The origin of allosteric functional modulation: multiple pre-existing pathways

Abstract

Although allostery draws increasing attention, not much is known about allosteric mechanisms. Here we argue that in all proteins, allosteric signals transmit through multiple, pre-existing pathways; which pathways dominate depend on protein topologies, specific binding events, covalent modifications, and cellular (environmental) conditions. Further, perturbation events at any site on the protein surface (or in the interior) will not create new pathways but only shift the pre-existing ensemble of pathways. Drugs binding at different sites or mutational events in disease shift the ensemble toward the same conformations; however, the relative populations of the different states will change. Consequently the observed functional, conformational, and dynamic effects will be different. This is the origin of allosteric functional modulation in dynamic proteins: allostery does not necessarily need to invoke conformational rearrangements to control protein activity and pre-existing pathways are always defaulted to during allostery regardless of the stimulant and perturbation site in the protein.

Figure 1

The two critical differences between the old and updated new view. In allostery, perturbation at the effector site causes some activity change at the substrate site. The allosteric stimulus and activity change are illustrated as binding events. Figure 1A presents the old view: an effector binds and causes an active site conformational change via a single propagation pathway, making an unfavorable substrate binding become favorable. Figures 1B and 1C highlight the two basic differences between the old and the updated new view. First, due to the nature of the thermodynamic equilibrium, the updated new view is based on population shift, and does not endorse the kinetic concept of propagating and releasing the strain energy created at the allosteric site via a single pathway; rather, it postulates that population shift from an off-state to an on-state (or vice-versa) must involve multiple pathways through network interactions. Figure 1B presents the multiple propagation pathways with an active site conformational change. Only pathways relating to such conformational change are shown. Second, in Figure 1C, no conformational change is associated with the population shift; here, allostery is entropy-dominated. Thermodynamically, population shift with an accompanied conformational change could be dominated by enthalpy or entropy; however, a free energy change without conformational change is entropy-dominated. In the case of allostery with enthalpy-dominated conformational change, all propagation pathways effectively lead to the required conformational change. In positive cooperativity, the unfavorable active site conformation becomes substrate-favorable. The opposite holds for negative cooperativity. For the case of entropy-dominated conformational change, consider an effector binding to disordered structure. This is positive cooperativity with entropy loss (disorder becomes order) prepaid by effector binding. Prepaid entropy via backbone and side-chain rigidification would be a case of entropy-dominated positive cooperativity without conformational change. In negative cooperativity without conformational change, effector binding enhances the dynamics; subsequent substrate-induced rigidification creates extra, non-additive entropy loss. Above we focus on enthalpy- or entropy-dominated allostery; not a combination of both (Tsai et al., 2008). The realization of these two critical differences impacts the understanding and prediction of the allosteric effects.

Figure 2

Examples of proteins with experimental data supporting the existence of multiple pathways for allosteric communications. Figure 2A shows two possible pathways for allosteric communications upon RA-GEF2 peptide binding mapped onto the structure of PDZ2-RA-GEF2 (PDB: code: 1D5G), with the RA-GEF2 peptide shown in yellow. In red are represented residues energetically-coupled to His71(Lockless and Ranganathan, 1999). The black line depicts a pathway formed by these residues, that starts at the peptide binding site residue His71, and splits into two sub-pathways that end at two residues located at the opposite side of the domain (A46 and V85). An alternative pathway comprised of residues whose side chain dynamics significantly change upon peptide binding (shown in blue) is represented with the white line (Fuentes et al., 2004). This pathway also starts at a peptide binding site residue (V26) and splits into two sub-pathways ending at two residues (A39 and T81) belonging to two distal surfaces from the peptide binding site (Fuentes et al., 2004), correspondingly. Distal surface 1 is comprised of Thr81, Val85, Val61, Val64, Leu66, and Leu69, whereas distance surface 2 contains Ala39 and Val40. Residues shown in brown are common to both pathways. Figure 2b shows two alternative possible pathways for the transmission of perturbation upon ligand binding (MLL) on the structure of the complex KIX-MLL-cMyb (PDB code: 2AGH). In red are residues which experience structural changes upon MLL binding (Bruschweiler et al., 2009). The black line depicts a pathway formed by these residues, which starts at the MLL binding site and ends at the c-Myb binding site. The yellow and pink helices correspond to MLL and c-Myb ligands, respectively. Residues colored in blue, which exhibit significant backbone chemical shift changes upon MLL binding (Goto et al., 2002), form a hydrophobic groove that links the MLL binding site with a distinct protein surface. This surface might be a novel binding site for other ligands. Residue F612 shown in brown is common to both networks of residues.

Figure 3

A schematic 2-D lattice chain illustrating allosteric pathways. The figure depicts a 15-residues chain with red bars indicating its backbone trace. Each residue is also given two exact interactions with its neighbors either via sidechain-sidechain or sidechain-backbone interaction. The long brown bar indicates a strong interaction and short blue bar stands for a weak interaction. A micro-pathway is drawn starting from the perturbation site down to the second (substrate) site via the residue-residue interaction linkage (8->7->6->5->13). A mutation at residue 10 will not alter the allosteric effect since not a single residue-residue interaction has been changed.