Foundations Of Allosteric Receptor Theory

The development of allosteric theory was basically driven by two interrelated concepts: the first being the discovery of cooperativity in the binding of some ligands to their target proteins, and the second being the realization that proteins naturally explore multiple conformations and that conformational changes can be transmitted across protein structures such that the binding of a ligand could affect the conformation of a distant binding pocket recognized by another ligand. The idea that certain proteins could bind more than one ligand was notably described early last century for the enzyme, hemoglobin, which can simultaneously bind up to four molecules of oxygen [4], and subsequently extended to other enzymes and ligand-gated ion channels [5, 6] . In most known instances of cooperativity, the binding of one ligand to its site has the potential to alter the affinity for subsequent equivalents of the same ligand at the remaining (unoccupied) sites [7]. In the case of hemoglobin, for instance, this corresponds to a 200-fold increase in the affinity of the fourth oxygen molecule bound compared to the first [8]. Around the middle of the last century, important studies were also underway that led to the concept of global protein conformational changes, whereby proteins were postulated to isomer-ize between multiple conformations that can show different affinities for ligands and/or different functional properties [9, 10] . In such a scheme, the process of ligand binding can be viewed as an event that biases certain protein conformational states toward those that show higher affinity for the ligand, at the expense of other possible protein states.

The term "allosteric" was first coined by Monod and colleagues in studies of enzyme inhibition [11, 12]. They noted that inhibitors that were structurally diverse from the enzyme substrate were likely to act at alternate binding sites that were somehow conformationally linked to that of the substrate binding pocket. The authors defined allosteric proteins on the following criteria: an oligomeric architecture, cooperativity in binding, and the ability of ligand binding to preferentially select for certain protein conformations that exist in equilibrium. This strict definition of allosteric proteins has since been relaxed somewhat, with the term being used to describe multisite interactions on proteins, irrespective of whether or not they are oligomeric, and/or to describe the transition of a protein between different conformational states, regardless of whether or not they possess multiple binding sites.

Although the phenomenon was initially studied on enzyme and ion channel systems, it should be noted that GPCRs are naturally allosteric proteins. Specifically, they are designed to recognize extracellular stimuli and then to transmit them across biological membranes to impart the signal to their cognate G protein(s) and other intracellular effectors. Thus, the endogenous orthosteric ligand binds to one site on the receptor that is usually extracellu-larly accessible, while the G protein and any other accessory proteins interact with interfaces that are present inside the cell, and therefore topographically distinct from the binding site of the endogenous ligand. Moreover, there is a wealth of evidence that the binding of G proteins, and other types of GPCR-protein interactions, can influence the conformational state of the receptor, as evidenced by altered ligand affinity and/or signaling [13-17] . Given this tremendous conformational plasticity, it is perhaps not surprising that many small molecules are now being discovered with increasing prevalence that can bind to sites other than the orthosteric site on a GPCR to promote conformational changes that have a profound effect on the binding and/or function of orthosteric ligands.

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