The protein kinase catalytic domain plays three fundamental roles: binding and orientation of the substrate peptide; binding and orientation of the ATP (or GTP) as a complex with a cation (Mg2+ or Mn2+); and transfer of the -phosphate from the ATP (or GTP) to the acceptor hydroxyl group of a serine, threonine or
tyrosine residue. There is no need for binding first substrate or ATP, but there is a thermodynamic preference for the nucleotide (Cook et al., 1982; Grant and Adams, 1996). The phosphorylated residue in the substrate peptide,
“phosphosite” (P-site), needs to be framed by a consensus recognition sequence with affinity for the catalytic site (Chan, Hurst and Graves, 1982; Pearson and Kemp, 1991).
The protein kinase catalytic domain structure (250-300 amino acids) is common to the entire eukaryotic tree including animals, plants, fungi and protozoa (Taylor and Radzio-Andzelm, 1994). It has 12 subdomains characterised by patterns of conserved residues nearly invariant throughout the protein kinase superfamily, these subdomains are never interrupted by large amino acid
insertions and fall into topologically similar core structures. The variable non- conserved insertions build up non-catalytically relevant features of the protein surface. The non-catalytic domain can modulate activity interacting with the protein kinase core, or binding other proteins that enhance or repress the catalytic function (Pawson and Gish, 1992; Newton, 1997; Sicheri and Kuriyan, 1997; Brushia and Walsh, 1999). Other factors affecting activation are fatty acid acylation (Blenis and Resh, 1993; Johnson and Cornell, 1999), isoprenylation (Inglese and Premont, 1996; Pitcher, Freedman and Lefkowitz, 1998), second messengers (Rasmussen, 1989; Liscovitch and Cantley, 1994; Lucas et al., 2000), subcellular localization (Pawson and Scott, 1997; Garrington and Johnson, 1999; Scott and Pawson, 2000), and phosphorylation (Shenolikar, 1988; Hunter, 1995). The first crystal structure of a catalytic protein kinase domain available was a mouse PKA with a substrate (Ben-David et al., 1991; Rossomando et al., 1992), soon obtained also with the complexes ATP-Mn2+ and ATP-Mg2+ (Pearson and Kemp, 1991). As new structures of other protein kinases were published it was confirmed that the topology of the catalytic domain was well conserved in eukaryotes. It is an overall two-lobe structure with a small NH2-terminal lobe
comprising subdomains I-IV, and a large COOH-terminal lobe including domains VIa –XI (Figure 1-10A and C). The hinge region is constituted by subdomain V, which protrudes towards both lobes creating the catalytic cleft. The NH2-
terminal lobe folds predominantly in antiparallel -sheet secondary structure and its main function is anchoring and orientation of the ATP (or GTP). The COOH-terminal lobe is mainly -helix configuration and is responsible for the phosphotransfer initiation. While the ATP nests deep into the pocket, the substrate remains in the periphery (Adams, 2001).
Figure 1-10. Protein kinase catalytic domain.
A. Ribbon diagram of PKA co-crystallized with ATP and a peptide inhibitor. NH2-
terminal lobe (mainly -sheet) and COOH-terminal lobe (predominantly -helix) are highlighted. Arrows point to the activation loop and its P-site, pThr-197, required for protein kinase activation. B. Key residue interactions in the active site of PKA. Mg1 and Mg2 represent the activating and inhibitory divalent metal ions, Mg2+, respectively (schematic representation based on A). Dotted lines indicate close contacts under 2.6 Å but are not drawn to scale. The hydroxyl of the substrate is shown based on a binary complex of PKA with a substrate peptide. C. ePK catalytic domain indicating the 12 conserved subdomains
(Roman numerals). Positions of amino-acid residues and motifs highly conserved throughout the ePK superfamily are indicated above, using single-letter amino- acid code with x as any amino acid. A and B, adapted from Adams 2001. C, adapted from Hanks 2003.
The structure-activity relationship of the different conserved motives has been reviewed in the literature (Hanks and Hunter, 1995; Adams, 2001). Using as reference residues in PKA, subdomain I constitutes a clamp anchoring the non- transferable phosphates on the ATP through H-bonds with Ser53, Phe54 and Gly55. Leu49 and Val47 form a hydrophobic space to fit the adenine ring. There is a consensus sequence throughout most of protein kinases at this domain: Gly50-x-Gly-x-x-Gly-x-Val.
The main feature at subdomain II is Lys72, a residue essential for maximal activity whose substitution is commonly used to produce inactive mutants (Hixson and Krebs, 1979; Hathaway, Zoller and Traugh, 1981; Buhrow et al., 1983; Weinmaster, Zoller and Pawson, 1986). It does not affect ATP binding but
NH2-lobe
COOH-lobe
A
B
is critical for correct orientation of the nucleotide. Lys72 forms a salt bridge with the invariable Glu91, which helps orientation and anchoring of ATP in the large -helix of subdomain III, Figure 1-10B.
Subdomain IV and VIa are apparently not involved in catalysis and do not have any invariable residues. Subdomain V articulates the hinge region with half - helix and half -sheet. The consensus Glu121, Val123, and Glu127, in the joint, anchor ATP forming H-bonds with adenine and the ribose ring. Met120, Tyr122 and Val123, also help forming a hydrophobic pocket. Glu127 forms in addition an ion pair with an arginine in the peptide recognition sequence.
Subdomain VIb contains the “catalytic loop”, the consensus His164-Arg-Asp-Leu- Lys-x-x-Asn. Asp166 may accept the proton from the substrate hydroxyl group, while Lys168 (sometimes an Arg) neutralize the negative charge of the
phosphate during the transference. Asp166 also stabilize the loop establishing a H-bond with Asn171, which chelates the Mg2+ that bridges - and -phosphates,
Figure 1-10B. Glu170 forms a H-bond with the ribose and an ion pair with an Arg in the peptide recognition consensus.
Subdomain VII has the consensus Asp184-Phe-Gly. Asp184 is essential as the kinase is not functional when replaced (Gibbs and Zoller, 1991). It forms an H- bond with Gly186 and chelates Mg2+ between - and -phosphates, assisting ATP orientation, Figure 1-10B.
Containing many peptide binding residues and a hydrophobic pocket made of Leu198, Cys199, Pro202 and Leu205, subdomain VIII has the characteristic
consensus Ala206-Pro207-Glu208 (APE). Frequently this domain contains residues that require phosphorylation before the protein kinase becomes active (Chan, Hurst and Graves, 1982; Luo, Zhou and Lodish, 1995; Songyang et al., 1995). Subdomain IX, contains the conserved Asp220 that forms H-bonds with Arg165 and Tyr164 in the “catalytic loop” contributing to its stability and to substrate recognition. Subdomain X is the less conserved, with all CMGC kinases presenting large insertions. Finally, subdomain XI has the invariable His-x-Aromatic-
Hydrophobic-10x-Arg280. This last residue forming an ion pair with Glu208 in the APE motive of subdomain VIII.