Figure 2. Molecular mechanism of substrate recognition and histidine methylation by SETD3.

(A) The β-actin peptide binds into a long groove on the surface of the SETD3 N-lobe region (right), with His73 of β-actin positioned into a hydrophobic pocket (left). His73 and AdoHcy are shown in yellow and cyan sticks, respectively, and their 2|Fo|–|Fc| σ-weighted maps are contoured at 1.2 σ. The His73-binding residues of SETD3 and are colored according to on regions in which they reside, as shown in Figure 1A. (B) Detailed interactions between His73me and SETD3 in the post- methyl transfer complex. His73me and AdoHcy are shown in yellow and cyan sticks, respectively, and their 2|Fo|–|Fc| σ-weighted maps are contoured at 1.2 σ. The His73me-binding residues of SETD3 are colored according to the scheme shown in Figure 2A. (C) Superposition of the two complexes on the histidine/methylhistidine binding pocket. For the SETD3–His73me complex, the His73me-binding residues of SETD3 are illustrated as in Figure 2A, with His73me and AdoHcy shown in yellow and cyan sticks, respectively. For the SETD3–His73 complex, the His73-binding residues and AdoHcy are shown in gray sticks, whereas His73 is shown in green sticks to show that it rotates 90 degrees after catalysis. After methylation, one new hydrogen bond is formed between Asn256 and the N¹ atom of His73me (red dashed line). (D) Lysine recognition by LSMT(PDB id: 1OZV) in the presence of AdoHcy. (E) Carnosine recognition by CARNMT1 (PDB id: 5YF1) in the presence of the AdoHcy mimics SINEFUNGIN (SFG). Histidine, lysine and carnosine are shown in yellow sticks, whereas the protein residues that are involved in binding are shown in red sticks. AdoHcy and SFG are shown in cyan sticks.

Figure 2—figure supplement 1. Detailed interactions of SETD3 and β-actin peptide(66–88).

The β-actin peptide mainly contacts with the N-terminal lobe of SETD3, while its N-terminal and C-terminal sides contact the SET and iSET domains, respectively. In the top panel, SETD3 is shown in ribbons and colored as in Figure 2A. β-actin peptide is shown in yellow sticks. Detailed interactions between SETD3 and Leu67-Glu72, and between SETD3 and Gly74 and Glu83, are shown in the bottom left and right panels, respectively. The SETD3 residues that are involved in β-actin-binding are shown in sticks.

Figure 2—figure supplement 2. Interactions between SETD3 and peptide.

Schematic of the detailed interactions (A) between SETD3 and unmodified β-actin and (B) between SETD3 and methylated β-actin. SETD3 residues are colored as in Figure 1A, while the peptide residues are colored in yellow. The hydrophobic interactions and hydrogen bonds between the proteins and peptides are shown in black solid arrows and black dashed arrows, respectively.

Figure 2—figure supplement 3. Actin undergoes conformational changes upon binding to SETD3.

β-actin peptide(66–88) (yellow cartoon) is overlaid with the same fragment in the structure of native β-actin (red cartoon) (PDB id: 1HLU). Native β-actin decomposes its local secondary structures upon SETD3 binding. Native β-actin and SETD3 are shown in red and blue ribbons, respectively.