Structural features underlying selective inhibition of protein kinase CK2 by ATP site-directed tetrabromo-2-benzotriazole

Abstract

Two novel crystal structures of Zea mays protein kinase CK2alpha catalytic subunit, one in complex with the specific inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) and another in the apo-form, were solved at 2.2 A resolution. These structures were compared with those of the enzyme in presence of ATP and GTP (the natural cosubstrates) and the inhibitor emodin. Interaction of TBB with the active site of CK2alpha is mainly due to van der Waals contacts, with the ligand fitting almost perfectly the cavity. One nitrogen of the five-membered ring interacts with two charged residues, Glu 81 and Lys 68, in the depth of the cavity, through two water molecules. These are buried in the active site and are also generally found in the structures of CK2alpha enzyme analyzed so far, with the exception of the complex with emodin. In the N-terminal lobe, the position of helix alphaC is particularly well preserved in all the structures examined; the Gly-rich loop is displaced from the intermediate position it has in the apo-form and in the presence of the natural cosubstrates (ATP/GTP) to either an upper (with TBB) or a lower position (with emodin). The selectivity of TBB for CK2 appears to be mainly dictated by the reduced size of the active site which in most other protein kinases is too large for making stable interactions with this inhibitor.

Fig. 1.

Changes in loop 42–50 position induced by different ligands. Superimposition of Gly-rich loop (residues 42–50) and of helix αC of CK2α in presence of cosubstrate ATP (red), in the Apo-form (yellow), and with the inhibitors TBB (green) and emodin (cyan). The main difference is found in loop 42–50, while helix αC is perfectly superimposed in all four structures.

Fig. 2.

Electron density map of TBB molecule. The |2F_O−F_C| electron density map at 1 sigma contour level is shown around the inhibitor. Most significant residues and the two water molecules W1 and W2 discussed in the text are depicted. Hydrogen bonds and electrostatic contacts are indicated by broken lines. In the box the chemical formula and the atom numbering of TBB are illustrated.

Fig. 3.

(A) Two clipped views of the active site showing the inhibitor (as cpk model) fitting the cavity are shown. The molecular surface of the protein is represented as a white mesh. Bromine atoms are in red, carbon atoms in green, and nitrogen atoms in blue. (B) Position of TBB with respect to that of ATP (cyan) and emodin (green) in the catalytic site is shown from different points of view. Inhibitor rings lay practically in the same plane of the purine moiety of the natural cosubstrates (bottom).