174 Self-association prompts proteins for new function: The role of altered dynamic properties

Local backbone flexibility along the chain was analyzed in pairs of monomer and homodimeric protein structures with identical sequence. We identified pairs, where highly flexible regions (corresponding to prominent peaks in rmsd’s or crystallographic B-factor values) clearly ‘migrate’ to a new location along the polypeptide in the homodimer. We present several systems, where the new flexible region can be linked to the recruitment of a 3rd binding partner, and where this recruitment is not reported for the corresponding monomer. Conformational sampling of these systems over microsecond timescales further confirms the flexibility migration phenomenon. Compelling evidence is provided that altered backbone flexibility in the homodimer state enables specific recruitment of the heterologous binding partner through the process of conformational selection. Our findings lend support to conformational selection as a general mechanism for protein–ligand binding and highlight the regulatory allosteric role played by the homomeric association event itself. There are many examples of allosteric behavior in homo-oligomers, but this is the first time that homomer association is shown to be the binding event that alters binding properties elsewhere in the protein.     

A structural comparison of ‘real’ and ‘model’ calmodulin clarified allosteric interactions regulating domain motion

Calmodulin (CaM) is a multifunctional calcium-binding protein, which regulates various biochemical processes. CaM acts via structural changes and complex forming with its target enzymes. CaM has two globular domains (N-lobe and C-lobe) connected by a long linker region. Upon calcium binding, the N-lobe and C-lobe undergo local conformational changes, after that, entire CaM wraps the target enzyme through a large conformational change. However, the regulation mechanism, such as allosteric interactions regulating the conformational changes, is still unclear. In order to clarify the allosteric interactions, in this study, experimentally obtained ‘real’ structures are compared to ‘model’ structures lacking the allosteric interactions. As the allosteric interactions would be absent in calcium-free CaM (apo-CaM), allostery-eliminated calcium-bound CaM (holo-CaM) models were constructed by combining the apo-CaM’s linker and the holo-CaM’s N- and C-lobe. Before the comparison, the ‘real’ and ‘model’ structures were clustered and cluster–cluster relationship was determined by a principal component analysis. The structures were compared based on the relationship, then, a distance map and a contact probability analysis clarified that the inter-domain motion is regulated by several groups of inter-domain contacting residue pairs. The analyses suggested that these residues cause inter-domain translation and rotation, and as a consequence, the motion encourage structural diversity. The resultant diversity would contribute to the functional versatility of CaM.     

The Concept of Allosteric Interaction and Its Consequences for the Chemistry of the Brain

Throughout this Reflections article, I have tried to follow up on the genesis in the 1960s and subsequent evolution of the concept of allosteric interaction and to examine its consequences within the past decades, essentially in the field of the neuroscience. The main conclusion is that allosteric mechanisms built on similar structural principles operate in bacterial regulatory enzymes, gene repressors (and the related nuclear receptors), rhodopsin, G-protein-coupled receptors, neurotransmitter receptors, ion channels, and so on from prokaryotes up to the human brain yet with important features of their own. Thus, future research on these basic cybernetic sensors is expected to develop in two major directions: at the elementary level, toward the atomic structure and molecular dynamics of the conformational changes involved in signal recognition and transduction, but also at a higher level of organization, the contribution of allosteric mechanisms to the modulation of brain functions.     

Neutralizing Antibodies Against Allosteric Proteins: Insights From a Bacterial Adhesin

Allosteric proteins transition between ‘inactive’ and ‘active’ states. In general, such proteins assume distinct conformational states at the level of secondary, tertiary and/or quaternary structure. Different conformers of an allosteric protein can be antigenically dissimilar and induce antibodies with a highly distinctive specificities and neutralizing functional effects. Here we summarize studies on various functional types of monoclonal antibodies obtained against different allosteric conformers of the mannose-specific bacterial adhesin FimH – the most common cell attachment protein of Escherichia coli and other enterobacterial pathogens. Included are types of antibodies that activate the FimH function via interaction with ligand-induced binding sites or by wedging between domains as well as antibodies that inhibit FimH through orthosteric, parasteric, or novel dynasteric mechanisms. Understanding the molecular mechanism of antibody action against allosteric proteins provides insights on how to design antibodies with a desired functional effect, including those with neutralizing activity against bacterial and viral cell attachment proteins.     

