Abstract
The α-kinases are a family of a typical protein kinases present in organisms ranging from protozoa to mammals. Here we report an autoinhibited conformation for the α-kinase domain of Dictyostelium myosin-II heavy chain kinase A (MHCK-A) in which nucleotide binding to the catalytic cleft, located at the interface between an N-terminal and C-terminal lobe, is sterically blocked by the side chain of a conserved arginine residue (Arg592). Previous α-kinase structures have shown that an invariant catalytic aspartic acid residue (Asp766) is phosphorylated. Unexpectedly, in the autoinhibited conformation the phosphoryl group is transferred to the adjacent Asp663, creating an interaction network that stabilizes the autoinhibited state. The results suggest that Asp766 phosphorylation may play both catalytic and regulatory roles. The autoinhibited structure also provides the first view of a phosphothreonine residue docked into the phospho-specific allosteric binding site (Pi-pocket) in the C-lobe of the α-kinase domain.
Highlights
The WD-repeat domain binds myosin-II filaments and is required for myosin-II heavy chain kinase A (MHCK-A) to effectively phosphorylate myosin-II in cells[19]
Adenine binds into the far left-hand portion of the catalytic cleft, with the ribose moiety and phosphoryl groups extending towards the middle of the cleft
In all nucleotide-bound structures of A-CAT the left-hand side of the catalytic cleft is in a closed conformation with the phosphate-binding loop (P-loop) clamped down over the adenine base, ribose moiety and phosphoryl groups (Fig. 4a)
Summary
The WD-repeat domain binds myosin-II filaments and is required for MHCK-A to effectively phosphorylate myosin-II in cells[19]. A-CAT can utilize both ATP and ADP to phosphorylate peptides and proteins and is able to remove all three phosphoryl groups from ATP to generate adenosine. These unusual catalytic activities can be attributed to distinctive active site features, such as the presence of an invariant basic residue (Arg[592]; numbering for MHCK-A) in the phosphate-binding loop (P-loop) in the N-lobe. Asp[766] is phosphorylated in structures of A-CAT bound to ADP, AMP or adenosine, suggesting that it is able to accept all three phosphoryl groups Conventional protein kinases, such as PKA, do not form a phosphoenzyme intermediate[33]. It is possible that Asp[766] phosphorylation serves a dual function, both as an intermediate in the catalytic mechanism and as a regulatory switch
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