Abstract
Histidine methylation serves as an intriguing strategy to introduce altered traits of target proteins, including metal ion chelation, histidine-based catalysis, molecular assembly, and translation regulation. As a newly identified histidine methyltransferase, METTL9 catalyzes N1-methylation of protein substrates containing the "His-x-His" motif (HxH, x denotes small side chain residue). Here our structural and biochemical studies revealed that METTL9 specifically methylates the second histidine of the "HxH" motif, while exploiting the first one as a recognition signature. We observed an intimate engagement between METTL9 and a pentapeptide motif, where the small "x" residue is embedded and confined within the substrate pocket. Upon complex formation, the N3 atom of histidine imidazole ring is stabilized by an aspartate residue such that the N1 atom is presented to S-adenosylmethionine for methylation. Moreover, METTL9 displayed a feature in preferred consecutive and "C-to-N" directional methylation of tandem "HxH" repeats that exist in many METTL9 substrates. Collectively, our work illustrates the molecular design of METTL9 in N1-specific methylation of the broadly existing "HxH" motifs, highlighting its importance in histidine methylation biology.