Side Chain Independent Recognition of Aminoacyl Adenylates by the Hint1 Transcription Suppressor

[Image: see text] Human Hint1 suppresses specific gene transcription by interacting with the transcription factor MITF in mast cells. Hint1 activity is connected to lysyl-tRNA synthetase (LysRS), a member of the universal aminoacyl tRNA synthetase family that catalyzes specific aminoacylation of their cognate tRNAs, through an aminoacyl adenylate (aa-AMP) intermediate. During immune activation, LysRS produces a side-product diadenosine tetraphosphate (Ap(4)A) from the condensation of Lys-AMP with ATP. The pleiotropic signaling molecule Ap(4)A then binds Hint1 to promote activation of MITF-target gene transcription. Earlier work showed that Hint1 can also bind and hydrolyze Lys-AMP, possibly to constrain Ap(4)A production. Because Ap(4)A can result from condensation of other aa-AMP's with ATP, the specificity of the Hint1 aa-AMP–hydrolysis activity is of interest. Here we show that Hint1 has broad specificity for adenylate hydrolysis, whose structural basis we revealed through high-resolution structures of Hint1 in complex with three different aa-AMP analogues. Hint1 recognizes only the common main chain of the aminoacyl moiety, and has no contact with the aa side chain. The α-amino group is anchored by a cation-pi interaction with Trp123 at the C-terminus of Hint1. These results reveal the structural basis for the remarkable adenylate surveillance activity of Hint1, to potentially control Ap(4)A levels in the cell.