Mixed, nonclassical behavior in a classic allosteric protein.

Allostery is a major driver of biological processes requiring coordination. Thus, it is one of the most fundamental and remarkable phenomena in nature, and there is motivation to understand and manipulate it to a multitude of ends. Today, it is often described in terms of two phenomenological models proposed more than a half-century ago involving only T(tense) or R(relaxed) conformations. Here, methyl-based NMR provides extensive detail on a dynamic T to R switch in the classical dimeric allosteric protein, yeast chorismate mutase (CM), that occurs in the absence of substrate, but only with the activator bound. Switching of individual subunits is uncoupled based on direct observation of mixed TR states in the dimer. This unique finding excludes both classic models and solves the paradox of a coexisting hyperbolic binding curve and highly skewed substrate-free T-R equilibrium. Surprisingly, structures of the activator-bound and effector-free forms of CM appear the same by NMR, providing another example of the need to account for dynamic ensembles. The apo enzyme, which has a sigmoidal activity profile, is shown to switch, not to R, but to a related high-energy state. Thus, the conformational repertoire of CM does not just change as a matter of degree depending on the allosteric input, be it effector and/or substrate. Rather, the allosteric model appears to completely change in different contexts, which is only consistent with modern ensemble-based frameworks.

A nonequilibrium allosteric model for receptor-kinase complexes: The role of energy dissipation in chemotaxis signaling.

The Escherichia coli chemotaxis signaling pathway has served as a model system for the adaptive sensing of environmental signals by large protein complexes. The chemoreceptors control the kinase activity of CheA in response to the extracellular ligand concentration and adapt across a wide concentration range by undergoing methylation and demethylation. Methylation shifts the kinase response curve by orders of magnitude in ligand concentration while incurring a much smaller change in the ligand binding curve. Here, we show that the disproportionate shift in binding and kinase response is inconsistent with equilibrium allosteric models. To resolve this inconsistency, we present a nonequilibrium allosteric model that explicitly includes the dissipative reaction cycles driven by adenosine triphosphate (ATP) hydrolysis. The model successfully explains all existing joint measurements of ligand binding, receptor conformation, and kinase activity for both aspartate and serine receptors. Our results suggest that the receptor complex acts as an enzyme: Receptor methylation modulates the ON-state kinetics of the kinase (e.g., phosphorylation rate), while ligand binding controls the equilibrium balance between kinase ON/OFF states. Furthermore, sufficient energy dissipation is responsible for maintaining and enhancing the sensitivity range and amplitude of the kinase response. We demonstrate that the nonequilibrium allosteric model is broadly applicable to other sensor-kinase systems by successfully fitting previously unexplained data from the DosP bacterial oxygen-sensing system. Overall, this work provides a nonequilibrium physics perspective on cooperative sensing by large protein complexes and opens up research directions for understanding their microscopic mechanisms through simultaneous measurements and modeling of ligand binding and downstream responses.

Revisiting the Immunoglobulin Intramolecular Signaling Hypothesis.

A generation ago, the immunoglobulin intramolecular signaling, or allosteric, hypothesis was abandoned in favor of the associative hypothesis, which posited that Fc receptor crosslinking produced the increased affinity of antigen-antibody complexes. This essay argues that there is sufficient evidence to resuscitate the allosteric hypothesis, at least for some antibodies.

Site-Mutation of Hydrophobic Core Residues Synchronically Poise Super Interleukin 2 for Signaling: Identifying Distant Structural Effects through Affordable Computations.

A superkine variant of interleukin-2 with six site mutations away from the binding interface developed from the yeast display technique has been previously characterized as undergoing a distal structure alteration which is responsible for its super-potency and provides an elegant case study with which to get insight about how to utilize allosteric effect to achieve desirable protein functions. By examining the dynamic network and the allosteric pathways related to those mutated residues using various computational approaches, we found that nanosecond time scale all-atom molecular dynamics simulations can identify the dynamic network as efficient as an ensemble algorithm. The differentiated pathways for the six core residues form a dynamic network that outlines the area of structure alteration. The results offer potentials of using affordable computing power to predict allosteric structure of mutants in knowledge-based mutagenesis.

Temperature and voltage coupling to channel opening in transient receptor potential melastatin 8 (TRPM8).

