Structural insights into SETD3-mediated histidine methylation on β-actin

Abstract

SETD3 is a member of the SET (Su(var)3–9, Enhancer of zeste, and Trithorax) domain protein superfamily and plays important roles in hypoxic pulmonary hypertension, muscle differentiation, and carcinogenesis. Previously, we identified SETD3 as the actin-specific methyltransferase that methylates the N3 of His73 on β-actin (Kwiatkowski et al., 2018). Here, we present two structures of S-adenosyl-L-homocysteine-bound SETD3 in complex with either an unmodified β-actin peptide or its His-methylated variant. Structural analyses, supported by biochemical experiments and enzyme activity assays, indicate that the recognition and methylation of β-actin by SETD3 are highly sequence specific, and that both SETD3 and β-actin adopt pronounced conformational changes upon binding to each other. In conclusion, this study is the first to show a catalytic mechanism of SETD3-mediated histidine methylation on β-actin, which not only throws light on the protein histidine methylation phenomenon but also facilitates the design of small molecule inhibitors of SETD3.

Structural basis for the target specificity of actin histidine methyltransferase SETD3

“…In order to further grasp the measurable activity of SETD3 on K73-containing peptides, we attempted to improve the enzyme affinity ( K m value) by increasing the peptide length to actin residues 66–88 (as used by Guo et al 23 ). Indeed, the lengthened peptide decreased the K m value (increased affinity) to 5.2 μM (pH 10.5) against K73 (66–88) peptide, which is about four-fold better than that of the H73 (66–80) peptide (Fig.…”

“…Previously, Guo et al crystallized SETD3 in a ternary complex with a pre-methylated actin peptide and SAH (PDB 6ICT), where the methylated imidazole ring rotated by ~90° relative to that of the unmodified His73 23 . The observations between the two complexes with the methylated peptides (whether through chemical synthesis or enzyme driven reaction as described here) are generally in agreement, including the rotation of imidazole ring (though the degree of the rotation is not exactly the same) and the hydrogen bond between the N 1 atom and Asn255.…”

“…The literature on SET-domain-catalyzed protein lysine methylation has demonstrated that these MTases frequently have high substrate specificity due to recognition of the various sequences surrounding the target lysine and distinct product specificity for the number of methyl groups (one, two, or three) being transferred to the target lysine 13–20 . Recently, SETD3 was discovered as the first metazoan protein (actin) histidine MTase 21–23 , representing a SET domain family member that methylates a target residue other than lysine, thus broadening the possible target residues that can be methylated by SET domain proteins. This raises the previously unappreciated question of target specificity, i.e., how do SET-domain MTases discriminate between histidine and lysine in the active-site pocket?…”

Deletion of mouseSetd4promotes the recovery of hematopoietic failure

“…The human genome encodes an estimated of 58 SET domain methyltransferases (20). Although most of the SET domain proteins have documented histone modifying activity, the SETD6 group of SET proteins (including SETD6, SETD3 and SETD4) represents a distinct class of non-histone methyltransferases (20), and they share a similar substrate-binding domain resembling that of the plant Rubisco methyltransferase (21)(22)(23)(24)(25)(26)(27). SETD6 methylates p65 (RelA) (24,28), and other non-histone proteins (29)(30)(31).…”

“…SETD6 methylates p65 (RelA) (24,28), and other non-histone proteins (29)(30)(31). SETD3 was initially reported to methylate histones (32,33), but recently found to a histidine residue in b-actin (25,26,34). However, little is known about the function of mammalian SETD4.…”