Vincent J. Hilser

The ensemble nature of allostery.

Allostery is the process by which biological macromolecules (mostly proteins) transmit the effect of binding at one site to another, often distal, functional site, allowing for regulation of activity. Recent experimental observations demonstrating that allostery can be facilitated by dynamic and intrinsically disordered proteins have resulted in a new paradigm for understanding allosteric mechanisms, which focuses on the conformational ensemble and the statistical nature of the interactions responsible for the transmission of information. Analysis of allosteric ensembles reveals a rich spectrum of regulatory strategies, as well as a framework to unify the description of allosteric mechanisms from different systems.

Intrinsic disorder as a mechanism to optimize allosteric coupling in proteins

Transcription factors and other allosteric cell signaling proteins contain a disproportionate number of domains or segments that are intrinsically disordered (ID) under native conditions. In many cases folding of these segments is coupled to binding with one or more of their interaction partners, suggesting that intrinsic disorder plays an important functional role. Despite numerous hypotheses for the role of ID domains in regulation, a mechanistic model has yet to be established that can quantitatively assess the importance of intrinsic disorder for intramolecular site-to-site communication, the hallmark property of allosteric proteins. Here, we present such a model and show that site-to-site allosteric coupling is maximized when intrinsic disorder is present in the domains or segments containing one or both of the coupled binding sites. This result not only explains the prevalence of ID domains in regulatory proteins, it also calls into question the classical mechanical view of energy propagation in proteins, which predicts that site-to-site coupling would be maximized when a well defined pathway of folded structure connects the two sites. Furthermore, in showing that the coupling mechanism conferred by intrinsic disorder is robust and independent of the network of interactions that physically link the coupled sites, unique insights are gained into the energetic ground rules that govern site-to-site communication in all proteins.

The magnitude of the backbone conformational entropy change in protein folding

The magnitude of the conformational entropy change experienced by the peptide backbone upon protein folding was investigated experimentally and by computational analysis. Experimentally, two different pairs of mutants of a 33 amino acid peptide corresponding to the leucine zipper region of GCN4 were used for high‐sensitivity microcalorimetric analysis. Each pair of mutants differed only by having alanine or glycine at a specific solvent‐exposed position under conditions in which the differences in stability could be attributed to differences in the conformational entropy of the unfolded state. The mutants studied were characterized by different stabilities but had identical heat capacity changes of unfolding (ΔCp), identical solvent‐related entropies of unfolding (ΔSsolv), and identical enthalpies of unfolding (ΔH) at equivalent temperatures. Accordingly, the differences in stability between the different mutants could be attributed to differences in conformational entropy. The computational studies were aimed at generating the energy profile of backbone conformations as a function of the main chain dihedral angles ϕ and ϱ. The energy profiles permit a direct calculation of the probability distribution of different conformers and therefore of the conformational entropy of the backbone. The experimental results presented in this paper indicate that the presence of the methyl group in alanine reduces the conformational entropy of the peptide backbone by 2.46 ± 0.2 cal/K · mol with respect to that of glycine, consistent with a 3.4‐fold reduction in the number of allowed conformations in the alanine‐containing peptides. Similar results were obtained from the energy profiles. The computational analysis also indicates that the addition of further carbon atoms to the side chain had only a small effect as long as the side chains were unbranched at position β. A further reduction with respect to Ala of only 0.61 and 0.81 cal/K · mol in the backbone entropy was obtained for leucine and lysine, respectively. β‐branching (Val) produces the largest decrease in conformational entropy (1.92 cal/K · mol less than Ala). Finally, the backbone entropy change associated with the unfolding of an α‐helix is 6.51 cal/K · mol for glycine. These and previous results have allowed a complete estimation of the conformational entropy changes associated with protein folding.

Structural and Energetic Basis of Allostery

Allostery is a biological phenomenon of fundamental importance in regulation and signaling, and efforts to understand this process have led to the development of numerous models. In spite of individual successes in understanding the structural determinants of allostery in well-documented systems, much less success has been achieved in identifying a set of quantitative and transferable ground rules that provide an understanding of how allostery works. Are there organizing principles that allow us to relate structurally different proteins, or are the determinants of allostery unique to each system? Using an ensemble-based model, we show that allosteric phenomena can be formulated in terms of conformational free energies of the cooperative elements in a protein and the coupling interactions between them. Interestingly, the resulting allosteric ground rules provide a framework to reconcile observations that challenge purely structural models of site-to-site coupling, including (a) allostery in the absence of pathways of structural distortions, (b) allostery in the absence of any structural change, and (c) the ability of allosteric ligands to act as agonists under some circumstances and antagonists under others. The ensemble view of allostery that emerges provides insights into the energetic prerequisites of site-to-site coupling and thus into how allostery works.

