Info

General Structural Features of Ser/Thr Protein Kinases Catalytic Domain

Ser/Thr protein kinase catalytic domains are structurally well conserved throughout evolution, from prokaryotes to mammals, while their amino acid sequences show a much lower degree of similarity (Manning et al. 2002a; Young et al. 2003). Nevertheless, residues essential for the function of the enzyme are conserved in all kinases that invariably share some characteristic properties (Hanks and Hunter 1995). Figure 1 shows an alignment between the sequences of cAPK, that is often considered as the reference prototype kinase, and the CK2 catalytic a-subunit from human and Zea mays, with the most important residues highlighted.

The overall three-dimensional structure comprises both b-strands and a-helices, with a large predominance of the former in the N-terminal lobe and of the latter in the C-terminal one (Fig. 2).

Between the two lobes an essentially hydrophobic deep cleft is formed, where the co-substrate (ATP or, less frequently, GTP) can bind. The two lobes are connected by a segment known as the hinge region, where the adenine moiety of ATP can be anchored by means of some hydrogen bonds.

In order to be active, the conformation of a kinase must guarantee the access of cosubstrate and substrate to the catalytic zone and the correct arrangement of the catalytic and binding residues; in particular, the proper orientation of ATP is crucial for catalysis. The binding of the ATP phosphate tail to the enzyme is favored by two metal ions, usually magnesium, that are coordinated by an asparagine residue of the so-called catalytic loop (Asn161 in CK2, 171 in cAPK) and an aspartate at the beginning of the activation segment (Asp175 in CK2, 184 in cAPK).

Two other structurally conserved important zones in kinases are the Gly-rich loop, or p-loop, and the helix aC (Fig. 2); the Gly-rich loop contributes

Fig. 1 Sequence alignment of cAPK and CK2 catalytic a-subunits from human and maize. Vertical arrows mark important residues not conserved between cAPK and CK2, as discussed in the text. The structurally and functionally important regions of CK2 are indicated. Numbering in the upper line refers to human CK2 sequence. Black background, residues identical in all three sequences; gray background, residues identical in only two sequences

Fig. 1 Sequence alignment of cAPK and CK2 catalytic a-subunits from human and maize. Vertical arrows mark important residues not conserved between cAPK and CK2, as discussed in the text. The structurally and functionally important regions of CK2 are indicated. Numbering in the upper line refers to human CK2 sequence. Black background, residues identical in all three sequences; gray background, residues identical in only two sequences to strengthen the ATP anchoring, while the orientation of the aC helix is essential for the activity of the enzyme. In Ser/Thr kinases that present inactive and active forms, as in the case of cAPK or CDK2, helix aC has been found with an orientation substantially different in the two arrangements (Jeffrey et al. 1995). So, when a transition from an inactive to an active structure occurs, there is usually a rearrangement of the relative orientation of the two lobes, to properly position essential residues of the catalytic site (for instance those corresponding to Lys72 and Asp166 in CDK2) and a concomitant correct positioning of the aC helix in the N-terminal domain. Another important structural change occurring for the activation is represented by the displacement of the activation segment, a long loop in the C-terminal domain, that moves away from the close position of the inactive form to an open one (Fig. 2), allowing the binding of the substrate and co-substrate to

Fig. 2 Three-dimensional structure of Apo-CK2. Regions important in all Ser/Thr kinases are indicated. The N-terminal region (upperpart) is rich in b-sheets while the C-terminal one (lower part) is rich in a-helical structure. ATP binds in the cleft between the two lobes, adjacent to the Gly-rich loop and the hinge region. The activation segment adopts an open active conformation

Fig. 2 Three-dimensional structure of Apo-CK2. Regions important in all Ser/Thr kinases are indicated. The N-terminal region (upperpart) is rich in b-sheets while the C-terminal one (lower part) is rich in a-helical structure. ATP binds in the cleft between the two lobes, adjacent to the Gly-rich loop and the hinge region. The activation segment adopts an open active conformation the enzyme (Johnson et al. 1996). This movement can be caused by a phosphorylation event, as in the case of cAPK when Thr197 is phosphorylated, sometimes coupled with an interaction with e regulatory subunit, as in the case of CDK2 with cyclin. The activation segment displacement is made possible principally by a rotation of a short stretch of three amino acids positioned at the beginning of the loop; this tripeptide, Asp-Phe-Gly, is highly conserved in kinases but not in CK2, where the phenylalanine is substituted by a tryptophan at position 176 (indicated by a vertical arrow in Fig. 1). The C-terminal end of the activation segment, the p+1 loop, is responsible for substrate recognition and binding.

It must be noted, however, that not all kinases show a dramatic transition from an active to an inactive form as exhibited by CDK2 and cAPK. As already mentioned, CK2 is one of the few examples being found solely in the active form.

Was this article helpful?

0 0

Post a comment