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Proteins interact with one another across a broad spectrum of affinities. Our understanding of the low end of this spectrum, as characterized by millimolar dissociation constants, relies on a handful of cases in which weak encounters have experimentally been identified. These weak interactions away from the specific target binding site can lead toward a higher-affinity complex. Recently, we detected weak encounters between two paralogous phosphotransferase pathways of Escherichia coli, which regulate various metabolic processes and stress responses. In addition to encounters that are known to occur between cognate proteins, i.e., those that can exchange phosphate groups with each other, surprisingly, encounters involving noncognates were also observed. It is not clear whether these “futile” encounters have a cooperative or competitive role. Using agent-based simulations, we find that the encounter complexes can be cooperative or competitive so as to increase or lower the effective binding affinity of the specific complex under different circumstances. This finding invites further questions into how organisms might exploit such low affinities to connect their signaling components.

There are two paralogous E. coli phosphotransferase systems, one for sugar import (PTS(sugar)) and one for nitrogen regulation (PTS(Ntr)), that utilize proteins Enzyme I(sugar) (EI(sugar)) and HPr, and Enzyme I(Ntr) (EI(Ntr)) and NPr, respectively. The Enzyme I proteins have similar folds, as do their substrates HPr and NPr, yet they show strict specificity for their cognate partner both in stereospecific protein-protein complex formation and in reversible phosphotransfer. Here, we investigate the mechanism of specific EI(Ntr):NPr complex formation by the study of transient encounter complexes. NMR paramagnetic relaxation enhancement experiments demonstrated transient encounter complexes of EI(Ntr) not only with the expected partner, NPr, but also with the unexpected partner, HPr. HPr occupies transient sites on EI(Ntr) but is unable to complete stereospecific complex formation. By occupying the non-productive transient sites, HPr promotes NPr transient interaction to productive sites closer to the stereospecific binding site and actually enhances specific complex formation between NPr and EI(Ntr). The cellular level of HPr is approximately 150 times higher than that of NPr. Thus, our finding suggests a potential mechanism for cross-regulation of enzyme activity through formation of competitive encounter complexes.

Paralogous enzymes arise from gene duplication events that confer a novel function, although it is unclear how cross-reaction between the original and duplicate protein interaction network is minimized. We investigated HPr:EIsugar and NPr:EINtr, the initial complexes of paralogous phosphorylation cascades involved in sugar import and nitrogen regulation in bacteria, respectively. Although the HPr:EIsugar interaction has been well characterized, involving multiple complexes and transient interactions, the exact nature of the NPr:EINtr complex was unknown. We set out to identify the key features of the interaction by performing binding assays and elucidating the structure of NPr in complex with the phosphorylation domain of EINtr (EINNtr), using a hybrid approach involving X-ray, homology, and sparse nuclear magnetic resonance. We found that the overall fold and active-site structure of the two complexes are conserved in order to maintain productive phosphorylation, however, the interface surface potential differs between the two complexes, which prevents cross-reaction.

In a variety of bacteria, the phosphotransferase protein IIA(Glc) plays a key regulatory role in catabolite repression in addition to its role in the vectorial phosphorylation of glucose catalyzed by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The lactose permease (LacY) of Escherichia coli catalyzes stoichiometric symport of a galactoside with an H(+), using a mechanism in which sugar- and H(+)-binding sites become alternatively accessible to either side of the membrane. Both the expression (via regulation of cAMP levels) and the activity of LacY are subject to regulation by IIA(Glc) (inducer exclusion). Here we report the thermodynamic features of the IIA(Glc)-LacY interaction as measured by isothermal titration calorimetry (ITC). The studies show that IIA(Glc) binds to LacY with a Kd of about 5 μM and a stoichiometry of unity and that binding is driven by solvation entropy and opposed by enthalpy. Upon IIA(Glc) binding, the conformational entropy of LacY is restrained, which leads to a significant decrease in sugar affinity. By suppressing conformational dynamics, IIA(Glc) blocks inducer entry into cells and favors constitutive glucose uptake and utilization. Furthermore, the studies support the notion that sugar binding involves an induced-fit mechanism that is inhibited by IIA(Glc) binding. The precise mechanism of the inhibition of LacY by IIA(Glc) elucidated by ITC differs from the inhibition of melibiose permease (MelB), supporting the idea that permeases can differ in their thermodynamic response to binding IIA(Glc).

Pairwise interactions are intuitive to our understanding on protein-ligand binding; however in vivo this is rarely true. Most intracellular proteins operate in a highly cooperative manner to perform tasks ranging from metabolic turnover to intricate signaling regulation. In some cases, one substrate needs to simultaneously interact with more than one binding partner to carry out faithful signal transductions. While one of these binding partners is the determinant of such signal transduction, it can share very similar tertiary structure with the other but might differ in functional role and abundance. Motivated by this observation, we explore the physical consequences of the mere steric presence of a non-specific ligand, the “competitor”, crowding the surface of a “target” ligand. The specific interaction occurs between the protein and the “target” ligand, which explores the same surface as well, albeit for its unique binding site. A simple lattice model incorporating these elements along with the natural rules of exclusion and hopping reveals the regimes for when recruitment (turnover) or residence (transition state stabilization) are favored. Exploration of the search dynamics of the two ligands along the protein surface provides further insight.

Carbohydrate uptake in certain bacteria is under the regulation of the phosphotransfer protein IIAGlc. IIAGlc regulates the expression and activity of various inducible transporters, including the Na+-coupled melibiose permease (MelB) and H+-lactose permease (LacY). We characterized the thermodynamic features of IIAGlc binding to both the permeases by applying chemical cross-link and isothermal titration calorimetry. Here, we show that IIAGlc directly binds to MelB or LacY in the absence or presence of melibiose, a sugar substrate for both permeases. The protein-protein interaction in both cases exhibits dissociation constant between 3-10 μM at a stoichiometry of one. Strikingly, the thermodynamic strategy for the binding is different. The binding to MelB, which is crystallized in an outside-open conformation, is driven by enthalpy with compensation of entropy; the binding to LacY, which is captured in an inside-open conformation, is driven by entropy with compensation of enthalpy; however, the binding in both cases restrains the conformational entropy of the permease. Consistently, we show that IIAGlc-bound MelB or LacY exhibits a largely decreased affinity for sugar substrate. The data allow us to conclude that IIAGlc regulates activity of MelB and LacY via restraining conformational dynamics and an induced-fit process. In all cases studied, IIAGlc blocks inducer entry into the cells thus preferentially favoring constitutive glucose uptake.