Molecular Basis for Cohesin Acetylation by Establishment of Sister Chromatid Cohesion N-Acetyltransferase ESCO1

Abstract

Protein acetylation is a prevalent posttranslational modification that is regulated by diverse acetyltransferase enzymes. Although histone acetyltransferases (HATs) have been well characterized both structurally and mechanistically, far less is known about non-histone acetyltransferase enzymes. The human ESCO1 and ESCO2 paralogs acetylate the cohesin complex subunit SMC3 to regulate the separation of sister chromatids during mitosis and meiosis. Missense mutations within the acetyltransferase domain of these proteins correlate with diseases, including endometrial cancers and Roberts syndrome. Despite their biological importance, the mechanisms underlying acetylation by the ESCO proteins are not understood. Here, we report the X-ray crystal structure of the highly conserved zinc finger-acetyltransferase moiety of ESCO1 with accompanying structure-based mutagenesis and biochemical characterization. We find that the ESCO1 acetyltransferase core is structurally homologous to the Gcn5 HAT, but contains unique additional features including a zinc finger and an ∼40-residue loop region that appear to play roles in protein stability and SMC3 substrate binding. We identify key residues that play roles in substrate binding and catalysis, and rationalize the functional consequences of disease-associated mutations. Together, these studies reveal the molecular basis for SMC3 acetylation by ESCO1 and have broader implications for understanding the structure/function of non-histone acetyltransferases.

Keywords: ESCO1; SMC3; X-ray crystallography; acetyltransferase; cell division; chromatin regulation; cohesin; crystal structure; enzyme structure; gene expression; posttranslational modification (PTM); protein acylation; protein structure; structure-function.

FIGURE 1.

Overall structure of ESCO1. A, schematic diagram of ESCO1 domain architecture. B, sequence alignment with ESCO1 orthologs with GCN5 for reference. Strictly conserved residues are highlighted in black, and highly conserved residues are boxed. C, overall structure, colored by domain, with zinc finger shown in orange, β4-β7 loop insert shown in yellow, and GNAT fold shown in green (as depicted in A) with Ac-CoA shown in sticks and the zinc ion shown as a gray sphere. D, Ac-CoA binding region, with interacting secondary structures labeled. F_o − F_c simulated annealing omit map is shown around the cofactor, contoured at 3.0 σ. E, superimposition of ESCO1 and GCN5 (wheat) bound to histone H3 substrate peptide.

FIGURE 2.

Functional analysis of ESCO1. A, C₂H₂ zinc finger (orange) with residues involved in zinc ligation or core domain interaction highlighted in wheat. The GNAT α2 core (dark green), C₂H₂ zinc finger contact residues (light green), and zinc ion (gray) are highlighted. B, results from thermal stability assays of ESCO1 WT and mutants. C, results from enzymatic activity assays of ESCO1 WT and mutants. Error bars are the standard deviation of triplicate assays. The dotted line represents values for ESCO1 WT. Blank represents reaction in the absence of enzyme. D, ESCO1 crystallographic dimer with interface residues shown as sticks in the same color scheme as in Fig. 1. E, analytical ultracentrifugation analysis of ESCO1 with and without Ac-CoA, which is most consistent with the monomer simulation (blue).

FIGURE 3.

Sedimentation equilibrium analytical ultracentrifugation of ESCO1. ESCO1 (residues 590–840) was analyzed at 4 °C using interference optics in the Apo, Ac-CoA, and CoA (1 mm) + SMC3 peptide (1 mm) states at three different centrifugation speeds (12,000, 18,000, and 26,000 rpm) and three different protein concentrations (30, 58, and 118 μm). The data for each speed were collected in quadruplicate. The viscosity of the samples was estimated using Sednterp (43, 44), and the most representative runs were included to calculate theoretical molecular masses using the program HeteroAnalysis. The data are summarized in Fig. 2E.

FIGURE 4.

ESCO1 kinetic assays and substrate binding pocket. A and B, Michaelis-Menten analysis of WT ESCO1 against substrate peptide derived from SMC3 and Ac-CoA co-factor. C, close-up view of the ESCO1 active site highlighting residues (light green) that could play catalytic roles, with Ac-CoA (yellow CPK). D, pH activity profile of ESCO1 WT. E, pH activity profile of mutants R771A (cyan) and D810N (dark blue). Error bars represent standard deviation of triplicate assays.

FIGURE 5.

Peptide binding and ESCO1-ESCO2 disease-causing mutations. A, surface representation of ESCO1 structure color-coded as in A. B, H3 peptide superimposed onto the structure of ESCO1 using coordinates of the GCN5·CoA H3 peptide structure (PDB ID 1QSN). C, ESCO1 and ESCO2 residues that are mutated in disease are highlighted in light green CPK coloring, and nearby interacting residues are shown.

FIGURE 6.

Superimposition of ESCO1 and αTAT1. A, overall structural superimposition with αTAT1 shown in blue. B, close-up view of the active sites of ESCO1 and αTAT1 highlighting the putative catalytic general base residue for activity. C, ESCO1 and αTAT1 electrostatic surface potential (calculated in PyMOL (45)) plotted from −65 kiloteslas (red) to +65 kiloteslas (blue) shows the large areas of positive charge on αTAT1 suitable for interacting with negatively charged substrates as compared with ESCO1.