Analysis of the domain structure of elongation factor‐2 kinase by mutagenesis

A number of elongation factor‐2 kinase (eEF‐2K) mutants were constructed to investigate features of this kinase that may be important in its activity. Typical protein kinases possess a highly conserved lysine residue in subdomain II which follows the GXGXXG motif of subdomain I. Mutation of two lysine residues, K340 and K346, which follow the GXGXXG motif in eEF‐2K had no effect on activity, showing that such a lysine residue is not important in eEF‐2K activity. Mutation of a conserved pair of cysteine residues C‐terminal to the GXGXXG sequence, however, completely inactivated eEF‐2K. The eEF‐2K CaM binding domain was localised to residues 77–99 which reside N‐terminal to the catalytic domain. Tryptophan 84 is an important residue within this domain as mutation of this residue completely abolishes CaM binding and eEF‐2K activity. Removal of approximately 130 residues from the C‐terminus of eEF‐2K completely abolished autokinase activity; however, removal of only 19 residues inhibited eEF‐2 kinase activity but not autokinase activity, suggesting that a short region at the C‐terminal end may be important in interacting with eEF‐2. Likewise, removal of between 75 and 100 residues from the N‐terminal end completely abolished eEF‐2K activity.