Proteins in our body have many functions. They are building material for tissues and organs, they transport substances, transmit and receive chemical signals, and are catalysts for biochemical reactions. Accurate control and regulation of protein function is vital. There is a biochemical mechanism in the body that allows one to fine-tune the functions of proteins, depending on the state of the system and external conditions. This mechanism is called allosteric regulation.
This is a diagram of allosteric regulation of an enzyme. A - Active site; B - Allosteric site; C -Substrate;D – Inhibitor; E – Enzyme.
The concept of allostery emerged when studying changes in enzyme activity during the binding of regulatory molecules (ligands) (1). Allosteric regulation of proteins is any form of regulation of their properties and functions in which an effector molecule (a regulator) binds to a protein (or protein complex) at a place that is physically different and independent of its functional centre (for example, a binding site with a substrate for enzymes or with a ligand for receptors) (1-5). Hence the name, derived from the Greek roots allos – 'other' and stereos – 'hard' (1,6). In other words, allosteric proteins are proteins that have at least one additional (allosteric) binding site in addition to the main (orthosteric) binding site. The binding of an effector molecule to an allosteric site that does not directly contact the orthosteric site nevertheless leads to changes in the latter (2-7). That is, a signal is transmitted from one site of the protein to another distant site, which allows the properties of the protein to be modulated and its activity to be regulated.
How then does signal transmission work and what are the molecular mechanisms underlying it? Scientists have been trying to find answers to these questions for more than half a century. Modern methods for studying protein structure, including computer modelling, have made significant progress in solving this problem. However, scientists have not yet succeeded in fully understanding how functional and regulatory sites located at a considerable distance from each other are interconnected.
Initially, it was believed that allostery is caused purely by conformational changes in the protein. In 1965, the first quantitative mathematical model of allostery was formulated – the Monod-Wyman-Changeux model or 'MWC model' (10) (1-3, 5, 8). According to this model (also called the 'concerted model'), there are at least two conformational states of protein subunits: relaxed (R) and tensed (T), reflecting functional activity (active and inactivated states). There is a thermodynamic equilibrium between these states, which can be shifted through the binding of an effector molecule. The transition from one conformation to another occurs in all subunits in concert, in other words proteins consist only of R or T subunits (8). This simplified model made it possible, for example, to kinetically describe the observed abrupt increase in the affinity of haemoglobin for oxygen as it saturates (1-3,5,7).
Illustration of the 'concerted model' of allosteric regulation.
(a) Haemoglobin model. The cooperative transition path from tensed (T) to relaxed (R) state is highlighted in red, haem groups are shown in light blue.
(b) Allosteric transition of tetrameric haemoglobin: tetrameric haemoglobin in the T state is depicted on the left with two α-subunits (blue) and two β-subunits (purple), each with its own haem group (light blue). The salt bridges – positive charges marked red and negative blue – keep the molecule in the T conformation. When oxygen is bound (orange circle), these salt bridges are disconnected, and a transition to the R conformation occurs. Source (8).
Haemoglobin was one of the first allosteric proteins to be investigated (1,3,7,8). It is a typical multimeric protein in that it consists of several subunits: each of the four subunits has a haem molecule that binds oxygen, and they are located at a considerable distance from each other. According to the model, in the case of haemoglobin, the relaxed and tensed states are characterised by high and low affinity for oxygen, respectively. Subsequently, structural studies have confirmed this phenomenon at a molecular level. It was demonstrated that oxygenation or deoxygenation causes significant changes in the spatial organisation of the functional centre of the protein and displacement of the subunits relative to one another. Thus, the addition or removal of one oxygen molecule in one subunit initiates the corresponding process in the remaining subunits. This is the so-called cooperative effect, one of the most famous examples of allosteric regulation, when a small increase in substrate concentration leads to a significant increase in the reaction rate (1,8).
