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

Abstract

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 (Ap4A) from the condensation of Lys-AMP with ATP. The pleiotropic signaling molecule Ap4A 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 Ap4A production. Because Ap4A 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 Ap4A levels in the cell.