Activation Loop Conformation

Phosphorylation of the activation loop appears to drive the equilibrium towards a catalytically active conformation due to the abUity of the phosphorylated residue to form stabilizing salt bridges with a series of lysine or arginine residues. However, it should be noted that, based on the evidence to date, the activation loop can occupy a range of conformations including one dose to an active conforma- 

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Figure 7.8 Binding of Gieevec to c-Abl (pdb code 1IEP) and the difference in activation loop conformation between Gieevec and PD-173955 bound c-Abl.

Loop motion Active site conformation adap- Nanoseconds (ns) to micro-... 

The insulin-like growth factor I receptor is closely related to the insulin receptor. The RTK activity of the IGF-I receptor is regulated by intermolecular autophosphorylation at three sites within the activation loop. The crystal structure of the trisphosphorylated form of IGF-I RTK domain with an ATP analog and a specific peptide substrate showed that autophosphorylation stabilizes the activation loop in a conformation that facilitates catalysis. Furthermore, the structure revealed how... 

Another group solved the crystal structure of the IGF-I RTK domain phosphorylated at two tyrosine residues within the activation loop and bound to an ATP analog. The ligand is not in a conformation compatible with phosphoryl transfer, and the activation loop is partially disordered. IGF-I RTK is trapped in a half-closed, previously unobserved conformation. This conformation may be intermediary between the open, inactive conformation and closed, active conformation of insulin and IGF-I RTKs. 

All isoforms of PKC are predominantly localized to the cytosol and, upon activation, undergo translocation to either plasma or nuclear membranes. However, newly synthesized PKCs are localized to the plasmalemma and are in an open conformation in which the auto inhibitory pseudosubstrate sequence is removed from the substrate binding domain. The maturation of PKC isoforms is effected by phosphoinositide-dependentkinase-I (PDK-I), which phosphorylates a conserved threonine residue in the activation loop of the catalytic (C4) domain [24]. This in turn permits the autophosphorylation of C-terminus threonine and serine residues in PKC, a step which is a prerequisite for catalytic activity (see also Chs 22 and 23). The phosphorylated enzyme is then released into the cytosol, where it is maintained in an inactive conformation by the bound pseudosubstrate. It was originally thought that 3-phosphoinositides such as PI(3,4)P2 and PI(3,4,5)P3 could directly activate PKCs. However, it now seems more likely that these lipids serve to activate PDK-1 (a frequent contaminant of PKC preparations). 

RPTK activation. The activity of RPTKs is normally suppressed in their quiescent state. This suppression is due to the numerous loose and unstructured conformations of the activation loop (A loop) within the catalytic domain the majority of these conformations interfere with substrate and ATP binding. However, a subset of these conformations is amenable to binding of substrate and ATP, resulting in activation of the RPTKs. Phosphorylation of the tyrosine residue(s) in the A loop shifts the equilibrium towards the active conformation. Because of steric hindrance, PTK catalytic domains appear to be unable to autophosphorylate tyrosine residue(s) in the A loop within the same molecule rather frans-autophosphoryla-tion between two different catalytic domains is necessary for their activation. As a consequence, ligand-induced dimerization is an important step in the activation of RPTKs (Fig. 24-7). 

Crystallography studies showed that imatinib binds to an inactive form of Abl [36,37]. In this bound conformation the activation loop of the Abl kinase domain is distinct from that of both the inactive and active forms of the SFKs, explaining why imatinib does not inhibit these kinases. The crystal structure also revealed that the Thr 315 residue was involved in a key hydrogen bonding interaction with the C-2 amino group of imatinib. 

A switch-clamp-latch mechanism is critical for the quiescent state of c-ABL normally. The switch refers to a flip that occurs in an activation loop from the C-lobe that goes between opened and closed conformations and represent active and inactive states of the enz5mie, respectively (Fig. 2). This clamp or loop limits the access of ATP and other substrates to the active binding site of the ABLl protein. A latch stabilizes... 

Fig. 2. Structure of the ABLl kinase portion of the BCR-ABLl protein. The activation loop (blue) is in the closed (inactive) conformation on the left and in the open (active) conformation on the right. A molecule of imatinib is positioned in the ATP-binding site and is in green. (Reprinted with permission from U S. Healthcare Communications, LLC. LitzowMR, Tefferi A. Chronic myeloid leukemia problems propel progress. Amer J Hematol/Oncol, 2007 6(5) supplement 7 19-22).

As discussed previously, the activation loop can also flip into an active state, and the pyridopyrimidine derivatives and dasatinib are able to bind ABLl whether the activation loop is in the closed or open position (inactivated or activated) (92). Thus, binding is not affected by the activation state. Dasatinib and related compounds are also smaller in size than IM, so the P-loop must undergo major conformational changes on binding with IM, whereas only minimal changes occur with dasatinib and related compounds. This dual activity of dasatinib also raises the question as to whether its broader activity may have broader effects, including potentially adverse effects in the treatment of patients. 

FIGURE 12-7 Activation of the insulin-receptor Tyr kinase by autophosphorylation. (a) In the inactive form of the Tyr kinase domain (PDB ID 11RK), the activation loop (blue) sits in the active site, and none of the critical Tyr residues (black and red ball-and-stick structures) are phosphorylated. This conformation is stabilized by hydrogen bonding between Tyr1162 and Asp"32, (b) When insulin binds to the a chains of insulin receptors, the Tyr kinase of each /3 subunit of the dimer phosphorylates three Tyr residues (Tyr"58, Tyr"62, and... 

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