DFG motif

Another very important conserved domain in kinases is the activation loop. This 20-30 amino acid region is positioned between a highly conserved DFG and APE motif (IDA region, inter DFG-APE region). In its activated state the activation loop is in an open, extended conformation, which allows substrate binding to the kinase. The aspartate residue (Asp-145) of the DFG motif interacts with one of the two magnesium ions in the active site. 

In the absence of cyclin-A, the C helix is twisted and the conserved Glu-51 residue on its surface faces the solvent and is unable to coordinate with Lys-33, which instead coordinates with Asp-145. The torsion angles of Phe-146 and Asp-145 in the DFG motif are typical for an inactive kinase [15] (Table 7.2) and show that the orientation of Asp-145 is unfit for catalysis. 

In some cases, mutation of surface or active site residues has been required to improve stability and solubility. Mutation of surface cysteines prevented the formation of disulfide-linked aggregates in FGFRl [54]. Mutation of highly conserved, active site residues, to create a kinase-dead mutant, was required for a number of kinases whose over-expression proved to be toxic to cells. The conserved aspartic acid in the DFG motif was mutated to an asparagine in CDK5 while the conserved lysine in PAR was mutated to an arginine [48, 51]. 

Pseudokinases are a protein family that constitute approximately 10% of the human kinome (for reviews on this topic, see Ref. 51-53). These proteins are characterized by the presence of a kinase-homology domain predicted to lack enzymatic activity due to the absence of at least one of the three conserved critical catalytic motifs (1) the Val-Ala-Ile-Lys (VAIK) motif in subdomain II, in which the side-chain of Lys interacts with the a and p phosphates of ATP (2) the His-Arg-Asp (HRD) motif in subdomain Ylb, in which the aspartic acid is the catalytic residue and (3) the Asp-Phe-Gly (DFG) motif in sub-domain VII, in which the carboxylic moiety of aspartic acid binds the Mg11 ion that coordinates the p and y phosphates of ATP. Owing to their lack of intrinsic phosphoryl-transfer catalytic activity, pseudokinase domain-containing... 

For the apo form of the p38 MAP kinase it was deduced that the absence of the amide peak for Phel69 in the DFG motif under all tested NMR conditions is consistent with a conformational in/out equilibrium taking place at an intermediate NMR timescale. Binding of the pyrazole-urea-based DFG-out inhibitor is not compatible with the DFG-in conformation therefore, the conformational exchange process of the DFG loop is directly interfered. 

The availabUity of the DFG-out pocket requires the kinase activation loop to adopt a catalytically deficient conformation in which the ATP binding site becomes partially occluded by the Phe side chain of the DFG motif. While the DFG-out conformation is more favorable in the unphosphorylated kinase, phosphorylation of the activation loop shifts conformational equUibria to the more active DFG-in conformation, increases kinase activity, and often reduces the affinity of type 11 and type 111 inhibitors [1]. Although the search for chemical scaffolds which have affinity for the DFG-out pocket is moving to the forefront of kinase inhibitor research, efforts have been constrained by the lack of high-throughput assay technologies which can identify and discriminate for hgands which bind to and stabihze enzymatically inactive kinase conformation. 

Fig. 1. The FLiK technology for the detection of allosteric Type ll/lll kinase Inhibitors. Kinases are regulated by an activation loop which can adopt active and inactive conformations, (a) The inactive conformation (DFG-out) presents an alternative binding site in which DFG-out ligands can bind and prevent the kinase from adopting and active conformation, (b) A Cys was mutated into the activation loop and (c) used for the attachment of acrylodan. (d) The DFG motif and activation loop adopt a different conformation in inactive kinases when compared to active kinase with ATP bound, resulting in a fluorescence change. Active kinases have an activation loop which is open and extended in contrast to the inactive DFG-out conformation. Reproduced from (7) with permission from Nature Publishing Group.

Negative Control Repeat for a Type I ATP-competitive inhibitor which is known to bind to the active DFG-in conformation and not induce movement of the DFG motif and/or activation loop. 

Due to the large number of available protein structures, kinases still have to be considered the best case for structure-based library design. This is even truer for targeting allosteric modulation [124]. Here, a group of inhibitors has been inden-tified that bind outside the ATP pocket and, thereby, stabilize the DFG motif. 

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