Molecular mechanisms and binding site locations for noncompetitive antagonists of nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors are pentameric proteins that belong to the Cys-loop receptor superfamily. Their essential mechanism of functioning is to couple neurotransmitter binding, which occurs at the extracellular domain, to the opening of the membrane-spanning cation channel. The function of these receptors can be modulated by structurally different compounds called noncompetitive antagonists. Noncompetitive antagonists may act at least by two different mechanisms: a steric and/or an allosteric mechanism. The simplest idea representing a steric mechanism is that the antagonist molecule physically blocks the ion channel. On the other hand, there exist distinct allosteric mechanisms. For example, noncompetitive antagonists may bind to the receptor and stabilize a nonconducting conformational state (e.g., resting or desensitized state), and/or increase the receptor desensitization rate. Barbiturates, dissociative anesthetics, antidepressants, and neurosteroids have been shown to inhibit nicotinic receptors by allosteric mechanisms and/or by open- and closed-channel blockade. Receptor modulation has proved to be highly complex for most noncompetitive antagonists. Noncompetitive antagonists may act by more than one mechanism and at distinct sites in the same receptor subtype. The binding site location for one particular molecule depends on the conformational state of the receptor. The mechanisms of action and binding affinities of noncompetitive antagonists differ among nicotinic receptor subtypes. Knowledge of the structure of the nicotinic acetylcholine receptor, the location of its noncompetitive antagonist binding sites, and the mechanisms of inhibition will aid the design of new and more efficacious drugs for treatment of neurological diseases.     

Integrins: bidirectional,allosteric signaling machines

In their roles as major adhesion receptors, integrins signal across the plasma membrane in both directions. Recent structural and cell biological data suggest models for how integrins transmit signals between their extracellular ligand binding adhesion sites and their cytoplasmic domains, which link to the cytoskeleton and to signal transduction pathways. Long-range conformational changes couple these functions via allosteric equilibria.     

Synaptically released zinc: neuromodulator and toxin

In addition to its familiar role as a component of metalloproteins, zinc is also sequestered in the presynaptic vesicles in ‘zinc‐containing’ neurons. The best‐established physiological role of synaptically released zinc is the tonic modulation of brain excitability through modulation of amino acid receptors; prominent pathological effects include acceleration of plaque deposition in Alzheimer's disease and exacerbation of excitotoxic neuron injury. Synaptically released zinc functions as a conventional synaptic neurotransmitter or neuromodulator being released into the cleft then recycled into the postsynaptic neurons during synaptic events, functioning analogously to calcium in this regard, as a transmembrane neural signal. To stimulate comparisons of zinc signals with calcium signals, we have compiled a list of the important parameters of calcium signals and zinc signals. More speculatively, we hypothesize that zinc signals may loosely mimic phosphate signals in the sense that signal zinc ions may commonly bind to proteins in a lasting manner, as a result changing their structure and function.     

Alpha(1)-adrenergic receptor subtypes: non-identical triplets with different dancing partners?

Alpha(1)-adrenergic receptors are one of the three subfamilies of G protein coupled receptors activated by epinephrine and norepinephrine to control important functions in many target organs. Three human subtypes (alpha(1A), alpha(1B), alpha(1D)) are derived from separate genes and are highly homologous in their transmembrane domains but not in their amino or carboxyl termini. Recent advances in our understanding of these "non-identical triplets" include development of knockout mice lacking single or multiple subtypes, new insights into subcellular localization and trafficking, identification of allosteric modulators, and increasing evidence for an important role in brain function. Although all three subtypes activate the same G(q/11) signaling pathway, they also appear to interact with different protein binding partners. Recent evidence suggests they may also form dimers, and may initiate independent signals through pathways yet to be clearly elucidated. Thus, this subfamily represents a common phenomenon of a group of similar but non-identical receptor subtypes activated by the same neurotransmitter, whose individual functional roles remain to be clearly established.     

