Protein kinase substrate recognition studied using the recombinant catalytic domain of AMP-activated protein kinase and a model substrate.
We have expressed a truncated form of the alpha1 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/alpha (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 gamma-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.
Scott
John W
JW
Division of Molecular Physiology, School of Life Sciences and Wellcome Trust Biocentre, Dundee University, Scotland, UK.
Norman
David G
DG
Hawley
Simon A
SA
Kontogiannis
Luke
L
Hardie
D Grahame
DG
D055372:AMP-Activated Protein Kinases; D000103:Acetyl-CoA Carboxylase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006639:Histidine; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D012694:Serine; D006639:Histidine; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D000103:Acetyl-CoA Carboxylase