Competing Ligands Can Both Obstruct and Enhance Protein-Complex Formation

Main Text

Specific molecular recognition is a basic requirement for life. In physical terms, the source of interaction specificity that favors a small set of specific interactions over the multitude of possibilities is evolutionary favored, but how this drives evolution is still an open question (1, 2, 3). Although nonspecific interactions are typically weak and short-lived, they are still expected to affect function because of their sheer numbers in the crowded milieu of the cell. Both specific and nonspecific interactions form weak encounters with other proteins. The difference is that specific interactions form fruitful encounters that are on pathway to complex formation, whereas nonspecific interactions form futile encounters that dissociate rapidly (4, 5). Fruitful encounters, which are formed at the proximity of the final binding site but before interface desolvation and the formation of short-range interactions, are an integral part of the association process. Fruitful encounters are followed by a docking step, which includes interface desolvation and the formation of short-range interactions that stabilize the complex. Competing interactions will form futile encounter complexes that will not develop toward a structurally defined complex. Therefore, it is assumed that these competing interactions will either be neutral or negatively affect the association kinetics.

In a study published in this issue (6), Kale et al. argue that in some cases, competing interactions may actually enhance binding kinetics. The authors analyzed, using agent-based simulations of experimental NMR paramagnetic relaxation enhancement measurements, the effect of a competing, noninteracting ligand on the rate and affinity of complex formation. The study was done on the complex between the N-terminal domain of enzyme I (EIN) of the bacterial phosphoenolpyruvate-dependent nitrogen phosphotransferase system and the phosphor-carrier protein NPr. The competing agent is Escherichia coli HPr, which is very similar to the E. coli NPr but does not bind EIN-dependent nitrogen phosphotransferase (EIN^Ntr) (7, 8).

Paramagnetic relaxation enhancement measurements on EIN^Ntr in the presence of a paramagnetically labeled NPr showed the formation of weak encounters between them. However, when HPr was introduced into the solution in up to 10-fold excess over NPr, it enhanced NPr binding. That is, the addition of HPr increased the population of the stereo-specific complexes between NPr and EIN^Ntr (9). Thus, they show that futile encounters between noncognate proteins can serve to stabilize the specific complex rather than competing with it (Fig. 1).

Figure 1.

NPr (green) can bind the specific interface on EIN (blue), whereas HPr (orange) can bind EIN at multiple sites. In this case, NPr encounters are driven toward the specific binding interface because of reduced search space. This will result in both increased residence time and rate of formation of the specific complex. To see this figure in color, go online.

To better understand this unusual cross talk, they used a physical model-guided simulation, showing that this will occur within a narrow range of relative affinities, stoichiometry between the proteins and rate of encounter exchanges. The mechanism suggested here is that having an excess of the competing ligand will limit the space of futile encounter complexes that can be formed by the specific ligand. By limiting the futile encounters, fruitful encounters of the specific partner are favored. Thus, limiting the futile encounters through the excess of competing ligand will enhance specific complex formation (Fig. 1). Although the effect on the binding of the competing ligand on the association of the specific one is small (few percent), it is still significant. As the effect of HPr emerges as a result of crowding, one wonders whether crowding in the cytosol could have a role as well. Here, a control experiment using ubiquitin, a protein absent in bacteria but with similar size to HPr, did not reproduce this effect (9). Thus, it is not merely crowding but some degree of attraction between the nonspecific protein surfaces that is necessary for encounter-driven stabilization. What is the fraction of proteins that form some degree of attraction between them within the cytosol is not known. Previous studies of manipulating nonspecific binding in the crowded milieu of the cell has shown that substitution of surface residues affected nonspecific attraction, which was explained by net charge density, surface hydrophobicity, and the electric dipole moment (10). Although Kale et al. (6) avoid addressing the wider problem of the effect of crowding on binding, this is a first step toward this direction. What mechanisms allow proteins to bind fast and specific in crowded environments is still an open question. This study raises the possibility that nonspecific binders may contribute toward this effect. Thus, weak, futile interactions in the crowded milieu could also enhance fruitful binding, in addition to their obstructive potential, as suggested by the model presented here.

Editor: Madan Babu Mohan.