Allostery: an illustrated definition for the 'second secret of life'

Abstract

Although allosteric regulation is the 'second secret of life', the molecular mechanisms that give rise to allostery currently elude understanding. In my opinion, experimental progress is hampered by a commonly used but misleading definition of allostery as protein structural changes that are elicited by the binding of a single ligand. Allostery is more strictly defined in functional terms as a comparison of how one ligand binds in the absence, versus the presence, of a second ligand. Therefore, as each of the two binding events involves two protein complexes, a study of allostery must consider four complexes and not just two. Such a comparison can distinguish allosteric from non-allosteric protein changes, the importance of which is frequently overlooked. When a study of all four complexes is not feasible, an alternative, albeit limited, strategy can identify subsets of allosteric-specific changes.

Figure 1

Thermodynamic energy cycle of allostery. Allostery can be analyzed as a thermodynamic energy cycle. This analysis demonstrates that a structure/function correlation aimed at understanding the allosteric mechanism must consider four enzyme complexes. Each enzyme complex may be a single protein conformation, an equilibrium of a limited number of conformational substates, or an ensemble of conformational substates (a dynamic structure). (a) The energy cycle for an enzyme (E) which binds one substrate (A) and one allosteric effector (X). b(i) Differences between the conformation/dynamics of the enzyme complexes within circles in (ii) and (iii) are due to binding. (ii) The structural/dynamic differences that occur when A binds in the absence vs. in the saturating presence of X are allosteric effects. (iii) The structural (conformational/dynamic) differences that occur when X binds in the presence vs. in the absence of A are allosteric effects. The two presentations of allosteric effects in (ii) and (iii) are due to reciprocity.

Figure 2

A schematic of the four protein complexes of Figure 1. These simplified illustrations demonstrate how some ligand-elicited changes in the protein structure will be relevant to allostery, but others will not. They also show why allostery is only realized in the ternary complex. Ligand dependent structural changes are indicated by arrows and change in the exterior boarder of the protein. Structural changes associated with A binding are blue and those associated with X binding are red. The region with a crucial allosteric role is in the middle of the protein. The heavy square in XEA highlights allosteric changes resulting from the representative steric clash of levers.

Figure 3

The comparison between the a) EX and b) EX’ complexes and between c) EA and d) EA’ complexes. As presented in the text, this comparison can be used to identify structural changes relevant to allostery. This alternative strategy has been developed due to common technical challenges associated with studying the ternary complex. X’ is a non-allosteric analog that binds competitively with the X ligand. A’ is a non-allosteric analog that binds competitively with the A ligand. See Figure 2 for other details.

Figure 4

A schematic of an allosteric drug (D) that alters substrate (A) binding using a pathway other than that used by the native effector. This schematic illustrates how an allosteric drug might use an allosteric mechanism that is different from that used by a native allosteric effector. It also demonstrates how rational drug design can target any region of the protein that is modified by the binding of A. This contrasts the example in Figure 2 (replacing X with D) which shows that allosteric drugs may target regions of the protein that are not directly modified by the binding of A. The interactions important to the allosteric function of the drug are contained in the bolded square at the bottom of the schematic. See Figure 2 for other details.

Box 2, Figure I. Model Data

This model data demonstrates potential changes that could result from modifying the allosteric effector, mutating or covalently modifying the protein, and/or changing temperature, pH, or other solution conditions. Curve A is the reference line. The allosteric coupling (Q_ax) for curve A is represented by the double headed arrow. Although compared to A, B has a 10-fold decrease in effector affinity in the absence of substrate and C has a 10-fold decrease in substrate affinity in the absence of effector, A, B, and C have equivalent allosteric coupling. D has a 10 fold change in allosteric coupling as compared to A.