The underappreciated role of allostery in the cellular network

Abstract

Allosteric propagation results in communication between distinct sites in the protein structure; it also encodes specific effects on cellular pathways, and in this way it shapes cellular response. One example of long-range effects is binding of morphogens to cell surface receptors, which initiates a cascade of protein interactions that leads to genome activation and specific cellular action. Allosteric propagation results from combinations of multiple factors, takes place through dynamic shifts of conformational ensembles, and affects the equilibria of macromolecular interactions. Here, we (a) emphasize the well-known yet still underappreciated role of allostery in conveying explicit signals across large multimolecular assemblies and distances to specify cellular action; (b) stress the need for quantitation of the allosteric effects; and finally, (c) propose that each specific combination of allosteric effectors along the pathway spells a distinct function. The challenges are colossal; the inspiring reward will be predicting function, misfunction, and outcomes of drug regimes.

Figure 1.

The complexity of signaling in the cell. (A) The figure illustrates extracellular stimuli transmitted through the membrane, cytoplasm to the nucleus, and cellular response via turning genes on/off. Signaling is (often) mediated by allosteric events through proteins, nucleic acids, and lipids, and their permanent and short-lived combinatorial associations. (B) An outline of the estrogen receptor (ER) signaling pathways as an example of the complexity of signaling in the cell. Estrogen receptors (Era and ERP) can be selectively activated by ligand binding (80). ER activation is controlled by extracellular signals, hormone and co-factor binding events (105). Extracellular signals lead to phosphorylation of the ER monomer. Examples of the extracellular signals are: (i) binding of dopamine and cAMP to GPCR can activate PKA; (ii) growth factors (GFs) activate their receptors with subsequent activation of the RAS- RAF-ERK pathway; and (iii) non-genomic 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. The shift in helix H12 triggered by antagonist ligands blocks subsequent co-factor binding, while agonist ligands allostericaly change the ER conformations to allow co-factor recruitment. Cofactors (42) like the nuclear receptor corepressor (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). Secondary co-activators (like CoCoA and PRMT) also bind ERS indirectly through association with SRCs. Thus, the network diagram provides an overview of the binding events.

Figure 2.

Schematic description of the shift in the free energy landscape following allosteric binding events. The activation of cyclin-dependent kinase 6 (CDK6) is controlled by the regulatory subunits of various cyclins. On the left in Figure 2A, the free energy shifts from the favorable inactive conformation of apo CDK6 (green) to active conformation (red) following binding to a virus-encoded cyclin (v-Cyclin). The conformational change of CDK6 due to the allosteric binding of v-Cyclin (orange ribbon) is highlighted in dark color on the right by superimposing the two CDK6s with the apo inactive CDK6 (PDB 3nux) depicted by a green ribbon and the cyclin-bound active form (PDB 1jow) in a red ribbon. In Figure 2B, the energy landscape adversely shifts from the active state (red) back to the favorable inactive state (green) after the p18 (yellow ribbon) binds to the CDK6 (green ribbon) which is in complex with k-Cyclin (yellow ribbon). As shown in the dark color part of the two superimposed CDK6s, the hallmarks of activated kinase conformation (red) were lost due to the conformational changes (green) which were caused by the allosteric p18 binding.

Figure 2.

Schematic description of the shift in the free energy landscape following allosteric binding events. The activation of cyclin-dependent kinase 6 (CDK6) is controlled by the regulatory subunits of various cyclins. On the left in Figure 2A, the free energy shifts from the favorable inactive conformation of apo CDK6 (green) to active conformation (red) following binding to a virus-encoded cyclin (v-Cyclin). The conformational change of CDK6 due to the allosteric binding of v-Cyclin (orange ribbon) is highlighted in dark color on the right by superimposing the two CDK6s with the apo inactive CDK6 (PDB 3nux) depicted by a green ribbon and the cyclin-bound active form (PDB 1jow) in a red ribbon. In Figure 2B, the energy landscape adversely shifts from the active state (red) back to the favorable inactive state (green) after the p18 (yellow ribbon) binds to the CDK6 (green ribbon) which is in complex with k-Cyclin (yellow ribbon). As shown in the dark color part of the two superimposed CDK6s, the hallmarks of activated kinase conformation (red) were lost due to the conformational changes (green) which were caused by the allosteric p18 binding.

Figure 3.

The extracellular Eph region is a long distance away from the intracellular region; and the signals are transmitted over these distances to control cell movement. The Eph contains a conserved N-terminal ligand-binding domain (LBD), an adjacent cysteine-rich domain (CRD), followed by two fibronectin repeats (FN3) in the extracellular Eph region; it is connected through trans-membrane domain (TM), and linked to a kinase domain, and a SAM domain (not shown). The LBD domain has multiple open and closed conformations recognized by different ligands. On the right hand-side, nine EphA4 ligand binding domain (LBD) conformations are in complex with the ligands. Recently, two new crystal structures of EphA4 revealed eight additional unique conformations in each crystal structure (97). These multiple conformations of the free EphA4 LDB illustrate how conformational dynamics of EphA4 and the Eph-ephrin can facilitate cross-subclass ephrin signaling. The crystal structures are taken from the PDB, codes: 3MX0.

Figure 4.

The transient binding of each ephrin ligand A1, A2, B1, B2 (α, β, γ, δ) to different conformations of the Eph receptor (A, B, C, D) leads to (slightly) different preferred conformation of the second site and/or altered binding site dynamics. These may lead to selecting different partners (Aα, Bβ, Cγ, Dδ) and thus pathways (I, II, III, IV) and functional outcomes (59; 84; 97)

Figure 5.

An illustration of how diversified signaling pathways are achieved via allostery in G protein-coupled receptors (GPCR), also known as seven-transmembrane spanning receptors (7TMR). The first step of the diversified GPCR signaling involves a process of conformational selection among the multiple active states of a single GPCR. The population shift toward one of the active GPCR conformations elicited by the bound ligand leads to two distinctive pathways, the agonist (G protein-dependent) pathway and the biased agonist (arrestin-dependent) pathway. The binding of an agonist referred as ‘the classical model’ results in the activation of heterotrimeric G proteins and promotes the consequent signaling of second messenger such as cyclic AMP. However, the activated conformations of GPCRs also bind and activate GPCR kinases (GRKs) to phosphorylate Ser/Thr in the cytoplasmic loops and tail of the GPCR. In turn, the phosphorylation enables the recruitment of β-arrestins to mediate receptor desensitization and internalization. The biased agonist pathway emphasizes that a distinct active GPCR conformation activates a different set of GRKs to create phosphorylation patterns (‘barcode’) on GPCR. It is the barcode that imparts distinct conformations (illustrated in different colors in the Figure) to the recruited arrestin either through orthosteric (at the binding site) or allosteric phosphorylation events, which in turn mediate different signaling pathways such as the ERK 1/2 activation. Note that the detailed cell/tissue- specific variability of regulated signaling pathways as well as those related to isoforms of GPCRs, GRKs, and arrestins, is not included in the Figure (65; 81).