There are ~500 kinases in the human genome, with specificity towards serine/threonine or tyrosine, (the histidine kinases, prevalent in bacteria, are excluded from our discussion) (Manning et al., 2002
). Most kinases have multiple regulatory domains that link kinase activity to unique signaling inputs and outputs (Pawson and Kofler, 2009
). In spite of this broad diversity, the phosphorylation reaction itself constitutes a highly conserved process, which is dependent on a set of structural features of the active site that are common to all kinases.
Much of what we know about the structure of the active state of protein kinases emerged from studies on c-AMP-dependent protein kinase (PKA), a serine/threonine kinase (Kornev and Taylor, 2010
). The active site is located between two lobes of the kinase domain (the N- and C-terminal lobes; ). In the case of PKA, a hinge motion changes the relative orientation of these two lobes from a more open state in the inactive conformation to a more closed state in the active conformation. The closure of the lobes results in the proper positioning of ATP and the substrate in the active site and is dependent on phosphorylation of a threonine residue (Thr 197; the residue numbering and secondary structure notation are from PKA, PDB code: 1ATP; this numbering will be used in the rest of the review, unless otherwise specified) in a centrally located activation segment or “loop” (Knighton et al., 1991
; Nolen et al., 2004
). The general features of the mechanism of PKA are modulated to yield the diverse set of regulatory mechanisms seen in other kinases (Huse and Kuriyan, 2002
Figure 1 Activation of protein kinases. (A) Crystal structure (PDB ID: 1ATP) and cartoon representation of the active state of the protein kinase A (PKA). The inset selectively displays the catalytically critical components of the kinase active site: the activation (more ...)
The key structural features required for catalysis were revealed by the analysis of the crystal structures of the active conformation of PKA and the insulin receptor tyrosine kinase bound to ATP (or ATP-analogs) and substrate peptide (or mimics) (Bossemeyer et al., 1993
; Hubbard, 1997
; Knighton et al., 1991
; Zheng et al., 1993
). These structures highlighted the importance of the activation loop in controlling the activation state of the kinase. The N-terminal region of the activation loop contains a conserved Asp-Phe-Gly (DFG) motif. The sidechain of the aspartate in the DFG motif points towards the phosphate groups of ATP and plays a critical role in coordinating a magnesium ion, which is required for ATP binding. The C-terminal region of the activation loop adopts an open conformation, serving as a platform for docking the substrate peptide (). In some kinases, such as the C-terminal Src kinase, Csk, substrate docking involves interactions elsewhere, and the C-terminal portion of the activation loop is disordered (Levinson et al., 2008
Another important structural element of the kinase active site is helix αC, in the N-lobe of the kinase, within which a conserved glutamate residue (Glu 91) is located. In the active conformation helix αC packs closely against the rest of the N-lobe of the kinase, allowing the glutamate residue to form a salt bridge with a conserved lysine residue (Lys 72) in strand β3 that coordinates the α- and β-phosphate groups of the substrate ATP molecule (). This lysine residue is commonly mutated to generate inactive forms of kinase domains (Robinson et al., 1996
). Two other important interactions in the active site involve the glycine-rich P-loop and the highly conserved HRD motif (YRD in PKA) located in the catalytic loop that directly precedes the activation loop. The glycine-rich P-loop is important for nucleotide binding in the active site by making interactions with the β and γ-phosphates of ATP. The HRD aspartate (Asp 166) serves as a catalytic base to accept the proton from the hydroxyl group of the substrate residue during the catalysis ().
Two highly conserved and functionally important intramolecular networks between the N-lobe and the C-lobe are correlated with the activity of protein kinases (Kornev et al., 2006
; Kornev et al., 2008
). These networks, or “spines”, involve hydrophobic residues that can be assembled or disassembled depending on the presence of ATP or the substrate peptide (). One of these, the “regulatory spine”, is involved primarily in substrate binding and its proper assembly depends on the conformation of the activation loop (Kornev et al., 2006
). There are four residues in the regulatory spine, Leu 106 in strand β4, Leu 95 in the catalytically important helix αC, Phe 185 in the conserved DFG motif and Tyr 164 in the conserved HRD motif. Upon activation, the change in activation loop conformation, which in most kinases is responsive to phosphorylation of the activation loop, changes the orientation of helix αC and the HRD motif, and results in the assembly of the regulatory spine. Alterations in the flexibility of the regulatory spine have been suggested to underlie the mechanism by which some of the frequently detected mutations in cancer patients mediate resistance to treatment with kinase inhibitors (Azam et al., 2008
The second spine, the “catalytic spine”, incorporates the adenine ring of ATP and establishes a connection between the N-lobe and the C-lobe upon nucleotide binding (Kornev et al., 2008
). The residues in the catalytic spine are located in helix αF (Met 231 and Leu 227) and helix αD (Met 128 and Leu 172) in the C-lobe, and the residues in the N-lobe: Ile 174 and Leu 173 in the β7 strand, Val 57 in β2 strand and Ala 70 in β3 strand. Helix αF also is also connected to the regulatory spine by an aspartate residue (Asp 220), which is highly conserved (Kornev et al., 2008
). Helix αF, which is the most buried helix in the structure, emerges as an essential structural element in kinases that integrates assembly of the two hydrophobic spines with kinase activation ().