Intrasteric inhibition of ATP binding is not required to prevent unregulated autophosphorylation or signaling by the insulin receptor.

Receptor tyrosine kinases may use intrasteric inhibition to suppress autophosphorylation prior to growth factor stimulation. To test this hypothesis we made an Asp1161Ala mutant in the activation loop that relieved intrasteric inhibition of the unphosphorylated insulin receptor (IR) and its recombinant cytoplasmic kinase domain (IRKD) without affecting the activated state. Solution studies with the unphosphorylated mutant IRKD demonstrated conformational changes and greater catalytic efficiency from a 10-fold increase in k(cat) and a 15-fold-lower K(m ATP) although K(m peptide) was unchanged. Kinetic parameters of the autophosphorylated mutant and wild-type kinase domains were virtually identical. The Asp1161Ala mutation increased the rate of in vitro autophosphorylation of the IRKD or IR at low ATP concentrations and in the absence of insulin. However, saturation with ATP (for the IRKD) or the presence of insulin (for the IR) yielded equivalent rates of autophosphorylation for mutant versus wild-type kinases. Despite a biochemically more active kinase domain, the mutant IR expressed in C2C12 myoblasts was not constitutively autophosphorylated. However, it displayed a 2.5-fold-lower 50% effective concentration for insulin stimulation of autophosphorylation and was dephosphorylated more slowly following withdrawal of insulin than wild-type IR. In tests of the regulation of the unphosphorylated basal state, these results demonstrate that neither intrasteric inhibition against ATP binding nor suppression of kinase activity is required to prevent premature autophosphorylation of the IR. Finally, the lower rate of dephosphorylation suggests invariant residues of the activation loop such as Asp1161 may function at multiple junctures in cellular regulation of receptor tyrosine kinases.

Conformational changes in the activation loop of the insulin receptor's kinase domain.

Low catalytic efficiency of basal-state protein kinases often depends on activation loop residues blocking substrate access to the catalytic cleft. Using the recombinant soluble form of the insulin receptor's kinase domain (IRKD) in its unphosphorylated state, activation loop conformation was analyzed by limited proteolysis. The rate of activation loop cleavage by trypsin is slow in the apo-IRKD. Bound Mg-adenine nucleoside di- and triphosphates increased the cleavage rate with half-maximal effects observed at 0.4-0.9 mM nucleotide. Adenosine monophosphate at concentrations up to 10 mM was not bound appreciably by the IRKD and had virtually no impact on activation loop cleavage. Amino-terminal and carboxy-terminal core-flanking regions of the IRKD had no statistically significant impact on the ligand-dependent or -independent activation loop cleavages. Furthermore, the core-flanking regions did not change the inherent conformational stability of the active site or the global stability of the IRKD, as determined by guanidinium chloride-induced denaturation. These measurements indicate that the intrasterically inhibitory conformation encompasses > or =90% of the ligand-free basal state kinase. However, normal intracellular concentrations of Mg-adenine nucleotides, which are in the millimolar range, would favor a basal-state conformation of the activation loop that is more accessible.

A Bacillus subtilis locus encoding several gene products affecting transport of cations.

A 3.6-kb DNA fragment from Bacillus subtilis was found to complement the K+ uptake-deficient Escherichia coli strain TK2420. Transformation with a pKLO61 plasmid harboring this fragment conferred the capacity to grow on a minimal medium containing only 10 mM K+. Insertional mutagenesis and subcloning identified a single gene responsible for the complementation. This gene coded for an apparent homolog of E. coli TrkA. Sequence analysis of the cloned region also revealed three additional open reading frames. These included: a gene encoding a homolog to the czcD gene product of Alcaligenes eutrophus, a lysR-type regulatory gene which was found to enhance Na+ resistance in E. coli NM81 (delta nhaA) in a separate complementation test, and an orfD with no significant similarity to sequences deposited in Genbank.

Activation of the insulin receptor's kinase domain changes the rate-determining step of substrate phosphorylation.

The insulin receptor and many other protein kinases are activated by relief of intrasteric inhibition that is regulated by reversible phosphorylation. The changes accompanying activation of the insulin receptor's kinase domain were analyzed using steady-state kinetics, viscometric analysis, and equilibrium binding measurements. Peptide phosphorylation catalyzed by the unphosphorylated basal-state kinase is limited by a slow rate of the chemical step, and the activated enzyme is limited by product release rates. Underlying these changes were a 36-fold increase in the rate constant for the chemical step of the enzyme-catalyzed reaction, a 5-fold increase in the affinity for MgATP, and an 8-fold increase in the affinity for peptide substrate. This results in binding of substrates that is 2.2 kcal/mol more favorable and a free energy barrier for transition state formation that is lowered by 2.1 kcal/mol in the activated enzyme. Therefore, the change in conformational free energy inherent in the protein after autophosphorylation [Bishop, S. M., Ross, J. B. A., and Kohanski, R. A. (1999) Biochemistry 38, 3079-3089] is equally distributed between formation of the substrate ternary complex and formation of the transition state complex.

