MgATP-induced conformational change of the catalytic subunit of cAMP-dependent protein kinase.
ABSTRACT Conformational changes of the cAMP-dependent protein kinase (PKA) catalytic (C) subunit are critical for the catalysis of gamma-phosphate transfer from adenosine 5'-triphosphate (ATP) to target proteins. Time-resolved fluorescence anisotropy (TRFA) was used to investigate the respective roles of Mg(2+), ATP, MgATP, and the inhibitor peptide (IP20) in the conformational changes of a 5,6-carboxyfluorescein succinimidyl ester (CF) labeled C subunit ((CF)C). TRFA decays were fit to a biexponential equation incorporating the fast and slow rotational correlation times phi(F) and phi(S). The (CF)C apoenzyme exhibited the rotational correlation times phi(F)=1.8+/-0.3 ns and phi(S)=20.1+/-0.6 ns which were reduced to phi(F)=1.1+/-0.2 ns and phi(S)=13.3+/-0.9 ns in the presence of MgATP. The reduction in rotational correlation times indicated that the (CF)C subunit adopted a more compact shape upon formation of a (CF)C.MgATP binary complex. Neither Mg(2+) (1-3 mM) nor ATP (0.4 mM) alone induced changes in the (CF)C subunit conformation equivalent to those induced by MgATP. The effect of MgATP was removed in the presence of ethylenediaminetetraacetic acid (EDTA). The addition of IP20 and MgATP to form the (CF)C x MgATP x IP20 ternary complex produced rotational correlation times similar to those of the (CF)C x MgATP binary complex. However, IP20 alone did not elicit an equivalent reduction in rotational correlation times. The results indicate that binding of MgATP to the C subunit may induce conformation changes in the C subunit necessary for the proper stereochemical alignment of substrates in the subsequent phosphorylation.
Article: Measurement of binding kinetics between PI3-K and phosphorylated IGF-1R using a surface plasmon resonance biosensor[show abstract] [hide abstract]
ABSTRACT: Quantitative measurement of biochemical and biophysical parameters of molecular recognition events in signaling pathways is very important for understanding biological function. Binding of insulin-like growth factor-1 receptor (IGF-1R) with IGF-1 activates receptor tyrosine phosphorylation and triggers several signaling pathways including phosphatidylinositol-3 kinase (PI3-K)/AKT and extracellular signal-regulated protein kinase (ERK)/mitogen-activated protein kinase (MAPK) pathways. We have analyzed the interactions between PI3-K and IGF-1R with or without IGF-1 stimulation. The results demonstrate that p85 subunits of PI3-K bind to IGF-1R with IGF-1 stimulation in intact cells. The binding kinetics between PI3-K and IGF-1R with or without IGF-1 stimulation were obtained using surface plasmon resonance biosensor. The affinity constant of the PI3-K to phosphorylated IGF-1R was (2.27 ± 0.12) × 108 M−1, which was about 20 times higher than that of PI3-K to unphosphorylated IGF-1R. Moreover, the kinetic effects of Mg2+, ATP and two kinase inhibitors, genistein and quercetin, on the binding between PI3-K and phosphorylated IGF-1R were studied. The data showed that Mg2+ increased the binding affinity of PI3-K with IGF-1R about 2-fold, while genistein decreased the affinity constant 2.7-fold. On the other hand, ATP and quercetin had no significant effects on the affinity constant, although both k a and k d values were increased or decreased by ATP or quercetin, respectively. This study implicated that the PI3-K binding sites on IGF-IR may be different from its phosphorylation and catalytic sites.Microchimica Acta 06/2008; 162(1):253-260. · 3.03 Impact Factor
Article: Conformational transitions upon ligand binding: holo-structure prediction from apo conformations.[show abstract] [hide abstract]
ABSTRACT: Biological function of proteins is frequently associated with the formation of complexes with small-molecule ligands. Experimental structure determination of such complexes at atomic resolution, however, can be time-consuming and costly. Computational methods for structure prediction of protein/ligand complexes, particularly docking, are as yet restricted by their limited consideration of receptor flexibility, rendering them not applicable for predicting protein/ligand complexes if large conformational changes of the receptor upon ligand binding are involved. Accurate receptor models in the ligand-bound state (holo structures), however, are a prerequisite for successful structure-based drug design. Hence, if only an unbound (apo) structure is available distinct from the ligand-bound conformation, structure-based drug design is severely limited. We present a method to predict the structure of protein/ligand complexes based solely on the apo structure, the ligand and the radius of gyration of the holo structure. The method is applied to ten cases in which proteins undergo structural rearrangements of up to 7.1 A backbone RMSD upon ligand binding. In all cases, receptor models within 1.6 A backbone RMSD to the target were predicted and close-to-native ligand binding poses were obtained for 8 of 10 cases in the top-ranked complex models. A protocol is presented that is expected to enable structure modeling of protein/ligand complexes and structure-based drug design for cases where crystal structures of ligand-bound conformations are not available.PLoS Computational Biology 01/2010; 6(1):e1000634. · 5.22 Impact Factor