Competition between electrostatic interactions and halogen bonding in the protein–ligand system: structural and thermodynamic studies of 5,6-dibromobenzotriazole-hCK2α complexes

Abstract

CK2 is a member of the CMGC group of eukaryotic protein kinases and a cancer drug target. It can be efficiently inhibited by halogenated benzotriazoles and benzimidazoles. Depending on the scaffold, substitution pattern, and pH, these compounds are either neutral or anionic. Their binding poses are dictated by a hydrophobic effect (desolvation) and a tug of war between a salt bridge/hydrogen bond (to K68) and halogen bonding (to E114 and V116 backbone oxygens). Here, we test the idea that binding poses might be controllable by pH for ligands with near-neutral pKa, using the conditionally anionic 5,6-DBBt and constitutively anionic TBBt as our models. We characterize the binding by low-volume Differential Scanning Fluorimetry (nanoDSF), Isothermal Calorimetry (ITC), Hydrogen/Deuterium eXchange (HDX), and X-ray crystallography (MX). The data indicate that the ligand pose away from the hinge dominates for the entire tested pH range (5.5–8.5). The insensitivity of the binding mode to pH is attributed to the perturbation of ligand pKa upon binding that keeps it anionic in the ligand binding pocket at all tested pH values. However, a minor population of the ligand, detectable only by HDX, shifts towards the hinge in acidic conditions. Our findings demonstrate that electrostatic (ionic) interactions predominate over halogen bonding.

Effect of histidine protonation state on ligand binding at the ATP-binding site of human protein kinase CK2

“…However, a slight opposite trend was observed for TBBt binding, for which anionic form predominates at all tested pH. We had assigned this apparent variation in TBBt binding by WT protein to the pH-induced (de)protonation of the neighboring histidine residue (putatively His160, with a theoretical pK a of ~ 7) 18 . To confirm this hypothesis, we analyzed a series of DSF data collected at different pH for both histidine mutants against the values previously reported for the WT protein (Table 4 and Fig.…”

“…Our recent study demonstrated that pH-dependent binding of 5,6-Br 2 Bt is due to forced ligand deprotonation upon the binding event 18 . However, a slight opposite trend was observed for TBBt binding, for which anionic form predominates at all tested pH.…”

“…According to the analysis of available structures of halogenated ligands with CK2, neutral ligands, irrespective of the organism (here maize or human), bind closer to the hinge, possibly forming halogen bonds with the backbone carbonyl oxygen atoms of residues from the hinge region 18 , 40 . However, for anionic ligands, there are some slight differences depending on the origin of CK2α.…”

“…We use the catalytic domain of human protein kinase CK2 (hCK2α) as the model protein in ligand binding studies 18 – 20 . CK2 is a serine/threonine protein kinase that plays an essential role in many processes, including cell growth, differentiation, death, and survival, by controlling several signaling pathways 21 .…”

“…Our recent studies demonstrated that electrostatic interactions predominate over possible halogen/hydrogen bonding with the hinge region of human CK2α 41 . We showed that the apparent binding affinity of titratable 5,6-dibromobenzotriazole (5,6-Br 2 Bt; pK a = 6.93) 42 to hCK2α varies non-linearly with pH, and the inflection point identifies the dissociation of the triazole proton upon ligand binding 18 . However, a slight inverse trend of pH-dependent changes was observed for 4,5,6,7-tetrabromobenzotriazole (TBBt), in which anionic form predominates in the tested pH range.…”