In the fields ofbiochemistry andpharmacology anallosteric regulator (orallosteric modulator) is a substance that binds to a site on anenzyme orreceptor distinct from theactive site, resulting in a conformational change that alters the protein's activity, either enhancing or inhibiting its function. In contrast, substances that bind directly to an enzyme's active site or the binding site of theendogenous ligand of a receptor are calledorthosteric regulators or modulators.
The site to which the effector binds is termed theallosteric site orregulatory site. Allosteric sites allow effectors to bind to the protein, often resulting in aconformational change and/or a change inprotein dynamics.[1][2] Effectors that enhance the protein's activity are referred to asallosteric activators, whereas those that decrease the protein's activity are calledallosteric inhibitors.
Allosteric regulations are a natural example of control loops, such asfeedback from downstream products orfeedforward from upstream substrates. Long-range allostery is especially important incell signaling.[3] Allosteric regulation is also particularly important in thecell's ability to adjustenzyme activity.
The termallostery comes from theAncient Greekallos (ἄλλος), "other", andstereos (στερεός), "solid (object)". This is in reference to the fact that the regulatory site of an allosteric protein is physically distinct from its active site. Allostery contrasts withsubstrate presentation which requires no conformational change for an enzyme's activation. The termorthostery comes from theAncient Greek orthós (ὀρθός) meaning "straight", "upright", "right" or "correct".
Many allosteric effects can be explained by theconcertedMWC model put forth byMonod,Wyman, andChangeux,[4] or by thesequential model (also known as the KNF model) described byKoshland, Nemethy, and Filmer.[5] Both postulate thatprotein subunits exist in one of twoconformations, tensed (T) or relaxed (R), and that relaxed subunits bind substrate more readily than those in the tense state. The two models differ most in their assumptions about subunit interaction and the preexistence of both states. For proteins in whichsubunits exist in more than twoconformations, the allostery landscape model described by Cuendet, Weinstein, and LeVine,[6] can be used. Allosteric regulation may be facilitated by the evolution of large-scale, low-energy conformational changes, which enables long-range allosteric interaction between distant binding sites.[7]
The concerted model of allostery, also referred to as the symmetry model orMWC model, postulates that enzyme subunits are connected in such a way that a conformational change in one subunit is necessarily conferred to all other subunits. Thus, all subunits must exist in the same conformation. The model further holds that, in the absence of any ligand (substrate or otherwise), the equilibrium favors one of the conformational states, T or R. The equilibrium can be shifted to the R or T state through the binding of oneligand (the allosteric effector or ligand) to a site that is different from the active site[citation needed]
The sequential model of allosteric regulation holds that subunits are not connected in such a way that a conformational change in one induces a similar change in the others. Thus, all enzyme subunits do not necessitate the same conformation. Moreover, the sequential model dictates that molecules of a substrate bind via aninduced fit protocol. While such an induced fit converts a subunit from the tensed state to relaxed state, it does not propagate the conformational change to adjacent subunits. Instead, substrate-binding at one subunit only slightly alters the structure of other subunits so that their binding sites are more receptive to substrate. To summarize:[citation needed]
Themorpheein model of allosteric regulation is a dissociative concerted model.[8]
A morpheein is a homo-oligomeric structure that can exist as an ensemble of physiologically significant and functionally different alternate quaternary assemblies. Transitions between alternate morpheein assemblies involve oligomer dissociation, conformational change in the dissociated state, and reassembly to a different oligomer. The required oligomer disassembly step differentiates the morpheein model for allosteric regulation from the classic MWC and KNF models.[citation needed]
Porphobilinogen synthase (PBGS) is the prototype morpheein.[citation needed]
Ensemble models of allosteric regulation enumerate an allosteric system'sstatistical ensemble as a function of itspotential energy function, and then relate specific statistical measurements of allostery to specific energy terms in the energy function (such as an intermolecular salt bridge between two domains).[9] Ensemble models like the ensemble allosteric model[10] and allosteric Ising model[11] assume that each domain of the system can adopt two states similar to the MWC model. The allostery landscape model introduced by Cuendet, Weinstein, and LeVine[6] allows for the domains to have any number of states and the contribution of a specific molecular interaction to a given allosteric coupling can be estimated using a rigorous set of rules.Molecular dynamics simulations can be used to estimate a system's statistical ensemble so that it can be analyzed with the allostery landscape model.[citation needed]
Allosteric modulation is used to alter the activity of molecules and enzymes in biochemistry and pharmacology. For comparison, a typical drug is made to bind to the active site of an enzyme which thus prohibits binding of a substrate to that enzyme causing a decrease in enzyme activity. Allosteric modulation occurs when aneffector binds to an allosteric site (also known as a regulatory site) of an enzyme and alters the enzyme activity. Allosteric modulators are designed to fit the allosteric site to cause a conformational change of the enzyme, in particular a change in the shape of the active site, which then causes a change in its activity. In contrast to typical drugs, modulators are notcompetitive inhibitors. They can be positive (activating) causing an increase of the enzyme activity or negative (inhibiting) causing a decrease of the enzyme activity. The use of allosteric modulation allows the control of the effects of specific enzyme activities; as a result, allosteric modulators are very effective in pharmacology.[12] In a biological system, allosteric modulation can be difficult to distinguish from modulation bysubstrate presentation.[citation needed]