G protein coupled receptors as allosteric proteins and the role of allosteric modulators

Seven transmembrane receptors (7TMRs) are proteins that convey signals through changes in conformation. These conformations are stabilized by external molecules (i.e. agonists, antagonists, modulators) and act upon other bodies (termed ‘guests’) which can be other molecules in the extracellular space, or proteins along the plane of the membrane (receptor oligomerization) or signaling proteins in the cytosol (i.e. G protein, β-arrestin). These elements comprise allosteric systems and a great deal of 7TMR pharmacology can be considered in terms of allosteric behavior. Allosteric ligands acting on 7TMRs possess four unique behaviors that can be valuable therapeutically; () the ability to alter the interaction of very large proteins, () probe dependence, () saturable effect, and () induction of separate changes in affinity and efficacy of other ligands. Two of these behaviors (namely probe dependence for CCR5-based HIV-1 entry inhibitors and functional selectivity for biased agonism) will be highlighted with examples.     

Functional Roles of Slow Enzyme Conformational Changes in Network Dynamics

Extensive studies from different fields reveal that many macromolecules, especially enzymes, show slow transitions among different conformations. This phenomenon is named such things as dynamic disorder, heterogeneity, hysteretic or mnemonic enzymes across these different fields, and has been directly demonstrated by single molecule enzymology and NMR studies recently. We analyzed enzyme slow conformational changes in the context of regulatory networks. A single enzymatic reaction with slow conformational changes can filter upstream network noises, and can either resonantly respond to the system stimulus at certain frequencies or respond adaptively for sustained input signals of the network fluctuations. It thus can serve as a basic functional motif with properties that are normally for larger intermolecular networks in the field of systems biology. We further analyzed examples including enzymes functioning against pH fluctuations, metabolic state change of Artemia embryos, and kinetic insulation of fluctuations in metabolic networks. The study also suggests that hysteretic enzymes may be building blocks of synthetic networks with various properties such as narrow-banded filtering. The work fills the missing gap between studies on enzyme biophysics and network level dynamics, and reveals that the coupling between the two is functionally important; it also suggests that the conformational dynamics of some enzymes may be evolutionally selected.     

Barbiturate and benzodiazepine modulation of GABA receptor binding and function

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) acts primarily on receptors that increase chloride permeability in postsynaptic neurons. These receptors are defined by sensitivity to the agonist muscimol and the antagonist bicuculline, and are also subject to indirect allosteric inhibition by picrotoxin-like convulsants and enhancement by the clinically important drugs, the benzodiazepines and the barbiturates. All of these drugs modulate GABA-receptor regulated chloride channels at the cellular level assayed by electrophysiological or radioactive ion tracer techniques. Specific receptor sites for GABA, benzodiazepines, picrotoxin/convulsants, and barbiturates can be assayed in vitro by radioactive ligand binding. Mutual chloride-dependent allosteric interactions between the four receptor sites indicate that they are all coupled in the same membrane macromolecular complex. Indirect effects of barbiturates on the other three binding sites define a pharmacologically specific, stereospecific receptor. All of the activities can be solubilized in the mild detergent 3-[(3-cholamidopropyl)-dimethylammonio]propane sulfonate (CHAPS) and co-purify as a single protein complex.     

Edgetic perturbation models of human inherited disorders

Cellular functions are mediated through complex systems of macromolecules and metabolites linked through biochemical and physical interactions, represented in interactome models as ‘nodes’ and ‘edges’, respectively. Better understanding of genotype‐to‐phenotype relationships in human disease will require modeling of how disease‐causing mutations affect systems or interactome properties. Here we investigate how perturbations of interactome networks may differ between complete loss of gene products (‘node removal’) and interaction‐specific or edge‐specific (‘edgetic’) alterations. Global computational analyses of ～50 000 known causative mutations in human Mendelian disorders revealed clear separations of mutations probably corresponding to those of node removal versus edgetic perturbations. Experimental characterization of mutant alleles in various disorders identified diverse edgetic interaction profiles of mutant proteins, which correlated with distinct structural properties of disease proteins and disease mechanisms. Edgetic perturbations seem to confer distinct functional consequences from node removal because a large fraction of cases in which a single gene is linked to multiple disorders can be modeled by distinguishing edgetic network perturbations. Edgetic network perturbation models might improve both the understanding of dissemination of disease alleles in human populations and the development of molecular therapeutic strategies.     

