PMC

Simple illustration of conformational selection in terms of the free energy landscape. Several crystal structures of the glucocorticoid receptors and their corresponding REs are used here as examples. GR samples the conformational space around the native state. Conformers lie in distinct local minima separated by low barriers, and their energies differ slightly. All conformers pre-exist prior to binding, and each selectively binds an RE (−). NMR has recently validated this theoretical proposition, illustrating that the bound conformers pre-exist in the unbound state (,−). Here five superimposed pairs of crystal conformers are shown presenting minor conformational changes. The conformers at the left and right have been manually slightly unfolded to depict higher energy states. At the bottom are the corresponding five superimposed GR REs with very similar DNA sequences. The corresponding PDB structures and REs (from left to right) are 3FYL-3G6P (DNA, CGT-FKBP5), 3G6Q-3G6R (DNA, FKBP5-FKBP5), 3G6T-3G6U (DNA, FKBP5-FKBP5), 3G8U-3G8X (DNA, GILZ-GILZ), and 3G97-3G99 (DNA, GILZ-PAL). The aligned RE sequences () are also presented, illustrating the minor base pair changes.

An illustration to explain why binding does not necessarily spell function. The response elements (REs, red boxes) of a given transcriptional control protein (TC) have very similar DNA sequences, with small base pair (bp) changes, yet they regulate genes whose functions can be vastly different. A key question is how the TC selectively binds a given RE among all the similar ones in the genome. A) Affinity of the TC to its REs is low. Binding of a cofactor (Co-F1) to the TC allosterically alters slightly the binding site of the TC to the DNA, leading to higher affinity binding to a specific RE. Which cofactor is available and binds depends on the cellular network. In this case, binding already implies specific function. B) The TC has high affinity to its REs; thus it binds to all chromatin-available ones. However, binding is insufficient. Since REs with even slightly different DNA sequences have slightly different conformations, binding to a specific RE allosterically leads to changes in the cofactor binding site of the TC, which now selectively binds a cofactor (Co-F2). Which cofactor binds again depends on the cellular network. C) Binding of a protein cofactor (Co-F3) has no functional consequences, since this binding event does not affect the allosteric communication pathways between the functional RE and transcriptionally relevant binding sites.

Two examples illustrating how binding at other sites−by an RE (A) or agonist/antagonist ligand (B)−can allosterically alter the respective co-regulator binding site conformation leading to activation or inhibition. In the left panel of A, three crystal structures () of the glucocorticoid receptor (GR) bound to three REs whose sequences are very similar to each other are superimposed. Binding allosterically leads to a conformational change at the co-regulator binding site (PDB ids: yellow, 3G99; blue, 3G6P; green, 3FYL). In the right panel, all 15 crystallized GR structures (3FYL, 3G6P, 3G6Q, 3G6R, 3G6T, 3G6U, 3G8U, 3G97, 3G8X, 3G99, 3G9I, 3G9J, 3G9M, 3G9O, and 3G9P) are superimposed and viewed from a different angle showing that the conformational change is far away from the DNA. Here only one RE (CGT in 3FYL) is shown, for clarity. Panel B illustrates the effects of the binding of an antagonist (left panel, PDB 1nde) and an agonist (right panel, PDB 1nde) on co-activator binding to the estrogen receptor (ER) ligand binding domain (LBD) (). The binding of the agonist and antagonist are at the same ER site; however, the antagonist leads to an allosteric displacement of H12 to occupy roughly the same position as the co-activator. Hence, co-activator binding to the LBD is blocked. Binding of the agonist exposes the co-regulator binding site.

Allosteric regulation underlies the complex binding−function relationship in cellular networks. The figure highlights the inadequacy of current cellular diagrams that depict series of binding events. As an example. we depict the estrogen receptor, for which there are experimental data. Estrogen receptors (Era and ERb) can be selectively activated by ligand binding, with allosteric control of ligand selectivity and function (). A) Schematic “textbook” network diagram of the estrogen receptor (ER) signaling pathways. ER activation is controlled by extracellular signals, hormone and cofactor binding events (). Extracellular signals lead to phosphorylation of the ER monomer. Examples of the extracellular signals are (i) dopamine and cAMP binding to GPCR can activate PKA; (ii) growth factors (GFs) activate their receptors with subsequent activation of the RAS-RAF-ERK pathway; and (iii) nongenomic action of ER in the membrane activates the PI3K-Akt pathway. Both antagonist and agonist ligands can prompt ER dimerization with different allosteric consequences for the helix H12 position (see , panel B). The shift in the H12 position triggered by antagonist ligands blocks subsequent cofactor binding, while agonist ligands allostericaly change the ER conformations to allow cofactor recruitment. Cofactors () like the nuclear receptor co-repressor (NCoR) and the repressor of the estrogen receptor activity (REA) lead to repression of ER response elements (ERE). Examples of direct activators are the thyroid hormone receptor (TRAP), steroid receptor activator (SRA), and steroid receptor co-activators (SRCs). The secondary co-activators (like CoCoA and PRMT) also bind ERS indirectly through association with SRCs. Thus, the network diagram provides simple binding events but misses the allosteric regulation of binding and function (), which are highlighted in panels B and C. B) Affinities between the central node (here the human ERa) and its binding partners (agonists, connected via the edges), where line thickness indicates the binding strength () thus specifying its rank. For example, the 17b-dihydroequilenin (17b-DHEquilenin) ranks third, and Equilenin ranks tenth in binding strength. C) The rank of human ERa functional activity (), where line thickness indicates the strength of the functional activity. Unlike the binding strength, 17b-DHEquilenin only ranks eighth and Equilenin ranks third in binding strength for ERa. For ERb, the Equilenin ranks last in binding strength but is the most active for ERb (). Comparison of the widths of corresponding edges between panels B and C illustrates that the extent of the affinity does not necessarily correspond to the degree of function. Thus the allosteric control of estrogen receptors strongly supports the notion that allostery should be considered when trying to understand how binding phenomena determine the functional outcome.