Three-Dimensional Structure of CK2 Catalytic a-Subunit

The first three-dimensional structure of a catalytic subunit of CK2 is that from Zea mays, appeared in 1998 (Niefind et al. 1998). Due to the high degree of identity with the human enzyme, CK2 from maize has been considered a suitable model for all the subsequent inhibition studies. This hypothesis has been validated by the recent determination of the crystal structure of the human holoenzyme (Niefind et al. 2001) and by those of the human catalytic subunit and a mutant independently determined by two different groups (Ermakova et al. 2003; Pechkcova et al. 2003). In all these structures the a-subunit shows essentially the same features of that from maize, particularly as far as the active site is concerned.

The CK2 catalytic subunit bears most of the sequence and structural features common to all Ser/Thr kinase. Regarding the primary structure, two notable exceptions are: (a) in the Gly-rich loop (residues 46-51 in CK2) the third glycine (indicated by a vertical arrow in Fig. 1) in the consensus sequence GXGX0G is missing (0 is usually a tyrosine, as in CK2, or a phenylalanine); (b) at the beginning of the activation segment a tryptophan (residue 176 in CK2) substitutes a phenylalanine in the otherwise conserved three-peptide Asp-Phe-Gly (Fig. 1).

Another distinctive feature of CK2 is the presence of a basic cluster at the beginning of helix aC (residues 74-80), where 6 out of 7 consecutive amino acids are basic (5 lysines and one arginine). The presence of this basic cluster, also known as a substrate recognition site, is supposed to be in relation with the attitude of CK2 to phosphorylate highly acidic substrates with the minimal consensus sequence Ser/Thr-X-X-Asp/Glu (Meggio et al. 1994). Other basic amino acids present in the p+1 loop at the C-terminal end of the activation segment, namely Arg191, Arg195 and Lys198, have been found relevant for substrate recognition (Sarno et al. 1996).

The elucidation of the three-dimensional structure of the catalytic domain of CK2 has unveiled the origin of the intrinsic activity of the enzyme (Niefind et al. 1998), showing that CK2 bears all the same structural properties responsible for the active state of cAPK, taken as the prototype of an active Ser/Thr kinase (Engh and Bossemeyer 2002). In CK2 many of the N-ter-minal thirty residues make several hydrophobic and polar interactions with some other important regions of the protein, located both in the N-terminal and C-terminal domains, contributing to the stabilization of the active conformation. Of particular interest are the interactions with the activation segment, which consequently is constrained in an open active state (Fig. 2). These interactions are unique among the protein kinases whose structure is known, and indeed they occur between residues highly conserved in CK2 s from different species but not among other kinases.

The presence of the tryptophan-176 instead of a phenylalanine, distinctive of CK2, contributes to block the activation segment in the open active conformation; that because a hydrogen bond between the nitrogen of the tryptophan indole ring and the backbone carbonyl of Leu173 hampers the possibility of the rotation necessary for the transition from an inactive to an active conformation, possible if a phenylalanine is present instead.

The active conformation of the enzyme is granted also by the exact relative orientation of the N- and C-terminal lobes, by the proper position of the substrate and co-substrate anchoring and catalytic residues of the active site and by the correct orientation of the aC helix, spanning residues from 74 to 89. In particular, Glu81 contributes, through an ionic interaction, to orient Lys68 in the optimal position for the proper alignment of the ATP phosphates for catalysis. The importance of Lys68 and Glu81 (numbering is referred to CK2 sequence) is underlined by their conservation in all kinase sequences known to date.

In the hinge region of CK2, three are the residues involved in the nucleo-tides binding: Glu114, Val116 and Asn118. In the case of ATP, two hydrogen bonds between N1 and N6 of adenosine and backbone carbonyl of Glu114 and amide of Val116 are present; these interactions are typical of many kinases. In the case of GTP, atoms N1 and O6 of the purine moiety are hydrogen bound to the amide and the carbonyl of the backbone of Val116. Aspara-gine 118 is involved in the coordination of both co-substrates, ATP and GTP, also with the involvement of a water molecule mediating the interaction with the co-substrates. The dual co-substrate specificity is achieved by means of two well-structured water molecules that allow the switching between the different coordination modes of ATP and GTP through an hydrogen bond frame shift (Niefind et al. 1999).

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