Protein kinase substrate recognition studied using the recombinant catalytic domain of AMP-activated protein kinase and a model substrate

• Published in: Journal of molecular biology 2002-03, Vol.317 (2), p.309-323
• Main Authors: Scott, John W, Norman, David G, Hawley, Simon A, Kontogiannis, Luke, Hardie, D.Grahame
• Format: Article
• Language: English
• Subjects: acetyl-CoA carboxylase; Acetyl-CoA Carboxylase - genetics; Acetyl-CoA Carboxylase - metabolism; Amino Acid Sequence; AMP-activated protein kinase; AMP-Activated Protein Kinases; Animals; Binding Sites; Catalytic Domain - genetics; Escherichia coli; Escherichia coli - genetics; Glutathione Transferase - genetics; Glutathione Transferase - metabolism; Histidine; kinase-substrate interaction; Male; Models, Molecular; molecular modelling; Molecular Sequence Data; Multienzyme Complexes - chemistry; Multienzyme Complexes - genetics; Multienzyme Complexes - metabolism; Mutation; Phosphorylation; Protein Conformation; Protein-Serine-Threonine Kinases - chemistry; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; Rats; Rats, Wistar; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Reproducibility of Results; Serine - metabolism; site-directed mutagenesis; Substrate Specificity
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.2001.5316

We have expressed a truncated form of the α1 kinase domain of AMP-activated protein kinase (AMPK) in Escherichia coli as a glutathione- S-transferase fusion (GST-KD). A T172D mutant version did not require prior phosphorylation and was utilized for most subsequent studies. We have also created a recombinant substrate (GST-ACC) by expressing 34 residues around the major phosphorylation site (Ser79) on rat acetyl-CoA carboxylase-1/α (ACC1) as a GST fusion. This was an excellent substrate that was phosphorylated with similar kinetic parameters to ACC1 by both native AMPK and the bacterially expressed kinase domain. We also constructed a structural model for the binding of the ACC1 sequence to the kinase domain, based on crystal structures for related protein kinases. The model was tested by making a total of 25 mutants of GST-ACC and seven mutants of GST-KD, and measuring kinetic parameters with different combinations. The results reveal that AMPK and ACC1 interact over a much wider region than previously realized (>20 residues). The features of the interaction can be summarised as follows: (i) an amphipathic helix from P − 16 to P − 5 on the substrate binds in a hydrophobic groove on the large lobe of the kinase; (ii) basic residues at P − 6 and P − 4 bind to two acidic patches (D215/D216/D217 and E103/D100/E143, respectively), on the large lobe; (iii) a histidine at P + 3 interacts with D56 on the small lobe; (iv) the side-chain of P + 4 leucine could not be precisely positioned, but a new finding was that asparagine or glutamine could replace a hydrophobic residue at this position. These interactions position the serine residue to be phosphorylated in close proximity to the γ-phosphate group of ATP. Although based on modelling rather than a determined structure, this represents one of the most detailed studies of the interaction between a kinase and its substrate achieved to date.