scholarly journals Analysis of High-Affinity Binding of Protein Kinase R to Double-Stranded RNA

Biochemistry ◽  
2012 ◽  
Vol 51 (44) ◽  
pp. 8764-8770 ◽  
Author(s):  
Bushra Husain ◽  
Ishita Mukerji ◽  
James L. Cole
Keyword(s):  
Protein Kinase ◽  
Affinity Binding ◽  
Protein Kinase R ◽  
High Affinity Binding ◽  
Double Stranded Rna ◽  
High Affinity

cAMP-dependent protein kinase defines a family of enzymes

1993 ◽  
Vol 340 (1293) ◽  
pp. 315-324 ◽  
Keyword(s):  
Protein Kinase ◽  
Consensus Sequence ◽  
Protein Kinase Inhibitors ◽  
Catalytic Subunit ◽  
Affinity Binding ◽  
Dependent Protein Kinase ◽  
High Affinity Binding ◽  
High Affinity ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

The structure of the recombinant mouse catalytic subunit of cAMP-dependent protein kinase is reviewed with particular emphasis on the overall features and specific amino acids that are shared by all members of the eukaryotic protein kinase family. The crystal structure of a ternary complex containing both MgATP and a twenty-residue inhibitor peptide defines the precise role of the conserved residues that are clustered at the active site. In addition to catalysing the post-translational modification of other proteins, the catalytic subunit is itself subject to covalent modifications. It is a phosphoprotein and is also myristylated at its amino terminus. The enzyme when crystallized in the presence of detergent shows a detergent molecule bound to an acyl pocket that is presumably occupied by the myristyl moiety in the mammalian enzyme. When expressed in E.coli , the catalytic subunit is autophosphorylated at four sites. Two stable phosphates at Ser338 and Thrl97 interact with multiple protein side chains thus explaining why they are inaccessible to phosphatases. Although all substrates and inhibitors of the catalytic subunit share a general minimum consensus sequence, the high affinity binding of protein inhibitors such as the regulatory subunits and the heat stable protein kinase inhibitors require additional determinants that lie beyond the consensus site. These two physiological inhibitors of the catalytic subunit appear to use different sites to achieve high-affinity binding.

Effect of metal ions on high-affinity binding of pseudosubstrate inhibitors to PKA

2008 ◽  
Vol 413 (1) ◽  
pp. 93-101 ◽  
Author(s):  
Bastian Zimmermann ◽  
Sonja Schweinsberg ◽  
Stephan Drewianka ◽  
Friedrich W. Herberg
Keyword(s):  
Protein Kinase ◽  
Metal Ions ◽  
Metal Binding ◽  
Molecular Mechanisms ◽  
Regulatory Subunit ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
C Subunit ◽  
Pseudosubstrate Inhibitors

Conformational control of protein kinases is an important way of modulating catalytic activity. Crystal structures of the C (catalytic) subunit of PKA (protein kinase A) in complex with physiological inhibitors and/or nucleotides suggest a highly dynamic process switching between open and more closed conformations. To investigate the underlying molecular mechanisms, SPR (surface plasmon resonance) was used for detailed binding analyses of two physiological PKA inhibitors, PKI (heat-stable protein kinase inhibitor) and a truncated form of the R (regulatory) subunit (RIα 92–260), in the presence of various concentrations of metals and nucleotides. Interestingly, it could be demonstrated that high-affinity binding of each pseudosubstrate inhibitor was dependent only on the concentration of divalent metal ions. At low micromolar concentrations of Mg2+ with PKI, transient interaction kinetics with fast on- and off-rates were observed, whereas at high Mg2+ concentrations the off-rate was slowed down by a factor of 200. This effect could be attributed to the second, low-affinity metal-binding site in the C subunit. In contrast, when investigating the interaction of RIα 92–260 with the C subunit under the same conditions, it was shown that the association rate rather than the dissociation rate was influenced by the presence of high concentrations of Mg2+. A model is presented, where the high-affinity interaction of the C subunit with pseudosubstrate inhibitors (RIα and PKI) is dependent on the closed, catalytically inactive conformation induced by the binding of a nucleotide complex where both of the metal-binding sites are occupied.

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