Abstract
Post-translational methylation plays a crucial role in regulating and optimizing protein function. Protein histidine methylation, occurring as the two isomers 1- and 3-methylhistidine (1MH and 3MH), was first reported five decades ago, but remains largely unexplored. Here we report that METTL9 is a broad-specificity methyltransferase that mediates the formation of the majority of 1MH present in mouse and human proteomes. METTL9-catalyzed methylation requires a His-x-His (HxH) motif, where “x” is preferably a small amino acid, allowing METTL9 to methylate a number of HxH-containing proteins, including the immunomodulatory protein S100A9 and the NDUFB3 subunit of mitochondrial respiratory Complex I. Notably, METTL9-mediated methylation enhances respiration via Complex I, and the presence of 1MH in an HxH-containing peptide reduced its zinc binding affinity. Our results establish METTL9-mediated 1MH as a pervasive protein modification, thus setting the stage for further functional studies on protein histidine methylation.
Highlights
Post-translational methylation plays a crucial role in regulating and optimizing protein function
To determine whether METTL9 is a protein MTase, we investigated the ability of a recombinant fusion protein between human METTL9 and glutathione-S-transferase (GST-hMETTL9) to methylate proteins in fractionated human cell extracts in the presence of [3H]AdoMet, and detected protein methylation by fluorography
WT GST-hMETTL9 methylated several distinct proteins in the different cellular fractions (Fig. 1a and Supplementary Fig. 2a). This was striking for the membrane fraction, where several proteins were methylated by the WT enzyme and showed a stronger signal in the KO extracts (Fig. 1a), indicating that these proteins represent bona fide cellular substrates of METTL9
Summary
Post-translational methylation plays a crucial role in regulating and optimizing protein function. Our results establish METTL9-mediated 1MH as a pervasive protein modification, setting the stage for further functional studies on protein histidine methylation. It was recently shown that the SET-domain MTase SETD3 is the long-sought enzyme responsible for the introduction of 3MH at His-73 in actin, and that SETD3-mediated methylation plays an important role in regulating actin function and muscle contractility[16,17]. Histidine is a common catalytic residue, acting as either a general base or acid It can be a sensor of local pH, and is involved in a variety of other interactions, frequently including coordination of metal cations[18,19]. Amino acid analysis of total protein hydrolysates shows that 1MH is a relatively abundant modification and that METTL9 introduces the majority of 1MH in human and mouse proteomes. The presence of 1MH modifications in an HxH-containing peptide is found to diminish its affinity towards zinc
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