An example of this model is seen with theMycobacterium tuberculosis, abacterium that is perfectly suited to adapt to living in the macrophages of humans. The enzyme's sites serve as a communication between different substrates. Specifically betweenAMP andG6P. Sites like these also serve as a sensing mechanism for the enzyme's performance.[13]
Positive allosteric modulation (also known asallosteric activation) occurs when the binding of oneligand enhances the attraction between substrate molecules and other binding sites. An example is the binding ofoxygen molecules tohemoglobin, where oxygen is effectively both thesubstrate and the effector. The allosteric, or "other", site is theactive site of an adjoiningprotein subunit. The binding of oxygen to one subunit induces a conformational change in that subunit that interacts with the remaining active sites to enhancetheir oxygen affinity.Another example of allosteric activation is seen in cytosolic IMP-GMP specific 5'-nucleotidase II (cN-II), where the affinity for substrate GMP increases upon GTP binding at the dimer interface.[citation needed]
Negative allosteric modulation (also known asallosteric inhibition) occurs when the binding of oneligand decreases the affinity for substrate at other active sites. For example, when2,3-BPG binds to an allosteric site on hemoglobin, the affinity for oxygen of all subunits decreases. This is when a regulator is absent from the binding site.[citation needed]
Direct thrombin inhibitors provides an excellent example of negative allosteric modulation.Allosteric inhibitors of thrombin have been discovered that could potentially be used as anticoagulants.[citation needed]
Another example isstrychnine, aconvulsant poison, which acts as an allosteric inhibitor of theglycine receptor.Glycine is a major post-synaptic inhibitoryneurotransmitter inmammalianspinal cord andbrain stem. Strychnine acts at a separate binding site on the glycine receptor in an allosteric manner; i.e., its binding lowers theaffinity of the glycine receptor for glycine. Thus, strychnine inhibits the action of an inhibitory transmitter, leading to convulsions.[citation needed]
Another instance in which negative allosteric modulation can be seen is betweenATP and the enzymephosphofructokinase within thenegative feedback loop that regulatesglycolysis. Phosphofructokinase (generally referred to asPFK) is an enzyme that catalyses the third step of glycolysis: thephosphorylation offructose-6-phosphate intofructose 1,6-bisphosphate.PFK can be allosterically inhibited by high levels ofATP within the cell. When ATP levels are high, ATP will bind to an allosteric site onphosphofructokinase, causing a change in the enzyme's three-dimensional shape. This change causes itsaffinity forsubstrate (fructose-6-phosphate andATP) at the active site to decrease, and the enzyme is deemed inactive. This causesglycolysis to cease when ATP levels are high, thus conserving the body'sglucose and maintaining balanced levels of cellular ATP. In this way, ATP serves as a negative allosteric modulator for PFK, despite the fact that it is also a substrate of the enzyme.[citation needed]
A homotropic allosteric modulator is asubstrate for its targetprotein, as well as a regulatory molecule of the protein's activity. It is typically an activator of the protein.[14] For example, O2 and CO are homotropic allosteric modulators of hemoglobin. Likewise, in IMP/GMP specific 5' nucleotidase, binding of one GMP molecule to a single subunit of the tetrameric enzyme leads to increased affinity for GMP by the subsequent subunits as revealed by sigmoidal substrate versus velocity plots.[14]
A heterotropic allosteric modulator is a regulatory molecule that is not the enzyme's substrate. It may be either an activator or an inhibitor of the enzyme. For example, H+, CO2, and2,3-bisphosphoglycerate are heterotropic allosteric modulators of hemoglobin.[15] Once again, in IMP/GMP specific 5' nucleotidase, binding of GTP molecule at the dimer interface in the tetrameric enzyme leads to increased affinity for substrate GMP at the active site indicating towards K-type heterotropic allosteric activation.[14]
As has been amply highlighted above, some allosteric proteins can be regulated by both their substrates and other molecules. Such proteins are capable of both homotropic and heterotropic interactions.[14]
Some allosteric activators are referred to as "essential", or "obligate" activators, in the sense that in their absence, the activity of their target enzyme activity is very low or negligible, as is the case with N-acetylglutamate's activity on carbamoyl phosphate synthetase I, for example.[16][17]
A non-regulatory allosteric site is any non-regulatory component of an enzyme (or any protein), that is not itself an amino acid. For instance, many enzymes require sodium binding to ensure proper function. However, the sodium does not necessarily act as a regulatory subunit; the sodium is always present and there are no known biological processes to add/remove sodium to regulate enzyme activity. Non-regulatory allostery could comprise any other ions besides sodium (calcium, magnesium, zinc), as well as other chemicals and possibly vitamins.[citation needed]
Allosteric modulation of a receptor results from the binding of allosteric modulators at a different site (a "regulatory site") from that of theendogenous ligand (an "active site") and enhances or inhibits the effects of the endogenous ligand. Under normal circumstances, it acts by causing aconformational change in a receptor molecule, which results in a change in thebinding affinity of the ligand. In this way, an allosteric ligand modulates the receptor's activation by its primaryorthosteric ligand, and can be thought to act like a dimmer switch in an electrical circuit, adjusting the intensity of the response.[citation needed]