Multiple activation loop conformations and their regulatory properties in the insulin receptor's kinase domain.

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 approximately D1132N > or = D1161A > Y1162F approximately K1085N > Q1208S > or = wild-type. 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 off-versus-on switch for kinase activation.

Partial activation of the insulin receptor kinase domain by juxtamembrane autophosphorylation.

Increased enzymatic activity of receptor tyrosine kinases occurs after trans-phosphorylation of one or two tyrosines in the activation loop, located near the catalytic cleft. Partial activation of the insulin receptor's kinase domain was observed at dilute concentrations of kinase, suggesting that cis-autophosphorylation was occurring. Autophosphorylation during partial activation mapped to the juxtamembrane (JM) tyrosines and not to activation loop tyrosines. Furthermore, a double JM Tyr-to-Phe mutant kinase (JMY2F) did not undergo partial activation but catalyzed substrate phosphorylation at a very low rate. Steady-state kinetics of peptide phosphorylation were determined with and without JM autophosphorylation. The JMY2F mutant was used to prevent concurrent cis-autophosphorylation and therefore to approximate the basal state apoenzyme in the kinetic analysis. Partial activation was dominated by a decreased Michaelis constant for peptide substrate, from KM,PEP >/= 2.5 mM in the basal state to 0.2 mM in the partially activated state; the KM,ATP remained virtually unchanged at approximately 1 mM, and kcat increased from 180 to 600 min-1. The high KM,PEP suggests weak binding of peptide substrates to the apoenzyme. This was confirmed by Ki > 1 mM for peptide substrates used as inhibitors of JM autophosphorylation. The absence of comparably large changes in kcat and KM,ATP suggests that the JM region is primarily a strong barrier to the peptide entry step of trans-phosphorylation reactions. The JM region therefore functions as an intrasteric inhibitor in the basal state of the insulin receptor's kinase domain.

Crystallographic and solution studies of an activation loop mutant of the insulin receptor tyrosine kinase: insights into kinase mechanism.

The tyrosine kinase domain of the insulin receptor is subject to autoinhibition in the unphosphorylated basal state via steric interactions involving the activation loop. A mutation in the activation loop designed to relieve autoinhibition, Asp-1161 --> Ala, substantially increases the ability of the unphosphorylated kinase to bind ATP. The crystal structure of this mutant in complex with an ATP analog has been determined at 2.4-A resolution. The structure shows that the active site is unobstructed, but the end of the activation loop is disordered and therefore the binding site for peptide substrates is not fully formed. In addition, Phe-1151 of the protein kinase-conserved DFG motif, at the beginning of the activation loop, hinders closure of the catalytic cleft and proper positioning of alpha-helix C for catalysis. These results, together with viscometric kinetic measurements, suggest that peptide substrate binding induces a reconfiguration of the unphosphorylated activation loop prior to the catalytic step. The crystallographic and solution studies provide new insights into the mechanism by which the activation loop controls phosphoryl transfer as catalyzed by the insulin receptor.

Mechanism-based design of a protein kinase inhibitor.

Protein kinase inhibitors have applications as anticancer therapeutic agents and biological tools in cell signaling. Based on a phosphoryl transfer mechanism involving a dissociative transition state, a potent and selective bisubstrate inhibitor for the insulin receptor tyrosine kinase was synthesized by linking ATPgammaS to a peptide substrate analog via a two-carbon spacer. The compound was a high affinity competitive inhibitor against both nucleotide and peptide substrates and showed a slow off-rate. A crystal structure of this inhibitor bound to the tyrosine kinase domain of the insulin receptor confirmed the key design features inspired by a dissociative transition state, and revealed that the linker takes part in the octahedral coordination of an active site Mg2+. These studies suggest a general strategy for the development of selective protein kinase inhibitors.

Probing the catalytic mechanism of the insulin receptor kinase with a tetrafluorotyrosine-containing peptide substrate.

The interaction of a synthetic tetrafluorotyrosyl peptide substrate with the activated tyrosine kinase domain of the insulin receptor was studied by steady-state kinetics and x-ray crystallography. The pH-rate profiles indicate that the neutral phenol, rather than the chemically more reactive phenoxide ion, is required for enzyme-catalyzed phosphorylation. The pK(a) of the tetrafluorotyrosyl hydroxyl is elevated 2 pH units on the enzyme compared with solution, whereas the phenoxide anion species behaves as a weak competitive inhibitor of the tyrosine kinase. A structure of the binary enzyme-substrate complex shows the tetrafluorotyrosyl OH group at hydrogen bonding distances from the side chains of Asp(1132) and Arg(1136), consistent with elevation of the pK(a). These findings strongly support a reaction mechanism favoring a dissociative transition state.