In fact, detailed inspection of several conformational degrees of freedom for the protein showed that possible halogen bond-promoting conformations are not realized in the CK2a -TBBt complex, as illustrated by the His160 residue, for which a flip of the aromatic ring would have enabled interaction of Ne2 with a central halogen atom. The only halogen-bond promoting conformation was found for the p-Br interaction involving the Ne of Arg47 and a peripheral halogen atom of TBBt, as also found for one of the two protein molecules located in the crystal asymmetric unit. The propensity of the CK2a binding pocket to bind a halogenated ligand must be considered in terms of the topology of potential halogen-bond acceptors and the internal flexibility of the protein. We have performed a detailed analysis of all of 21 accessible structures of complexes of CK2 with ligands carrying at least one halogen atom. All of them were inspected using a 4 A ° threshold, to identify the distribution of distances between halogen atoms and proximal halogen bond acceptors. The resulting distribution of Acc…X distances revealed the existence of a broad local maximum corresponding to a normal distribution with a mean value of 3.34 A ° and a standard deviation of 0.28 A °. The substantial break observed for longer distances strictly corresponds to the sum of the VdW radii of dominant bromine and oxygen atom pairs, thus clearly setting the upper limit for eventual halogen bonding. It should also be emphasized that the normal distribution is somewhat perturbed, displaying a visible shoulder centered at,3.0 A °. Although, according to the Anderson-Darling test, the assumption of normality of the observed distance distribution could not be rejected, superposition with an additional normal distribution visibly improves the agreement between the model and the observed data. The additional extremely narrow distribution confirms overrepresentation of some types of donor-acceptor pairs. Interestingly, the potential donors of a strong halogen bond, with the sole exception of the Arg47 side-chain nitrogen engaged in the complex with TBBT, are oxygen atoms of Bortezomib either a backbone carbonyl or an isolated water molecule. Amongst theese, the preferred topology of C-X…Acc-C remains acceptor-specific, significantly differentiating between carbonyl and hydroxyl acceptors. Such a strong geometric preference for these interatomic interactions denotes them as halogen bonds, while the accompanying broad distribution describes a rather highly non-specific balance of VdW and electrostatic interactions. It should further be noted that the distribution of donor-acceptor distances remains identical for protein carbonyl and water hydroxyl oxygen atoms. This pattern of interactions is in accord with our previous simulations for a series of symmetrically substituted Bt and 5- substituted derivatives of Bt and 4,6,7-Br3Bt, and is also consistent with the interaction scheme proposed by Battistutta et al., with the exception of Val45, shown to make contacts preferentially with ligands bound in an orientation analogous to that of TBBz, but not that of TBBt. The order of importance of interactions controlling ligand activity agrees with our experimental IC50 data, as well as with the energy terms derived from molecular modeling. Our overall results point to hydrophobic interactions as the main force driving ligand-CK2a interactions, but electrostatic contributions also appear important. Thus, for isomers carrying the same number of halogen atoms on the benzene ring, the inhibitory activity depends on the location of bromination sites. Moreover, a general correlation is observed between pKa for dissociation of the triazole proton and inhibitory activity. On the other hand, TBBz is 4-fold less active than TBBt, pointing to the importance of the negatively charged triazole ring for efficient binding to CK2a. In this view, our finding that 5,6- Br2Bt is the most active dibromo isomer points to the role of hydrogen bonding properties of the triazole ring itself rather than the effect of the formal negative charge located on it. The foregoing is also supported by the moderate activity of 4,5,6,7-tetrachloro-benzotriazole, which is less active than TBBt, because substitution of bromine by chlorine decreases the hydrophobicity, but does not significantly change the pKa, and the low, but detectable, activity of 4,5,6,7-tetramethylbenzotriazole, which is much less polar, and in the neutral form at physiological pH. It may be concluded that the balance of hydrophobic and electrostatic interactions are the main forces driving the binding of brominated benzotriazoles to CK2a.