cAMP_Dependent

cAPK is one of the simplest protein kinases due in part to its small size (350 residues). It has a molecular weight of 43643.8 Da and an isoelectric point of 8.95 as determined from ExPASy, a collection of bioinformatics databases capable of calculating may protein parameters (3). The biological significance of cAPK lies in a central feature of protein kinases. Protein kinases are enzymes responsible for regulating phosphorylation of a very large number of molecules and proteins. Though they are a very diverse family of proteins, they share the same catalytic core (2), which is also found in the catalytic subunit of cAMP Dependent Protein Kinase. Since the catalytic subunit is very simple in structure and is crystallized easily, it has become a very good model protein for studying the properties of the catalytic site that is conserved in all protein kinases (2, 4).

The secondary structure of the catalytic subunit can be split with a cleft into two distinct lobes. There is a small lobe associated with the N terminus of the protein, and a large lobe associated with the C-terminus of the protein. The N-terminus of the molecule contains an amphipathic alpha helix, however the exact residues making up the helix were not determined from the crystal structure (4). The N-terminus portion lies near the surface of the large lobe and is mainly hydrophobic indicating a potential site for a myristol group, attached to N-terminal glycine, which is seen in similar mammalian enzymes (4). The small lobe (residues 40-125) consists of five antiparallel beta sheets at the core with two alpha helices, separated by a few residues, wedged between two of the beta sheets (4) and is related mainly to MgATP binding. The adenine ring of the ATP molecule is enclosed within a hydrophobic pocket of the small lobe with the primary hydrogen bond being that between the backbone carbonyl of Glu-121 and the N6 amino group of ATP (2). A dramatic loss in binding is seen, if the N6 amino group is replaced with a hydroxyl group, indicating that the hydrogen bond between the N6 amino and Glu-121 is the primary bond holding the adenine ring in place (2). The primary phosphate that is used to phosphorylate peptides is the gamma phosphate which explains why the small loop has mechanisms to hold in place the alpha and beta phosphates. Lys-72 and Glu-91 together hold the alpha phosphate in place, while Lys-72 also interacts with the beta phosphate along with the amide groups of Gly-55 and Phe-54 (2). In the overall tertiary structure, the ATP molecule lies just underneath one of the beta sheets with the adenine ring near the base of the cleft under the beta sheet, and the phosphate groups facing out (4). This orientation is consistent with the observation that the gamma phosphate, or the outermost phosphate on ATP, is the one that is actually transferred during catalysis.

The large lobe is involved in the actual catalysis as well as much of the peptide recognition aspects of cAPK. The secondary structure of the larger lobe is primarily alpha helical with seven alpha helices. Two helices, both of which are hydrophobic, run antiparallel through the core of the lobe (4). In addition, there are also four antiparallel beta strands on the surface of the cleft between the two lobes (4). Residues 281-350 run the whole length of the structure and the residues near the cleft of cAPK participate in recognition of both ATP and the peptide substrate. Residues 120-127, which link the two lobes together, also participate in peptide recognition (4). Cys-199 interacts with the gamma phosphate of ATP in the cleft, and modification of this residue results in significant loss of MgATP binding (4). The major feature of the large lobe is the catalytic loop, consisting of Arg-165, Asp-166, Leu-167, Lys-168, Pro-169, Glu-170, and Asn-171, which facilitates the actual catalytic event, including transfer of the phosphate group as well as positioning of the peptide substrate. This loop is present between two adjacent beta sheets, of four total beta sheets, in the large lobe (5). Lys-168 interacts with the gamma phosphate of ATP in order to put it in the correct orientation for catalysis, while Asn-171 can interact with the alpha carbonyl of Asp-166, which is the catalytic base in the reaction (2,4). Thus, the catalytic loop facilitates both the locking in place of the gamma phosphate group as well as the catalytic base. The side chain of Asp-166 is near the beta carbon of the Ala residue at the phosphorylation position in the inhibitor complex. If the inhibitor was not present, then a hydroxyl group on a peptide would be present to serve as the phosphate acceptor (2).