The present and the future of protein biosensor engineering

Protein biosensors play increasingly important roles in cell and neurobiology and have the potential to revolutionise the way clinical and industrial analytics are performed. The gradual transition from multicomponent biosensors to fully integrated single chain allosteric biosensors has brought the field closer to commercial applications. We evaluate various approaches for converting constitutively active protein reporter domains into analyte operated switches. We discuss the paucity of the natural receptors that undergo conformational changes sufficiently large to control the activity of allosteric reporter domains. This problem can be overcome by constructing artificial versions of such receptors. The design path to such receptors involves the construction of Chemically Induced Dimerisation systems (CIDs) that can be configured to operate single and two-component biosensors.     

Dynamics-based community analysis and perturbation response scanning of allosteric interaction networks in the TRAP1 chaperone structures dissect molecular linkage between conformational asymmetry and sequential ATP hydrolysis

Allosteric interactions of the Hsp90 chaperones with cochaperones and diverse protein clients can often exhibit distinct asymmetric features that determine regulatory mechanisms and cellular functions in many signaling networks. The recent crystal structures of the mitochondrial Hsp90 isoform TRAP1 in complexes with ATP analogs have provided first evidence of significant asymmetry in the closed dimerized state that triggers independent activity of the chaperone protomers, whereby preferential hydrolysis of the buckled protomer is followed by conformational flipping between protomers and hydrolysis of the second protomer. Despite significant insights in structural characterizations of the TRAP1 chaperone, the atomistic details and mechanics of allosteric interactions that couple sequential ATP hydrolysis with asymmetric conformational switching in the TRAP1 protomers remain largely unknown. In this work, we explored atomistic and coarse-grained simulations of the TRAP1 dimer structures in combination with the ensemble-based network modeling and perturbation response scanning of residue interaction networks to probe salient features underlying allosteric signaling mechanism. This study has revealed that key effector sites that orchestrate allosteric interactions occupy the ATP binding region and N-terminal interface of the buckled protomer, whereas the main sensors of allosteric signals that drive functional conformational changes during ATPase cycle are consolidated near the client binding region of the straight protomer, channeling the energy of ATP hydrolysis for client remodeling. The community decomposition analysis of the interaction networks and reconstruction of allosteric communication pathways in the TRAP1 structures have quantified mechanism of allosteric regulation, revealing control points and interactions that coordinate asymmetric switching during ATP hydrolysis.     

Identification of new allosteric sites and modulators of AChE through computational and experimental tools

Allosteric sites on proteins are targeted for designing more selective inhibitors of enzyme activity and to discover new functions. Acetylcholinesterase (AChE), which is most widely known for the hydrolysis of the neurotransmitter acetylcholine, has a peripheral allosteric subsite responsible for amyloidosis in Alzheimer’s disease through interaction with amyloid β-peptide. However, AChE plays other non-hydrolytic functions. Here, we identify and characterise using computational tools two new allosteric sites in AChE, which have allowed us to identify allosteric inhibitors by virtual screening guided by structure-based and fragment hotspot strategies. The identified compounds were also screened for in vitro inhibition of AChE and three were observed to be active. Further experimental (kinetic) and computational (molecular dynamics) studies have been performed to verify the allosteric activity. These new compounds may be valuable pharmacological tools in the study of non-cholinergic functions of AChE.     

Functionally opposite receptors on neurones.

According to the second general principle of chemical neurotransmission, formulated by Eccles, a neurotransmitter released from the axonal terminals of a neurone has only one and the same effect on all the follower cells: all the follower cells are either excited or inhibited by it. The evidence reviewed in this paper suggests that there may be some exceptions to this general pattern of synaptic organisation. The co-existence of both excitatory and inhibitory receptors for the same transmitter substance on the same neuronal membrane has been described in invertebrates: ‘opposite receptors’ can occur for acetylcholine, dopamine and glutamate. There is also evidence suggesting the existence of opposite receptors for acetylcholine and nonadrenaline on neurones in the mammalian brain. The activation of functionally opposite receptors results in ‘antagonistic agonism’: the net pharmacological effect is obtained by the algebraic summation of the two opposing component effects. Both the time-course and the polarity of neuronal responses to a given neurotransmitter is influenced by the relationship between the two kinds of receptor. It is suggested that both pre-synaptic and post-synaptic factors may modify this relationship.     

