Substrate selectivity of human histidine methyltransferase METTL9

Methylation of histidine residues in zinc transporters by histidine methyltransferase METTL9 plays an important role in modulating their metal-binding properties. Here, we report synthetic, enzymatic, and computational studies on human METTL9-catalyzed Nπ-methylation of His375 in zinc transporter SLC39A5-derived peptides in which the histidine substrate and its neighboring residues are substituted by chemically and structurally diverse proteinogenic and nonproteinogenic amino acids. Our work reveals that the xHxH motif (residues 372-375) is essential for efficient Nπ-histidine methyltransferase METTL9 catalysis. We demonstrate that human METTL9 has an exceptionally narrow substrate scope towards His375 and does not catalyze methylation of simple histidine mimics in the SLC39A5 peptide. Moreover, METTL9 recognizes only 2-pyridylalanine, 3-(4-thiazolyl)alanine and backbone N-methylated histidine residues at position 373 in addition to the natural His373 residue. METTL9 has also a capacity to recognize a few simplest mimics of Ser374 and Gly372 residues in the SLC39A5 sequence. Molecular dynamics simulations support the experimental findings and deliver a comprehensive structural basis on the significance of the xHxH motif for stable METTL9-SLC39A5 complex formation and efficient METTL9 catalysis. Overall, this study provides evidence for different substrate selectivities of human histidine methyltransferase METTL9, the knowledge important from basic molecular and biomedical perspectives.