Electrostatic coupling to pH‐titrating sites as a source of cooperativity in protein‐ligand binding

Abstract

This paper describes an alternative mechanism for the cooperative binding of charged ligands to proteins. The ligandbinding sites are electrostatically coupled to protein side chains that can undergo protonation and deprotonation. The binding of one ligand alters the protein's protonation equilibrium in a manner that makes the the binding of the second ligand more favorable. This mechanism requires no conformational change to produce a cooperative effect, although it is not exclusive of conformational change. We present a theoretical description of the mechanism, and calculations on three kinds of systems: A model system containing one protonation site and two ligand-binding sites; a model system containing two protonation sites and two ligand-binding sites; and calbindin Dgk, which contains two Ca2+-binding sites and 30 protonation sites. For the one-protonation-site model, it is shown that the influence of the protonation site can only be cooperative. The competition of this effect with the anticooperative effect of ligand-ligand repulsion is studied in detail. For the two-protonation site model, the effect can be either cooperative or, in special cases, anticooperative. For calbindin Dgkr the calculations predict that six protonation sites in or near the ligand-binding sites make a cooperative contribution that approximately cancels the anticooperative effect of Ca2+-Ca2+ repulsion, accounting for more than half of the total cooperative effect that is needed to overcome repulsion and produce the net cooperativity observed experimentally. We argue that cooperative mechanisms of the kind described here are likely when there is more than one ligand-binding site in a protein domain.Keywords: allostery, calbindin Dgk cooperativity, electrostatics, linked equilibrium, multisite titration, pH titration of proteins, pK, values in proteins This paper demonstrates that cooperativity in the binding of charged ligands by proteins can arise from electrostatic couplings of the ligand-binding sites to sites of protonation/deprotonation, such as the ionizable side chains of the protein. We provide a theoretical framework for cooperative effects of this kind, and we present calculations on some simple model systems and on the Ca2+-binding protein, calbindin Dgk. In contrast to traditional models of cooperativity (Monod et al., 1965;Koshland et al., 1966), conformational changes in the protein are not required in our model, and therefore are not included in the present calculations. We study the conditions that cause the effects of electrostatic coupling to protonation sites to be cooperative or anticooperative, and of greater or lesser magnitude. The conditions under which this mechanism can produce a cooperative effect strong enough to overcome the anticooperative effect of direct ligand-ligand repulsion are given particular attention.We believe that effects of the kind described here are present in many cases of cooperative binding of charged ligands to proteins, Reprint requests to: Donald Bashford, Department of M...

Coupling of Protonation, Reduction, and Conformational Change in azurin from Pseudomonas aeruginosa Investigated with Free Energy Measures of Cooperativity

“…A decrease of the effective repulsion between some pairs of protonation reactions can arise from the release of protons from nearby third sites. 19,75,76 This effect can even lead to an apparent attractive interaction between protons bound to pairs of interacting protonatable sites as in the case of His-35 and Asp-11 or Lys-74 and Arg-79. Also the favorable effective interaction between the protonation of almost all sites and the reduction of the copper center could be expected on the basis of the attractive electrostatic interaction between the complementary charges of the ligands.…”

Electrostatic correlations: from plasma to biology

“…While these models are often sufficient to grasp the underlying physics, it is quite easy to push the models too far. This is particularly the case when one deals with specific structural properties of biomolecules [226,227,228]. As soon as the length scales on the order of few angstroms become important, approximation of water as a uniform dielectric medium is no longer sufficient [229,230].…”

Acid destabilization of the solution conformation of Bcl‐XL does not drive its pH‐dependent insertion into membranes

“…Typically, such changes in protonation state are accompanied by conformational changes; however, as demonstrated here, such conformational changes may not derive from protonation alone. Indeed, pH dependent cooperativity can be quantitatively described without invoking conformational changes for cases when strong electrostatic interactions exist between ligand binding sites and protonation sites (Spassov and Bashford 1998). It appears the Bcl-x L may be an example of this phenomenon.…”