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Autoinhibition plays a key role in the control of protein kinase activity. ErbB2 is a unique receptor-tyrosine kinase that does not bind ligand but possesses an extracellular domain poised to engage other ErbBs. Little is known about the molecular mechanism for ErbB2 catalytic regulation. Here we show that ErbB2 kinase is strongly autoinhibited, and a loop connecting the alphaC helix and beta4 sheet within the kinase domain plays a major role in the control of kinase activity. Mutations of two Gly residues at positions 776 and 778 in this loop dramatically increase ErbB2 catalytic activity. Kinetic analysis demonstrates that mutational activation is due to approximately 10- and approximately 7-fold increases in ATP binding affinity and turnover number, respectively. Expression of the activated ErbB2 mutants in cells resulted in elevated ligand-independent ErbB2 autophosphorylation, ErbB3 phosphorylation, and stimulation of mitogen-activated protein kinase. Molecular modeling suggests that the ErbB2 kinase domain is stabilized in an inactive state via a hydrophobic interaction between the alphaC-beta4 and activation loops. Importantly, many ErbB2 human cancer mutations have been identified in the alphaC-beta4 loop, including the activating G776S mutation studied here. Our findings reveal a new kinase regulatory mechanism in which the alphaC-beta4 loop functions as an intramolecular switch that controls ErbB2 activity and suggests that loss of alphaC-beta4 loop-mediated autoinhibition is involved in oncogenic activation of ErbB2.

The epidermal growth factor receptor (EGFR) kinase catalyzes phosphorylation of tyrosines in its C terminus and in other cellular targets upon epidermal growth factor (EGF) stimulation. Here, by using peptides derived from EGFR autophosphorylation sites and cellular substrates, we tested the hypothesis that ligand may function to regulate EGFR kinase specificity by modulating the binding affinity of peptide sequences to the active site. Measurement of the steady-state kinetic parameters, Km and kcat, revealed that EGF did not affect the binding of EGFR peptides but increased the binding affinity for peptides corresponding to the major EGFR-mediated phosphorylation sites of the adaptor proteins Gab1 (Tyr-627) and Shc (Tyr-317), and for peptides containing the previously identified optimal EGFR kinase substrate sequence EEEEYFELV (3–7-fold). Conversely, EGF stimulation increased kcat ∼5-fold for all peptides. Thus, ligand changed the relative preference of the EGFR kinase for substrates as evidenced by EGF increases of ∼5-fold in the specificity constants (kcat/Km) for EGFR peptides, whereas ∼15–40-fold increases were observed for other peptides, such as Gab1 Tyr-627. Furthermore, we demonstrate that EGF (i) increased the binding affinity of EGFR to Gab1 Tyr-627 and Shc Tyr-317 sites in purified GST fusion proteins ∼4–6-fold, and (ii) EGF significantly enhanced the phosphorylation of these sites, relative to EGFR autophosphorylation, in cell lysates containing the full-length Gab1 and Shc proteins. Analysis of peptides containing amino acid substitutions indicated that residues C-terminal to the target tyrosine were critical for EGF-stimulated increases in substrate binding and regulation of kinase specificity. To our knowledge, this represents the first demonstration that ligand can alter specificity of a receptor kinase toward physiologically relevant targets.

Creatine kinase (CK) is a dimeric enzyme important in ATP regeneration in cells where energy demands are high. The folding of CK under equilibrium and transient conditions has been studied in detail and is found to be complex. At equilibrium in 0.8 M GuHCl, 90% of CK molecules are in the form of a partially structured, monomeric intermediate. We exploit this property to measure kinetics of refolding and unfolding to and from this equilibrium intermediate (EI), using far-UV circular dichroism and intrinsic fluorescence as structural probes. We are thus able to compare the properties of EI and the kinetic intermediate formed during the burst phase in refolding. Native CK and EI unfold with rate constants in seconds and milliseconds, respectively. As is observed for refolding of fully-denatured CK, refolding from EI to the native state shows a burst phase followed by two exponential phases. The burst phase refolding intermediate is inferred to have more structure and greater stability than the equilibrium intermediate. When refolding from the fully-denatured state in 0.8 M GuHCl, the equilibrium intermediate is formed within the dead-time of mixing in the stopped-flow apparatus. The equilibrium intermediate may thus represent a kinetic intermediate formed early during folding.

This work is aimed at understanding how protein structure and conformation regulate activity and allosteric communication in the tryptophan synthase α2β2 complex from Salmonella typhimurium. Previous crystallographic and kinetic results suggest that both monovalent cations and a salt bridge between α subunit Asp56 and β subunit Lys167 play allosteric roles. Here we show that mutation of either of these salt bridging residues produced deleterious effects that could be repaired by increased temperature in combination with CsCl or with NaCl plus an α subunit ligand, α-glycerol 3-phosphate. Arrhenius plots of the activity data under these conditions were nonlinear. The same conditions yielded temperature-dependent changes in the equilibrium distribution of enzyme-substrate intermediates and in primary kinetic isotope effects. We correlate the results with a model in which the mutant enzymes are converted by increased temperature from a low activity, “open” conformation to a high activity, “closed” conformation under certain conditions. The allosteric ligand and different monovalent cations affected the equilibrium between the open and closed forms. The results suggest that α subunit Asp56 and β subunit Lys167 are not essential for catalysis and for allosteric communication between the α and β subunits but that their mutual interaction is important in stabilization of the active, closed form of the α2β2 complex.

Changes in the activity and conformation of Chinese hamster dihydrofolate reductase (ch-DHFR) were studied in reverse micelles of cetyltrimethylammonium bromide (CTAB) and dodecylammonium butyrate (DAB) at various water contents and denaturant concentrations. The ch-DHFR entrapped in CTAB and DAB reverse micelles shows very low activity at a lower ratio of water to surfactant (omega(0)). The activity was enhanced in the presence of low concentrations of guanidine hydrochloride. Emission fluorescence spectra of ch-DHFR in aqueous medium and in reverse micelles in the presence of low concentrations of denaturants were compared. Only a slight change in emission intensity of ch-DHFR accompanies enzyme activation in the presence of low concentrations of guanidine hydrochloride in CTAB and DAB reverse micelles. There was no detectable change in the average diameters of CTAB and DAB reverse micelles in the presence of low concentrations of denaturants measured by laser light scattering, indicating that the enzyme activation in the presence of denaturant is not due to a size change in the reverse micelles. Our results suggest that the reduced activity of ch-DHFR in reverse micelles at low omega(0) is due to the restrictions in enzyme conformation caused by an environment with low water content. The reduction in enzyme activity was restored by the presence of low concentrations of denaturant or increased water content, indicating that conformation flexibility is important for full expression of enzyme activity.

The effect of cationic surfactants on the fluorescence of the zirconium-morin system is described. In sulphuric acid medium, the fluorescence intensity of this complex can be enhanced strongly by the cationic surfactant cetyltrimethylammonium bromide (CTAB). It was found that the decisive factor is the change in the composition of the analyte complex in the presence of CTAB. When the concentration of CTAB is near its critical micelle concentration, the absorptiometric measurement is favoured by the formation of a 1:3 [Zr(IV):morin] complex. On increasing the CTAB concentration, a mixed-ligand complex with the composition of Zr(IV):morin:SO 2− 4 = 1:1:2 is formed, and the fluorescence intensity is enhanced dramatically. The role of CTAB in the formation of the mixed-ligand complex and the enhancement of the fluorescence is discussed.