located in helix αC. Subsequent crystal structures showed that helix αC is flexible
and can dislodge from the active site (αC out). The VIAK lysine/αC glutamate
salt bridge is therefore a hallmark of the active kinase conformation. (4) A tripeptide
motif DFG (Asp-Phe-Gly) is located between strands β8 and β9 in the C-lobe
and marks the beginning of the activation segment (A-loop). The conserved Asp
interacts with Mg
2+ itself coordinating to the ATP phosphates [18].
Protein kinases phosphorylate their substrates on Ser/Thr or Tyr residues
after binding of substrate sequences to the substrate binding site in an extended
conformation. The substrate interaction site can be described as a shallow surface
groove that is formed by side chains located in the four main C-terminal lobe helices
(αD–αH) as well as the A-loop APE (Ala-Pro-Glu) motif. Usually the A-loop is
20–30 residues long, and it is usually unstructured in kinases that are regulated
by A-loop phosphorylation [19]. The unstructured inactive A-loop conformation
partially impedes substrate interactions, but does not necessarily affect substrate
binding [20]. Phosphorylation of the A-loop locked this flexible motif in a defined
conformation resulting in kinase activation as described below.
A hallmark of the active kinase conformation is the accurate spatial
arrangement of the conserved catalytic domain motifs for efficient catalysis.
The active conformation is therefore structurally well-defined as exemplified for
CDK2 in Fig. 1b. Active protein kinases harbour a highly conserved Y/HRD
(Tyr/His-Arg-Asp) motif between strand β6 and β7. While some variations of
the Y/HRD motif exist in active kinases, the aspartate of this motif is strictly required
for catalytic activity of the phosphoryl transfer reaction. The Tyr/His in Y/HRD
typically forms hydrogen bond interactions with the DFG backbone linking
these two key elements. A salt bridge network between Y/H in Y/HRD to the
phosphate moiety of the activation loop phosphorylation site (pThr160 in CDK2)
stabilizes the activation segment and further links this segment to the catalytic loop.
The “R” in the HRD motif is not strictly conserved. The presence of this residue has
been thought to be indicative of the requirement of A-loop phosphorylation
(so-called RD kinases) which seems not always be the case [21]. However,
in RD kinases the polar interactions of the HRD arginine with the A-loop
phosphorylation site largely contribute to the conformation and stability of the
A-loop. Finally, the Asp (D127 in CDK2) of the HRD motif acts as a catalytic
base deprotonation the hydroxyl group of the peptide substrate Ser/Thr or
Tyr residue promoting the nucleophilic attack onto the γ-phosphate of ATP.
The Asp is further stabilized by a conserved hydrogen bond with an Asn from the
activation loop (N132 in CDK2) [22].
Overall the kinase active state is characterized by a structured A-loop with
a “DFG-in” conformation and an αC-helix in closed proximity to ATP site.
A characteristic canonical salt bridge between the VIAK motif lysine and the
conserved αC glutamate residue stabilizes the active form. Normally the N- and
C-lobes adopt a closed conformation with a structured P-loop, although some of
these structural features may not be present in crystal structures of active kinases.
4
S. Röhm et al.
and can dislodge from the active site (αC out). The VIAK lysine/αC glutamate
salt bridge is therefore a hallmark of the active kinase conformation. (4) A tripeptide
motif DFG (Asp-Phe-Gly) is located between strands β8 and β9 in the C-lobe
and marks the beginning of the activation segment (A-loop). The conserved Asp
interacts with Mg
2+ itself coordinating to the ATP phosphates [18].
Protein kinases phosphorylate their substrates on Ser/Thr or Tyr residues
after binding of substrate sequences to the substrate binding site in an extended
conformation. The substrate interaction site can be described as a shallow surface
groove that is formed by side chains located in the four main C-terminal lobe helices
(αD–αH) as well as the A-loop APE (Ala-Pro-Glu) motif. Usually the A-loop is
20–30 residues long, and it is usually unstructured in kinases that are regulated
by A-loop phosphorylation [19]. The unstructured inactive A-loop conformation
partially impedes substrate interactions, but does not necessarily affect substrate
binding [20]. Phosphorylation of the A-loop locked this flexible motif in a defined
conformation resulting in kinase activation as described below.
A hallmark of the active kinase conformation is the accurate spatial
arrangement of the conserved catalytic domain motifs for efficient catalysis.
The active conformation is therefore structurally well-defined as exemplified for
CDK2 in Fig. 1b. Active protein kinases harbour a highly conserved Y/HRD
(Tyr/His-Arg-Asp) motif between strand β6 and β7. While some variations of
the Y/HRD motif exist in active kinases, the aspartate of this motif is strictly required
for catalytic activity of the phosphoryl transfer reaction. The Tyr/His in Y/HRD
typically forms hydrogen bond interactions with the DFG backbone linking
these two key elements. A salt bridge network between Y/H in Y/HRD to the
phosphate moiety of the activation loop phosphorylation site (pThr160 in CDK2)
stabilizes the activation segment and further links this segment to the catalytic loop.
The “R” in the HRD motif is not strictly conserved. The presence of this residue has
been thought to be indicative of the requirement of A-loop phosphorylation
(so-called RD kinases) which seems not always be the case [21]. However,
in RD kinases the polar interactions of the HRD arginine with the A-loop
phosphorylation site largely contribute to the conformation and stability of the
A-loop. Finally, the Asp (D127 in CDK2) of the HRD motif acts as a catalytic
base deprotonation the hydroxyl group of the peptide substrate Ser/Thr or
Tyr residue promoting the nucleophilic attack onto the γ-phosphate of ATP.
The Asp is further stabilized by a conserved hydrogen bond with an Asn from the
activation loop (N132 in CDK2) [22].
Overall the kinase active state is characterized by a structured A-loop with
a “DFG-in” conformation and an αC-helix in closed proximity to ATP site.
A characteristic canonical salt bridge between the VIAK motif lysine and the
conserved αC glutamate residue stabilizes the active form. Normally the N- and
C-lobes adopt a closed conformation with a structured P-loop, although some of
these structural features may not be present in crystal structures of active kinases.
4
S. Röhm et al.