For example, theGABAA receptor has two active sites that the neurotransmittergamma-aminobutyric acid (GABA) binds, but also hasbenzodiazepine andgeneral anaesthetic agent regulatory binding sites. These regulatory sites can each produce positive allosteric modulation,potentiating the activity of GABA.Diazepam is apositive allosteric modulator at the benzodiazepine regulatory site, and its antidoteflumazenil is areceptor antagonist.[citation needed]
More recent examples of drugs that allosterically modulate their targets include the calcium-mimickingcinacalcet and the HIV treatmentmaraviroc.[citation needed]
Allosteric proteins are involved in, and are central in many diseases,[18][19] and allosteric sites may represent a noveldrug target. There are a number of advantages in using allosteric modulators as preferred therapeutic agents over classic orthosteric ligands. For example,G protein-coupled receptor (GPCR) allosteric binding sites have not faced the same evolutionary pressure asorthosteric sites to accommodate an endogenous ligand, so are more diverse.[20] Therefore, greater GPCR selectivity may be obtained by targeting allosteric sites.[20] This is particularly useful for GPCRs where selective orthosteric therapy has been difficult because of sequence conservation of the orthosteric site across receptor subtypes.[21] Also, these modulators have a decreased potential for toxic effects, since modulators with limited co-operativity will have a ceiling level to their effect, irrespective of the administered dose.[20] Another type of pharmacological selectivity that is unique to allosteric modulators is based on co-operativity. An allosteric modulator may display neutral co-operativity with an orthosteric ligand at all subtypes of a given receptor except the subtype of interest, which is termed "absolute subtype selectivity".[21] If an allosteric modulator does not possess appreciable efficacy, it can provide another powerful therapeutic advantage over orthosteric ligands, namely the ability to selectively tune up or down tissue responses only when the endogenous agonist is present.[21] Oligomer-specific small molecule binding sites are drug targets for medically relevantmorpheeins.[22]
There are many synthetic compounds containing severalnoncovalent binding sites, which exhibit conformational changes upon occupation of one site. Cooperativity between single binding contributions in suchsupramolecular systems is positive if occupation of one binding site enhances the affinity ΔG at a second site, and negative if the affinity isn't highered. Most synthetic allosteric complexes rely on conformational reorganization upon the binding of one effector ligand which then leads to either enhanced or weakened association of second ligand at another binding site.[23][24][25] Conformational coupling between several binding sites is in artificial systems usually much larger than in proteins with their usually larger flexibility. The parameter which determines the efficiency (as measured by the ratio of equilibrium constants Krel = KA(E)/KA in presence and absence of an effector E ) is the conformational energy needed to adopt a closed or strained conformation for the binding of a ligand A.[26]
In many multivalentsupramolecular systems[27] direct interaction between bound ligands can occur, which can lead to large cooperativities. Most common is such a direct interaction between ions in receptors for ion-pairs.[28][29] This cooperativity is often also referred to as allostery, even though conformational changes here are not necessarily triggering binding events.[citation needed]
Allostery is a direct and efficient means for regulation of biological macromolecule function, produced by the binding of a ligand at an allosteric site topographically distinct from the orthosteric site. Due to the often high receptor selectivity and lower target-based toxicity, allosteric regulation is also expected to play an increasing role in drug discovery and bioengineering.The AlloSteric Database (ASD)[30] provides a central resource for the display, search and analysis of the structure, function and related annotation for allosteric molecules. Currently, ASD contains allosteric proteins from more than 100 species and modulators in three categories (activators, inhibitors, and regulators). Each protein is annotated with detailed description of allostery, biological process and related diseases, and each modulator with binding affinity, physicochemical properties and therapeutic area. Integrating the information of allosteric proteins in ASD should allow the prediction of allostery for unknown proteins, to be followed with experimental validation. In addition, modulators curated in ASD can be used to investigate potential allosteric targets for a query compound, and can help chemists to implement structure modifications for novel allosteric drug design.[citation needed]
Not all protein residues play equally important roles in allosteric regulation. The identification of residues that are essential to allostery (so-called "allosteric residues") has been the focus of many studies, especially within the last decade.[31][32][33][34][35][36][37][38] In part, this growing interest is a result of their general importance in protein science, but also because allosteric residues may be exploited inbiomedical contexts. Pharmacologically important proteins with difficult-to-target sites may yield to approaches in which one alternatively targets easier-to-reach residues that are capable of allosterically regulating the primary site of interest.[39] These residues can broadly be classified as surface- and interior-allosteric amino acids. Allosteric sites at the surface generally play regulatory roles that are fundamentally distinct from those within the interior; surface residues may serve as receptors or effector sites in allosteric signal transmission, whereas those within the interior may act to transmit such signals.[40][41]
