We have revealed that bifunctional AceK kinase/phosphatase utilizes a stepwise addition–elimination mechanism in its dephosphorylation reaction. This work explains how AceK enables opposite kinase and phosphatase activities with Asp477 and a single Mg2+ ion.

The mammalian target of rapamycin, mTOR, forms various protein-protein complexes to regulate cell growth in response to the nutrient and energy status of the cell. Recently, the first crystal structure of large HEAT repeat protein mTOR revealed that the FAT domain interacts with the kinase domain through electrostatic effects and hydrophobic interactions. Based on the structure, the previous researches on how FAT domain regulates mTOR activity are reviewed. DEPTOR is currently known as an endogenous mTOR inhibitor, which may interact with mTOR FAT domain to suppress mTOR activity in vivo. The possible interactions of DEPTOR with the mTOR FAT domain are analyzed, too. In addition, the inhibition mechanism of DEPTOR may be similar to members of HEAT-involved RanGTP complex family, providing new mechanistic insights into mTOR kinase regulation.

•Human WWP2 binds PPXY motif substrates only via WW domains.•WW domains bind substrate in a priority dependent manner, displaying a novel direct binding mode.•Modeled WW domain structure substantiated by results in alanine scanning.WW domains harbor substrates containing proline-rich motifs, but the substrate specificity and binding mechanism remain elusive for those WW domains less amenable for structural studies, such as human WWP2 (hWWP2). Herein we have employed multiple techniques to investigate the second WW domain (WW2) in hWWP2. Our results show that hWWP2 is a specialized E3 for PPxY motif-containing substrates only and does not recognize other amino acids and phospho-residues. The strongest binding affinity of WW2, and the incompatibility between each WW domain, imply a novel relationship, and our SPR experiment reveals a dynamic binding mode in Class-I WW domains for the first time. The results from alanine-scanning mutagenesis and modeling further point to functionally conserved residues in WW2.

We have revealed that bifunctional AceK kinase/phosphatase utilizes a stepwise addition–elimination mechanism in its dephosphorylation reaction. This work explains how AceK enables opposite kinase and phosphatase activities with Asp477 and a single Mg2+ ion.