NORMAL GENOMIC

• Klingbeil CK, Gill GN
• A basic residue, Lys 782, composes part of the ATP-binding site on the epidermal growth factor receptor tyrosine kinase.
• Arch Biochem Biophys. 1999; 363: 27-32
• Display abstract

• To identify amino acids specific for tyrosine kinase activity, the role of several conserved basic residues in kinase function was tested. Modeling of the epidermal growth factor receptor tyrosine kinase domain based on the crystal structure of cyclic AMP-dependent protein kinase and insulin receptor revealed several basic residues present on the surface of epidermal growth factor receptor. Using the molecular modeling program, GRASP, the basic residues Arg 779, Lys 782, and Lys 855 were shown to provide an area of positive charge to the surface of the molecule. To deduce the role of these residues in ATP and substrate binding, site-directed mutants were prepared and kinetic constants were measured. Mutation of Lys 855 to Ala destabilized the enzyme and caused partial inactivation. Mutation of either Arg 779 or Lys 782 had little effect on the Km value for peptide substrate. However, alteration of Lys 782 increased the Km value for ATP 28-fold, indicating a role for Lys 782 in binding ATP. Because residues similar to Lys 782 in the sequences of mitogen-activated protein kinase and insulin receptor make contact with a ribose hydroxyl of ATP, it is proposed that Lys 782 may be one of the residues composing the ribose-binding site of epidermal growth factor receptor.

• Teplyakov A et al.
• Crystal structure of bacteriophage T4 deoxynucleotide kinase with its substrates dGMP and ATP.
• EMBO J. 1996; 15: 3487-97
• Display abstract

• NMP kinases catalyse the phosphorylation of the canonical nucleotides to the corresponding diphosphates using ATP as a phosphate donor. Bacteriophage T4 deoxynucleotide kinase (DNK) is the only member of this family of enzymes that recognizes three structurally dissimilar nucleotides: dGMP, dTMP and 5-hydroxymethyl-dCMP while excluding dCMP and dAMP. The crystal structure of DNK with its substrate dGMP has been determined at 2.0 A resolution by single isomorphous replacement. The structure of the ternary complex with dGMP and ATP has been determined at 2.2 A resolution. The polypeptide chain of DNK is folded into two domains of equal size, one of which resembles the mononucleotide binding motif with the glycine-rich P-loop. The second domain, consisting of five alpha-helices, forms the NMP binding pocket. A hinge connection between the domains allows for large movements upon substrate binding which are not restricted by dimerization of the enzyme. The mechanism of active centre formation via domain closure is described. Comparison with other P-loop-containing proteins indicates an induced-fit mode of NTP binding. Protein-substrate interactions observed at the NMP and NTP sites provide the basis for understanding the principles of nucleotide discrimination.