THE ENTHALPY CHANGE IN PROTEIN FOLDING AND BINDING : REFINEMENT OF PARAMETERS FOR STRUCTURE-BASED CALCULATIONS

Two effects are mainly responsible for the observed enthalpy change in protein unfolding: the disruption of internal interactions within the protein molecule (van der Waals, hydrogen bonds, etc.) and the hydration of the groups that are buried in the native state and become exposed to the solvent on unfolding. In the traditional thermodynamic analysis, the effects of hydration have usually been evaluated using the thermodynamic data for the transfer of small model compounds from the gas phase to water. The contribution of internal interactions, on the other hand, are usually estimated by subtracting the hydration effects from the experimental enthalpy of unfolding. The main drawback of this approach is that the enthalpic contributions of hydration, and those due to the disruption of internal interactions, are more than one order of magnitude larger than the experimental enthalpy value. The enthalpy contributions of hydration and disruption of internal interactions have opposite signs and cancel each other almost completely resulting in a final value that is over 10 times smaller than the individual terms. For this reason, the classical approach cannot be used to accurately predict unfolding enthalpies from structure: any error in the estimation of the hydration enthalpy will be amplified by a factor of 10 or more in the estimation of the unfolding enthalpy. Recently, it has been shown that simple parametric equations that relate the enthalpy change with certain structural parameters, especially changes in solvent accessible surface areas have considerable predictive power. In this paper, we provide a physical foundation to that parametrization and in the process we present a system of equations that explicitly includes the enthalpic effects of the packing density between the different atoms within the protein molecule. Using this approach, the error in the prediction of folding/unfolding enthalpies at 60°C, the median temperature for thermal unfolding, is better than ±3% (standard deviation = 4 kcal/mol).

Direct access to the cooperative substructure of proteins and the protein ensemble via cold denaturation.

The modern view of protein thermodynamics predicts that proteins undergo cold-induced unfolding. Unfortunately, the properties of proteins and water conspire to prevent the detailed observation of this fundamental process. Here we use protein encapsulation to allow cold denaturation of the protein ubiquitin to be monitored by high-resolution NMR at temperatures approaching −35 °C. The cold-induced unfolding of ubiquitin is found to be highly noncooperative, in distinct contrast to the thermal melting of this and other proteins. These results demonstrate the potential of cold denaturation as a means to dissect the cooperative substructures of proteins and to provide a rigorous framework for testing statistical thermodynamic treatments of protein stability, dynamics and function.

An Ensemble View of Allostery

Proteins existing as ensembles of conformations may be key to understanding signal transduction processes. Allostery is the process by which biological macromolecules (mostly proteins) transmit the regulatory effects induced by the binding of a ligand at one site to a different, often distant, functional site. This process governs the function of almost all metabolism and gene regulation (1, 2). Despite the importance of this process, a general understanding of the structural and energetic basis for allostery has been elusive. On page 685 of this issue, Bai et al. (3) shed light on a key obstacle to understanding allosteric control by showing how proteins existing as ensembles of multiple conformations affect the allosteric signal transduction process.

Predicting the equilibrium protein folding pathway: Structure-based analysis of staphylococcal nuclease

The equilibrium folding pathway of staphylococcal nucleas (SNase) has been approximated using a statistical thermodynamic formalism that utilizes the high‐resolution structure of the native state as a template to generate a large ensemble of partially folded states. Close to 400,000 different states ranging from the native to the completely unfolded states were included in the analysis. The probability of each state was estimated using an empirical structural parametrization of the folding energetics. It is shown that this formalism predicts accurately the stability of the protein, the cooperativity of the folding/unfolding transition observed by differential scanning calorimetry (DSC) or urea denaturation and the thermodynamic parameters for unfolding. More importantly, this formalism provides a quantitative account of the experimental hydrogen exchange protection factors measured under native conditions for SNase. These results suggest that the computer‐generated distribution of states approximates well the ensemble of conformations existing in solution. Furthermore, this formalism represents the first model capable of quantitatively predicting within a unified framework the probability distribution of states seen under native conditions and its change upon unfolding.

The role of protein conformational fluctuations in allostery, function, and evolution

It is now well-known that proteins exist at equilibrium as ensembles of conformational states rather than as unique static structures. Here we review from an ensemble perspective important biological effects of such spontaneous fluctuations on protein allostery, function, and evolution. However, rather than present a thorough literature review on each subject, we focus instead on connecting these phenomena through the ensemble-based experimental, theoretical, and computational investigations from our laboratory over the past decade. Special emphasis is given to insights that run counter to some of the prevailing ideas that have emerged over the past 40 years of structural biology research. For instance, when proteins are viewed as conformational ensembles rather than as single structures, the commonly held notion of an allosteric pathway as an obligate series of individual structural distortions loses its meaning. Instead, allostery can result from energetic linkage between distal sites as one Boltzmann distribution of states transitions to another. Additionally, the emerging principles from this ensemble view of proteins have proven surprisingly useful in describing the role of intrinsic disorder in inter-domain communication, functional adaptation mediated by mutational control of fluctuations, and evolutionary conservation of the energetics of protein stability.

Agonism/antagonism switching in allosteric ensembles

Ligands for several transcription factors can act as agonists under some conditions and antagonists under others. The structural and molecular bases of such effects are unknown. Previously, we demonstrated how the folding of intrinsically disordered (ID) protein sequences, in particular, and population shifts, in general, could be used to mediate allosteric coupling between different functional domains, a model that has subsequently been validated in several systems. Here it is shown that population redistribution within allosteric systems can be used as a mechanism to tune protein ensembles such that a given ligand can act as both an agonist and an antagonist. Importantly, this mechanism can be robustly encoded in the ensemble, and does not require that the interactions between the ligand and the protein differ when it is acting either as an agonist or an antagonist. Instead, the effect is due to the relative probabilities of states prior to the addition of the ligand. The ensemble view of allostery that is illuminated by these studies suggests that rather than being seen as switches with fixed responses to allosteric activation, ensembles can evolve to be “functionally pluripotent,” with the capacity to up or down regulate activity in response to a stimulus. This result not only helps to explain the prevalence of intrinsic disorder in transcription factors and other cell signaling proteins, it provides important insights about the energetic ground rules governing site-to-site communication in all allosteric systems.