Statistics and physical origins of pK and ionization state changes upon protein-ligand binding

Abstract

This work investigates statistical prevalence and overall physical origins of changes in charge states of receptor proteins upon ligand binding. These changes are explored as a function of the ligand type (small molecule, protein, and nucleic acid), and distance from the binding region. Standard continuum solvent methodology is used to compute, on an equal footing, pK changes upon ligand binding for a total of 5899 ionizable residues in 20 protein-protein, 20 protein-small molecule, and 20 protein-nucleic acid high-resolution complexes. The size of the data set combined with an extensive error and sensitivity analysis allows us to make statistically justified and conservative conclusions: in 60% of all protein-small molecule, 90% of all protein-protein, and 85% of all protein-nucleic acid complexes there exists at least one ionizable residue that changes its charge state upon ligand binding at physiological conditions (pH = 6.5). Considering the most biologically relevant pH range of 4-8, the number of ionizable residues that experience substantial pK changes (DeltapK > 1.0) due to ligand binding is appreciable: on average, 6% of all ionizable residues in protein-small molecule complexes, 9% in protein-protein, and 12% in protein-nucleic acid complexes experience a substantial pK change upon ligand binding. These changes are safely above the statistical false-positive noise level. Most of the changes occur in the immediate binding interface region, where approximately one out of five ionizable residues experiences substantial pK change regardless of the ligand type. However, the physical origins of the change differ between the types: in protein-nucleic acid complexes, the pK values of interface residues are predominantly affected by electrostatic effects, whereas in protein-protein and protein-small molecule complexes, structural changes due to the induced-fit effect play an equally important role. In protein-protein and protein-nucleic acid complexes, there is a statistically significant number of substantial pK perturbations, mostly due to the induced-fit structural changes, in regions far from the binding interface.

Figure 1

Flowchart of the overall computational methodology. (Thick color lines running from the top to the bottom of the diagram) Three procedures employed to obtain unligated protein structures. (Solid red lines) Overall procedure (1); (dashed green lines) electrostatics-only procedure (2); and (dotted blue lines) structural-changes-only procedure (3). (Thin horizontal lines at the bottom part of the diagram) Pairs of protein structures used to compute ΔpK values corresponding to each computational procedure.

Figure 2

Distance distribution of ionizable residues with substantial and biologically relevant pK change upon binding of the ligand in protein-protein, protein-small molecule, and protein-nucleic acid complexes. (Solid red bars) Overall procedure; (cross-hatched green bars) electrostatics-only procedure; and (striped blue bars) structural-changes-only procedure. The percentage reported for each region is relative to the total number of ionizable residues located in that region.

Figure 3

Distance distribution of ionizable residues with substantial pK change in the biologically relevant pH range. (Solid red bars) Overall procedure in protein-protein complexes; (open bars) false-positive pK changes due to structural noise. The percentage in each region is relative to the number of residues located in that region.