The second model of allostery, which was dominant for decades, was Koshland Nemethy and Filmer's 'sequential model' (KNF model) (11) (1-3, 5, 8). This model leaves open the possibility of finding protein subunits in different conformational states simultaneously, expanding its capabilities. The model assumes that the subunits within the multimer change their conformation alternately. That is to say, the binding of a regulatory molecule by one subunit changes its state, which, in turn, shifts the equilibrium for the neighbouring subunit and so on. In other words, there is a sequential change in the conformation of protein subunits. Although the models were to some extent successful in describing allostery, both are phenomenological, and do not therefore provide an exhaustive explanation of the allosteric phenomenon (8). Further studies have shown that the molecular mechanisms of allosteric regulation are complex and varied, and that it can occur without structural changes in proteins (3,4,8). In 1984, about 20 years after the concept appeared, the possibility of allostery without conformational changes was theoretically predicted: signal transmission from the allosteric site to other parts of the protein during regulatory molecule binding may be based on a change in the frequency and amplitude of thermal vibrations in the protein. This phenomenon is called 'dynamic allostery' (12) (3,4,8).
The current understanding of allosteric systems is increasingly influenced by the so-called 'ensemble allosteric model' (EAM) (2,8). According to this model, the allosteric behaviour of a macromolecular system arises from the properties of the original free energy landscape of the system and how it is influenced by regulatory molecule binding. The allosteric effect is due to the redistribution of the conformational states of proteins, which are complex assemblies of conformers in constant motion. Allosteric ligand binding leads to a population shift in the conformational ensemble, reconstructing the energy landscape. One can conclude, in accordance with this redistribution of the conformational ensemble, that allostery is not only associated with cooperativity between enzyme subunits but is also a property of all dynamic proteins. In the absence of a regulatory molecule, a tensed or relaxed state dominates the conformational ensemble, while in its presence the conformational equilibrium shifts towards the active state (2,8).
In general, the study of allosteric systems can be divided into thermodynamic and structural approaches. According to the former, a biomolecule can have more than one state with minimum free energy (for example, R and T), and the binding of an allosteric ligand shifts the equilibrium towards one of them. According to the latter, the binding of the regulator causes a series of changes in the interacting elements of the biomolecule (their bonds, mobility, and conformation) along a certain allosteric pathway (allosteric network) (8). In general, transmission of allosteric signals strongly depends on the general topology of proteins (7). A study was conducted into which structural features of proteins are conducive to allostery. It was found that less than 1% of allosteric proteins have an immunoglobulin-like structure. The alpha-beta plaits type, however, is typical of allosteric domains. Allosteric sites are most often located between protein subunits or domains, although a significant number are also present in one domain (7).
It is now known that not only low molecular weight ligands, but also protein-protein interactions, phosphorylation, protonation, and even point mutations can exert an allosteric regulatory effect. What is more, allosteric effectors can be both inhibitors and activators of protein function (8). Allosteric regulation can be affected not only by polysubunit proteins, but also by monomeric proteins (7). Moreover, there is reason to believe that allostery can be a property of almost all proteins, with the one probable exception of fibrillar proteins (such as collagen and keratins) which have a rigid structure (13). It is therefore unsurprising that allosteric regulation plays a key role in almost all biochemical processes: metabolic reactions, signal transmission, gene expression regulation, and many more (3,4,8). Virtually all enzymes, the four main classes of receptors (ligand and voltage-gated ion channels, G protein-coupled receptors, receptor tyrosine kinases and nuclear receptors) are allosteric proteins (3,9).
The ability to modulate the function of proteins using allostery is an extremely promising approach for the development of new drugs. To date, there are already various drugs that interact with the regulatory sites of proteins. For example, a large number of drugs allosterically bind to various types of membrane receptors (9). The list of drugs based on allosteric activators and inhibitors includes etravirine, maraviroc (HIV therapy); trastuzumab and pertuzumab (in cancer therapy); benzodiazepines, topiramate and lamotrigine (in the treatment of epilepsy); galantamine (therapy for cognitive and neurodegenerative disorders), verapamil (treatment of hypertension), etc. In addition, drugs for general anaesthesia (isoflurane and propofol), many neurotoxins, and ethanol have an allosteric mechanism of action (9).
