Multiple Activation Loop Conformations and Their Regulatory Properties in the Insulin Receptor's Kinase Domain

Abstract

Low catalytic efficiency of protein kinases often results from intrasteric inhibition caused by the activation loop blocking the active site. In the insulin receptor's kinase domain, Asp-1161 and Tyr-1162 in the peptide substrate-like sequence of the unphosphorylated activation loop can interact with four invariant residues in the active site: Lys-1085, Asp-1132, Arg-1136, and Gln-1208. Contributions of these six residues to intrasteric inhibition were tested by mutagenesis, and the unphosphorylated kinase domains were characterized. The mutations Q1208S, K1085N, and Y1162F each relieved intrasteric inhibition, increasing catalytic efficiency but without changing the rate-limiting step of the reaction. The mutants R1136Q and D1132N were virtually inactive. Steric accessibility of the active site was ranked by relative changes in iodide quenching of intrinsic fluorescence, and A-loop conformation was ranked by limited tryptic cleavage. Together these ranked the openness of the active site cleft as R1136Q Ϸ D1132N > D1161A > Y1162F Ϸ K1085N > Q1208S > wildtype. These findings demonstrate the importance of specific invariant residues for intrasteric inhibition and show that diverse activation loop conformations can produce similar steady-state kinetic properties. This suggests a broader range of regulatory properties for the activation loop than expected from a simple offversus-on switch for kinase activation.

Structural and Biochemical Evidence for an Autoinhibitory Role for Tyrosine 984 in the Juxtamembrane Region of the Insulin Receptor

“…The overall structure of IRK D1132N is similar to the structure of another IRK mutant, Asp-1161 3 Ala, in which loss of four hydrogen bonds mediated by Asp-1161 in the activation loop is responsible for switching the activation loop conformation from gate-closed to gate-open (15). The gate-open conformation observed in the IRK D1132N crystal structure is in agreement with solution studies that monitored the accessibility of the active site in this and other IRK mutants (23).…”

“…2B). IRK D1132N has negligible catalytic activity, however, because of the critical role of Asp-1132 in the phosphoryl transfer mechanism (23). The overall structure of IRK D1132N is similar to the structure of another IRK mutant, Asp-1161 3 Ala, in which loss of four hydrogen bonds mediated by Asp-1161 in the activation loop is responsible for switching the activation loop conformation from gate-closed to gate-open (15).…”

Insulin and its receptor: structure, function and evolution

“…While the amount of relevant information regarding living systems generated by such approaches is invaluable, information is not knowledge. Ron Kohanski (who has made important contributions to our understanding of the insulin receptor kinase enzyme kinetics (73,74) ) and I recently reflected that we are in danger of being regarded as dinosaurs for persisiting with our biochemical approaches to the insulinreceptor interaction. I do hope that our kind of dinosaurs will not become extinct.…”

Effect of Synthetic Cell-Penetrating Peptides on TrkA Activity in PC12 Cells

“…In the kinase domain of the insulin receptor, the unphosphorylated activation loop works as a pseudosubstrate to the catalytic site (20). Although the crystal structure of the intracellular portion of TrkA has not yet been revealed, homologous residues in the insulin receptor superfamily (e.g., insulin receptor, TrkA) lead to the speculation that the unphosphorylated activation loop of TrkA also interacts with the catalytic site and contributes to the stability of catalytic activity in TrkA.…”