Abstract
Most characterized SET domain (SETD) proteins are protein lysine methyltransferases, but SETD3 was recently demonstrated to be a protein (i.e. actin) histidine-N3 methyltransferase. Human SETD3 shares a high structural homology with two known protein lysine methyltransferases-human SETD6 and the plant LSMT-but differs in the residues constituting the active site. In the SETD3 active site, Asn255 engages in a unique hydrogen-bonding interaction with the target histidine of actin that likely contributes to its >1300-fold greater catalytic efficiency (kcat/Km ) on histidine than on lysine. Here, we engineered active-site variants to switch the SETD3 target specificity from histidine to lysine. Substitution of Asn255 with phenylalanine (N255F), together with substitution of Trp273 with alanine (W273A), generated an active site mimicking that of known lysine methyltransferases. The doubly substituted SETD3 variant exhibited a 13-fold preference for lysine over histidine. We show, by means of X-ray crystallography, that the two target nitrogen atoms-the N3 atom of histidine and the terminal ϵ-amino nitrogen of lysine-occupy the same position and point toward and are within a short distance of the incoming methyl group of SAM for a direct methyl transfer during catalysis. In contrast, SETD3 and its Asn255 substituted derivatives did not methylate glutamine (another potentially methylated amino acid). However, the glutamine-containing peptide competed with the substrate peptide, and glutamine bound in the active site, but too far away from SAM to be methylated. Our results provide insight into the structural parameters defining the target amino acid specificity of SET enzymes.
Highlights
Most characterized SET domain (SETD) proteins are protein lysine methyltransferases, but SETD3 was recently demonstrated to be a protein histidine-N3 methyltransferase
SETD3 was identified as the first metazoan histidine MTase that works on an actin histidine residue, which promotes signal-induced smooth muscle contraction and in a catalytic-independent manner is important for virulence of enteroviruses [13,14,15]
With substitution of Asn255 to alanine (N255A), valine (N255V), or phenylalanine (N255F), SETD3 loses the ability to form this hydrogen bond; this results in reduced activity for His73 methylation with slower kcat values (N255A and N255V), and surprisingly for N255F, no measurable activity was observed for histidine methylation (Fig. 1, B–D)
Summary
Most characterized SET domain (SETD) proteins are protein lysine methyltransferases, but SETD3 was recently demonstrated to be a protein (i.e. actin) histidine-N3 methyltransferase. The vast majority of characterized histone lysine MTases contain an ϳ130-residue SET domain that possesses the methylation activity [5, 6], with the exception of DOT1L (on H3K79) [7,8,9] and KMT9 (on H4K12) [10]. SETD3 shares a high degree of global similarity with two characterized SET domain proteins [14, 16, 17], namely human SETD6 and rubisco LSMT, which act respectively on lysine residues of the RelA subunit of nuclear factor NF-B and the large subunit of rubisco (18 –22). We investigate the structural and molecular determinant(s) of target specificity of histidine versus lysine versus glutamine in the active site of SETD3
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