One of the main advantages of allosteric ligands as drugs is their high specificity: the ability to affect certain receptor subtypes, which is often impossible to achieve by acting on conservative orthosteric sites that are often similar among different receptor subtypes (6,7). For example, rifampicin – the 'first choice' antibiotic for tuberculosis – selectively inhibits the transcription of the tubercle bacillus (Mycobacterium tuberculosis) by interacting with the allosteric center of the bacterial DNA-dependent RNA polymerase without affecting human enzymes (14). Another important factor is that allosteric modulators do not compete with the orthosteric ligand for the binding site, and can therefore act in synergy with it, enhancing or weakening its effect.
It has been suggested that allosteric regulation of proteins may be one of the possible mechanisms of action of drugs based on ultra-high dilutions (UHD), as well as homeopathic preparations (6). By taking a thermodynamic approach and invoking the idea of changing the free energy landscape of the system, the allosteric regulation of proteins has been speculatively compared with homeopathy's 'principle of similarity'. According to the authors, the homeopathic principle in this case lies in the similarity of the 'normal' configurations arising from the allosteric ligand binding and the 'abnormal' configurations typical of pathology. A drug that follows the 'principle of similarity' acts in a sick state as an 'artificial' disturbance, which is 'stronger' than the current pathological configuration, leading to its change to a 'normal' configuration. The therapeutic effect is stronger because the ability of the ligand to act on the protein through the allosteric bond evolved as a natural way to achieve an energetically favourable state. The latter effect depends not so much on the dose of the drug as on the sensitivity of the target system. The authors suggest that by binding to the protein, the drugs in the UHD change the free energy landscape of the system, reducing the energy barrier for the transition from a pathological local energy minimum (pathological attractor) to a physiological, energetically favorable one. When a protein is 'near' or 'on top' of the energy barrier between two conformational states, its unstable position can be considered a bifurcation point, where even minimal energy and informational influences, such as UHD, can control the 'switching' between active and inactive states (6).
Graphic summary of how allostery works. Allostery is considered from the thermodynamic standpoint, in terms of the energy landscape of population shift. A: Allosteric two-state model presenting a balance between the inactive state (left) and the active state (right) binding to an allosteric ligand; B: detachment of the ligand destabilizes the protein energy, and the population balance shifts to the left. Source (6).
There is experimental evidence indicating that homeopathic medicines may act by means of allosteric receptor regulation. For example, the anxiolytic-like activity of Gelsemium s. in rat neurons is inhibited by strychnine, an alkaloid that acts as an allosteric inhibitor of glycine receptors (15,16). Homeopathic dilutions of Ignatia a., which is based on strychnine, have a small but statistically significant anxiolytic activity in laboratory mice (17).
There is every reason to believe that allostery is a fundamental and universal property of biological macromolecules. It has been discovered that not only proteins, but also DNA-protein complexes and even various RNAs can exhibit allosteric properties (18-20). The public-domain Allosteric Database (ASD) publishes the identified allosteric sites of biomolecules, allosteric regulators, mutations, and many other data related to the phenomenon of allosteria. The ASD currently contains almost 2,000 allosteric sites (although not all information is supported by crystallographic information). It is not surprising that the phenomenon of allostery can influence most, if not all, biological processes and has even been called the "second secret of life" (after the genome) (21). The molecular mechanisms of allostery are not yet fully understood, but research into the phenomenon is important for a deeper understanding of the general principles of protein function, and its potential is of utmost interest for the development of new materials, biomolecules (for example, biocatalysts for industrial use), and drugs. It is also possible that understanding the phenomenon of allostery will be the key to the mechanism of action of low- and ultra-low dose drugs.
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