The final part of the crystallized cAPK structure is the 20 residue peptide inhibitor (PKI). The N-terminus of the inhibitor from Thr-1 to Ala-8 forms an amphipathic alpha helix (5). Additionally, there are two Gly-Arg residues at positions 10/11 and 13/14 which may serve as binding sites for PKI to the catalytic subunit (5). The rest of the protein is an extended conformation and contains the region for substrate recognition (5). The hydrophobic portion of the amphipathic helix buries itself in the hydrophobic core of the large lobe of the catalytic unit and can be used to explain the extremely high affinity binding of PKI to the catalytic subunit (5). The residue at the 17 position in the inhibitor would act as the site of phosphorylation. This residue is near Asp-166, the catalytic base, in the catalytic subunit (2,4,5). Recognition of the peptide near the catalytic site is facilitated by electrostatic interactions between hydrophilic residues, while interactions far from the site are facilitated by hydrophobic interactions between alpha helices. Specifically concerning PKI, important residues in the extended region are Arg-14 and Arg-15 (5). The guanidium nitrogen in Arg-14 in PKI is fixed in place by interactions with Glu-127, Glu-331, and Asp-329 along with the hydroxyl groups in the ribose ring in ATP. Multiple residues on the catalytic subunit fix Arg-15 as well (5). Proper orientation of the residue at the phosphorylation site is also dependent upon a hydrophobic residue directly adjacent on the C-terminal side (5). The alpha helices of the catalytic subunit are involved in peptide recognition mainly due to their ability to be amphipathic and thus have a hydrophobic core that can interact with hydrophobic residues on the peptide (5). In contrast, the beta sheets on the catalytic unit seem to mainly be involved in the catalysis itself due to their abilities to house the catalytic loop and the MgATP binding site in the beta turns while leaving adequate room for the ligands and substrates to bind (5).

There are two ligand components to cAPK: ATP and Mn2+. In the natural form, magnesium ions are actually present (2, 4), however in one instance, Manganese (II) was used in order to better crystallize cAPK, since it binds much more tightly to the enzyme (1). The role of the ATP has been discussed in detail above, however the two metal ions also play a significant role in catalysis. The primary magnesium ion site is located near the beta and gamma phosphates on ATP where it holds the gamma phosphate in place for catalysis (1). The second magnesium ion also interacts with the ATP molecule, however it serves as an inhibitor as it binds ADP more tightly and does not allow for the release of the products once the reaction is complete(1).

Protein Kinase B/Akt (1GZN) from Homo sapiens, or simply PKB, is another protein kinase in the same family as cAPK that has many conserved structural features albeit a different function (6). Dali Server generated a list of proteins similar in tertiary structure to cAPK and gave PKB a Z-score of 31.1 (7) The Dali Server compares intramolecular distances in order to find proteins with similar tertiary structures. A Z-score greater than two means that the given protein has a similar tertiary structure to the protein of interest. A protein specific integrated basic local alignment search tool query (PSI-BLAST) of cAPK gave PKB an E-value of 1e-86 (8). PSI-BLAST compares the sequences of proteins in order to find those with homologous sequences. It finds gaps in sequence homology and quantifies them into an e-value. As sequence homology increases, the E-value decreases and an E-value of less that 0.05 indicates that the protein has significant homology to the protein of interest. Functionally, cAPK and PKB are quite similar in they are protein kinases involved in phosphotransfers, however PKB differs slightly in its secondary and tertiary structure (6). The tertiary structure of PKB can be split into two separate lobes just as cAPK, however the small lobe of PKB has less helical characteristics than cAPK. While cAPK has three helices, two in the actual catalytic portion of the molecule, and one at the N-terminus whose surface is mainly hydrophobic (2, 4), PKB lacks the amphipathic helix at the N-terminus, and the two other helices in the small lobe are not as large as in cAPK (6). A major feature of PKB that distinguishes both its function and its activity from cAPK is that PKB must be phosphorylated at two sites in order to be active: Thr-309 and Ser-474. cAPK cannot be regulated in this way, and thus the activity of PKB can be turned on and off. Ser 474 phosphorylation occurs in a hydrophobic region of the molecule which promotes the formation of an interaction between two hydrophobic portions of the molecule, causing a comformational change (6). The new conformation causes an alpha helix to form in the small lobe of PKB (6). The sequence homology (8) of cAPK and PKB can be explained by the fact that they are both protein kinases. As was previously discussed, all protein kinases in the same family as cAPK share the same catalytic core (2). PKB, is a slightly larger protein due to extra regulatory units (6), but still contains a very similar catalytic loop and ATP binding mechanism as cAPK (2).