Characterizing and predicting the functional and conformational diversity of seven-transmembrane proteins

The activation of seven-transmembrane receptors (7TMRs) allows cells to sense their environment and convert extracellular signals (like hormone binding) into intracellular signals (through G protein-coupled and/or β arrestin-coupled pathways). A single 7TMR is capable of transducing a wide spectrum of physiological responses inside a cell by coupling to these pathways. This intracellular pleiotropic action is enabled by multiple conformations exhibited by these receptors. Developments in membrane protein structure determination technologies have led to a rapid increase in crystal structures for many 7TMRs. Majority of these receptors have been crystallized in their inactive conformation and, for some, one of the many active conformations has also been crystallized. Given the topological constraints of a lipid bilayer that results in a single fold of seven almost parallel TM helices connected by mostly unstructured loops, these structures exhibit a diversity of conformations not only across the receptors but also across the different functional forms for receptors with structures for one of the functionally active conformations. Here we present a method to characterize this conformational diversity in terms of transmembrane helix topology (TMHTOP) parameters and how to use these helix orientation parameters to predict functionally-distinct multiple conformations for these receptors. The TMHTOP parameters enable a quantification of the structural changes that underlie 7TMR activation and also sheds a unique mechanistic light on the pleiotropic nature of these receptors. It provides a common language to describe the 7TMR activation mechanisms as well as differences across many receptors in terms of visually intuitive structural parameters. Protein structure prediction methods can use these parameters to describe 7TMR conformational ensembles, which coupled to experimental data can be used to develop testable hypotheses for the structural basis of 7TMR functions.     

Recombinant FimH Adhesin Demonstrates How the Allosteric Catch Bond Mechanism Can Support Fast and Strong Bacterial Attachment in the Absence of Shear

The FimH protein of Escherichia coli is a model two-domain adhesin that is able to mediate an allosteric catch bond mechanism of bacterial cell attachment, where the mannose-binding lectin domain switches from an ‘inactive’ conformation with fast binding to mannose to an ‘active’ conformation with slow detachment from mannose. Because mechanical tensile force favors separation of the domains and, thus, FimH activation, it has been thought that the catch bonds can only be manifested in a fluidic shear-dependent mode of adhesion. Here, we used recombinant FimH variants with a weakened inter-domain interaction and show that a fast and sustained allosteric activation of FimH can also occur under static, non-shear conditions. Moreover, it appears that lectin domain conformational activation happens intrinsically at a constant rate, independently from its ability to interact with the pilin domain or mannose. However, the latter two factors control the rate of FimH deactivation. Thus, the allosteric catch bond mechanism can be a much broader phenomenon involved in both fast and strong cell-pathogen attachments under a broad range of hydrodynamic conditions. This concept that allostery can enable more effective receptor-ligand interactions is fundamentally different from the conventional wisdom that allostery provides a mechanism to turn binding off under specific conditions.     

Regulation of signal transduction at M2 muscarinic receptor

Muscarinic acetylcholine receptors mediate transmission of an extracellular signal represented by released acetylcholine to neuronal or effector cells. There are five subtypes of closely homologous muscarinic receptors which are coupled by means of heterotrimeric G-proteins to a variety of signaling pathways resulting in a multitude of target cell effects. Endogenous agonist acetylcholine does not discriminate among individual subtypes and due to the close homology of the orthosteric binding site the same holds true for most of exogenous agonists. In addition to the classical binding site muscarinic receptors have one or more allosteric binding sites at extracellular domains. Binding of allosteric modulators induces conformational changes in the receptor that result in subtype-specific changes in orthosteric binding site affinity for both muscarinic agonists and antagonists. This overview summarizes our recent experimental effort in investigating certain aspects of M2 muscarinic receptor functioning concerning i) the molecular determinants that contribute to the binding of allosteric modulators, ii) G-protein coupling specificity and subsequent cellular responses and iii) possible functional assays that exploit the unique properties of allosteric modulators for characterization of muscarinic receptor subtypes in intact tissue. A detailed knowledge of allosteric properties of muscarinic receptors is required to permit drug design that will modulate signal transmission strength of specific muscarinic receptor subtypes. Furthermore, allosteric modulation of signal transmission strength is determined by cooperativity rather than concentration of allosteric modulator and thus reduces the danger of overdose.