Autophosphorylation Activates Dictyostelium Myosin II Heavy Chain Kinase A by Providing a Ligand for an Allosteric Binding Site in the α-Kinase Domain*

By Scott W. Crawley, Mojdeh Samimi Gharaei, Q. Ye, Yidai Yang, Barak Raveh, N. London, O. Schueler‐Furman, Z. Jia, G. Côté. 2010

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Dictyostelium discoideum myosin II heavy chain kinase A (MHCK A), a member of the atypical α-kinase family, phosphorylates sites in the myosin II tail that block filament assembly. Here we show that the catalytic activity of A-CAT, the α-kinase domain of MHCK A (residues 552–841), is severely inhibited by the removal of a disordered C-terminal tail sequence (C-tail; residues 806–841). The key residue in the C-tail was identified as Thr825, which was found to be constitutively autophosphorylated. Dephosphorylation of Thr825 using shrimp alkaline phosphatase decreased A-CAT activity. The activity of a truncated A-CAT lacking Thr825 could be rescued by Pi, phosphothreonine, and a phosphorylated peptide, but not by threonine, glutamic acid, aspartic acid, or an unphosphorylated peptide. These results focused attention on a Pi-binding pocket located in the C-terminal lobe of A-CAT. Mutational analysis demonstrated that the Pi-pocket was essential for A-CAT activity. Based on these results, it is proposed that autophosphorylation of Thr825 activates ACAT by providing a covalently tethered ligand for the Pi-pocket. Ab initio modeling studies using the Rosetta FloppyTail and FlexPepDock protocols showed that it is feasible for the phosphorylated Thr825 to dock intramolecularly into the Pi-pocket. Allosteric activation is predicted to involve a conformational change in Arg734, which bridges the bound Pi to Asp762 in a key active site loop. Sequence alignments indicate that a comparable regulatory mechanism is likely to be conserved in Dictyostelium MHCK B-D and metazoan eukaryotic elongation factor-2 kinases.

Subfields: Cell Biology, Molecular Biology

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Sampling alternative conformational states of transporters and receptors with AlphaFold2 (2022, methodology): “Moreover, this approach showed limited success when applied to transporters whose structures were used to train AF2, hinting at the possibility that traditional methods may still be required to capture alternative conformers ( Crawley et al., 2011 ; Ollikainen et al., 2013 ).”