Expressed in somatosensory neurons of the dorsal root and trigeminal ganglion, the transient receptor potential melastatin 8 (TRPM8) channel is a Ca(2+)-permeable cation channel activated by cold, voltage, phosphatidylinositol 4,5-bisphosphate, and menthol. Although TRPM8 channel gating has been characterized at the single channel and macroscopic current levels, there is currently no consensus regarding the extent to which temperature and voltage sensors couple to the conduction gate. In this study, we extended the range of voltages where TRPM8-induced ionic currents were measured and made careful measurements of the maximum open probability the channel can attain at different temperatures by means of fluctuation analysis. The first direct measurements of TRPM8 channel temperature-driven conformational rearrangements provided here suggest that temperature alone is able to open the channel and that the opening reaction is voltage-independent. Voltage is a partial activator of TRPM8 channels, because absolute open probability values measured with fully activated voltage sensors are less than 1, and they decrease as temperature rises. By unveiling the fast temperature-dependent deactivation process, we show that TRPM8 channel deactivation is well described by a double exponential time course. The fast and slow deactivation processes are temperature-dependent with enthalpy changes of 27.2 and 30.8 kcal mol(-1). The overall Q10 for the closing reaction is about 33. A three-tiered allosteric model containing four voltage sensors and four temperature sensors can account for the complex deactivation kinetics and coupling between voltage and temperature sensor activation and channel opening.

Tertiary and quaternary effects in the allosteric regulation of animal hemoglobins.

In the last decade, protein allostery has experienced a major resurgence, boosted by the extension of the concept to systems of increasing complexity and by its exploitation for the development of drugs. Expansion of the field into new directions has not diminished the key role of hemoglobin as a test molecule for theory and experimental validation of allosteric models. Indeed, the diffusion of hemoglobins in all kingdoms of life and the variety of functions and of quaternary assemblies based on a common tertiary fold indicate that this superfamily of proteins is ideally suited for investigating the physical and molecular basis of allostery and firmly maintains its role as a main player in the field. This review is an attempt to briefly recollect common and different strategies adopted by metazoan hemoglobins, from monomeric molecules to giant complexes, exploiting homotropic and heterotropic allostery to increase their functional dynamic range. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

Assessing Allostery in Intrinsically Disordered Proteins With Ensemble Allosteric Model.

Intrinsically disordered (ID) proteins have been shown to play a major role in signaling in a broad range of proteins, using a process known as allostery, wherein the protein can integrate one or a number of inputs to regulate its function. The disorder-mediated allostery can be understood energetically with ensemble allosteric model (EAM). In this model, the molecule without effectors is considered as an ensemble of preexisting conformations, and effector binding is treated as an energetic perturbation of the ensemble to redistribute the microstates that are favorable or unfavorable to the second binding partner. As it only considers the intrinsic energetics of the system and does not depend on a crystallographic structure, it can be applied to both structured proteins, ID proteins, and mixed proteins with both structured and ID domains. Simulation with EAM on the basis of experimental data can help quantitatively explain experimental observations, as well as to make predictions to direct future research. This has recently been illustrated with the case of human glucocorticoid receptor, a multidomain transcription factor that contains both structured and disordered regions. In this chapter, we describe the assays for measuring the transcriptional activity, binding affinity to cognate DNA, conformational stability, either on single domain or tandem coupled domains in the GR two-domain isoforms. We then explain how these data are utilized as input parameters or constraints in the EAM for quantitative estimates of stabilities and coupling energies for each domain through global minimization method.

Understanding allosteric and cooperative interactions in enzymes.

The paper that introduced biochemists to the idea of allosteric feedback inhibition [Monod J, Changeux J-P & Jacob F (1963) J Mol Biol 6, 306-329] is now 50 years old, and the two papers on models for enzyme cooperativity that followed it [Monod J, Wyman J & Changeux J-P (1965) J Mol Biol 12, 88-118; Koshland DE, Némethy G & Filmer D (1966) Biochemistry 5, 365-385] are almost as old. All of these papers continue to be heavily cited today - more in the 21st century than they were in the last two decades of the 20th. This is because they continue to be central for understanding enzyme regulation, and increasingly important in the age of systems biology.