary mixtures of interacting ligands. These experimentally determined responses are compared with the responses calculated using a theoretical model that considers both direct competition and allosteric ligand interaction modes. This study demonstrates that the allosteric ternary complex model, which enables calculation of the fractional occupancy of the protein by each ligand in such ternary systems, is well suited for the theoretical calculation of these types of responses. For all of the ternary systems considered in this work, the experimental and calculated responses in the chosen concentration ratio range were identical within a five‐σ confidence interval when the calculations considered the correct interaction mode of the ligands (direct competition or different types of allosteric regulation), and in case of allosteric modulation, also the correct strength of this effect. This study also demonstrates that the allosteric ternary complex model‐based calculations are well suited to predict the ideal concentration ratio range or even single concentration ratios that can serve as hot spots for discrimination, and such hot spots can drastically reduce the numbers of measurements needed for discrimination between direct competition and distinct modulation modes (neutral, positive or negative allostery). Copyright © 2015 John Wiley & Sons, Ltd.     

Differentiating a ligand's chemical requirements for allosteric interactions from those for protein binding. Phenylalanine inhibition of pyruvate kinase

The isoform of pyruvate kinase from brain and muscle of mammals (M(1)-PYK) is allosterically inhibited by phenylalanine. Initial observations in this model allosteric system indicate that Ala binds competitively with Phe, but elicits a minimal allosteric response. Thus, the allosteric ligand of this system must have requirements for eliciting an allosteric response in addition to the requirements for binding. Phe analogues have been used to dissect what chemical properties of Phe are responsible for eliciting the allosteric response. We first demonstrate that the l-2-aminopropanaldehyde substructure of the amino acid ligand is primarily responsible for binding to M(1)-PYK. Since the allosteric response to Ala is minimal and linear addition of methyl groups beyond the beta-carbon increase the magnitude of the allosteric response, we conclude that moieties beyond the beta-carbon are primarily responsible for allostery. Instead of an all-or-none mechanism of allostery, these findings support the idea that the bulk of the hydrophobic side chain, but not the aromatic nature, is the primary determinant of the magnitude of the observed allosteric inhibition. The use of these results to direct structural studies has resulted in a 1.65 A structure of M(1)-PYK with Ala bound. The coordination of Ala in the allosteric amino acid binding site confirms the binding role of the l-2-aminopropanaldehyde substructure of the ligand. Collectively, this study confirms that a ligand can have chemical regions specific for eliciting the allosteric signal in addition to the chemical regions necessary for binding.     

Dynamically driven protein allostery

Allosteric interactions are typically considered to proceed through a series of discrete changes in bonding interactions that alter the protein conformation. Here we show that allostery can be mediated exclusively by transmitted changes in protein motions. We have characterized the negatively cooperative binding of cAMP to the dimeric catabolite activator protein (CAP) at discrete conformational states. Binding of the first cAMP to one subunit of a CAP dimer has no effect on the conformation of the other subunit. The dynamics of the system, however, are modulated in a distinct way by the sequential ligand binding process, with the first cAMP partially enhancing and the second cAMP completely quenching protein motions. As a result, the second cAMP binding incurs a pronounced conformational entropic penalty that is entirely responsible for the observed cooperativity. The results provide strong support for the existence of purely dynamics-driven allostery.     

Revealing Atomic-Level Mechanisms of Protein Allostery with Molecular Dynamics Simulations

Molecular dynamics (MD) simulations have become a powerful and popular method for the study of protein allostery, the widespread phenomenon in which a stimulus at one site on a protein influences the properties of another site on the protein. By capturing the motions of a protein’s constituent atoms, simulations can enable the discovery of allosteric binding sites and the determination of the mechanistic basis for allostery. These results can provide a foundation for applications including rational drug design and protein engineering. Here, we provide an introduction to the investigation of protein allostery using molecular dynamics simulation. We emphasize the importance of designing simulations that include appropriate perturbations to the molecular system, such as the addition or removal of ligands or the application of mechanical force. We also demonstrate how the bidirectional nature of allostery—the fact that the two sites involved influence one another in a symmetrical manner—can facilitate such investigations. Through a series of case studies, we illustrate how these concepts have been used to reveal the structural basis for allostery in several proteins and protein complexes of biological and pharmaceutical interest.     

A coupled equilibrium shift mechanism in calmodulin-mediated signal transduction

We used nuclear magnetic resonance data to determine ensembles of conformations representing the structure and dynamics of calmodulin (CaM) in the calcium-bound state (Ca(2+)-CaM) and in the state bound to myosin light chain kinase (CaM-MLCK). These ensembles reveal that the Ca(2+)-CaM state includes a range of structures similar to those present when CaM is bound to MLCK. Detailed analysis of the ensembles demonstrates that correlated motions within the Ca(2+)-CaM state direct the structural fluctuations toward complex-like substates. This phenomenon enables initial ligation of MLCK at the C-terminal domain of CaM and induces a population shift among the substates accessible to the N-terminal domain, thus giving rise to the cooperativity associated with binding. Based on these results and the combination of modern free energy landscape theory with classical allostery models, we suggest that a coupled equilibrium shift mechanism controls the efficient binding of CaM to a wide range of ligands.     

Kinetic and allosteric consequences of mutations in the subunit and domain interfaces and the allosteric site of yeast pyruvate kinase.

The mechanism by which pyruvate kinase (PK) is allosterically activated by fructose-1,6-bisphosphate (FBP) is poorly understood. To identify residues key to allostery of yeast PK, a point mutation strategy was used. T403E and R459Q mutations in the FBP binding site caused reduced FBP affinity. Introducing positive charges at the 403, 458, and 406 positions in the FBP binding site had little consequence. The mutation Q299N in the A [bond] A subunit interface caused the enzyme response to ADP to be sensitive to FBP. The T311M A [bond] A interface mutant has a decreased affinity for PEP and FBP, and is dependent on FBP for activity. The R369A mutation in the C [bond] C interface only moderately influenced allostery. Creating an E392A mutation in the C [bond] C subunit interface eliminated all cooperativity and allosteric regulation. None of the seven A [bond] C domain interface mutations altered allostery. A model that includes a central role for E392 in allosteric regulation of yeast PK is proposed.     

Role of water-bridged interactions in metal ion coupled protein allostery

Allosteric communication between distant parts of proteins controls many cellular functions, in which metal ions are widely utilized as effectors to trigger the allosteric cascade. Due to the involvement of strong coordination interactions, the energy landscape dictating the metal ion binding is intrinsically rugged. How metal ions achieve fast binding by overcoming the landscape ruggedness and thereby efficiently mediate protein allostery is elusive. By performing molecular dynamics simulations for the Ca²⁺ binding mediated allostery of the calmodulin (CaM) domains, each containing two Ca²⁺ binding helix-loop-helix motifs (EF-hands), we revealed the key role of water-bridged interactions in Ca²⁺ binding and protein allostery. The bridging water molecules between Ca²⁺ and binding residue reduces the ruggedness of ligand exchange landscape by acting as a lubricant, facilitating the Ca²⁺ coupled protein allostery. Calcium-induced rotation of the helices in the EF-hands, with the hydrophobic core serving as the pivot, leads to exposure of hydrophobic sites for target binding. Intriguingly, despite being structurally similar, the response of the two symmetrically arranged EF-hands upon Ca²⁺ binding is asymmetric. Breakage of symmetry is needed for efficient allosteric communication between the EF-hands. The key roles that water molecules play in driving allosteric transitions are likely to be general in other metal ion mediated protein allostery.