Abstract
Cohesin is a protein complex whose core subunits, Smc1, Smc3, Scc1, and SA1/SA2 form a ring-like structure encircling the DNA. Cohesins play a key role in the expression, repair, and segregation of eukaryotic genomes. Following a catalytic mechanism that is insufficiently understood, Esco1 and Esco2 acetyltransferases acetylate the cohesin subunit Smc3, thereby inducing stabilization of cohesin on DNA. As a prerequisite for structure-guided investigation of enzymatic activity, we determine here the crystal structure of the mouse Esco2/CoA complex at 1.8 Å resolution. We reconstitute cohesin as tri- or tetrameric assemblies and use those as physiologically-relevant substrates for enzymatic assays in vitro. Furthermore, we employ cell-based complementation studies in mouse embryonic fibroblast deficient for Esco1 and Esco2, as a means to identify catalytically-important residues in vivo. These analyses demonstrate that D567/S566 and E491/S527, located on opposite sides of the murine Esco2 active site cleft, are critical for catalysis. Our experiments support a catalytic mechanism of acetylation where residues D567 and E491 are general bases that deprotonate the ε-amino group of lysine substrate, also involving two nearby serine residues - S566 and S527- that possess a proton relay function.
Highlights
Cohesin is a protein complex whose core subunits, Smc[1], Smc[3], Scc[1], and SA1/SA2 form a ring-like structure encircling the DNA
We crystallized MmEsco2368–592 protein consisting of the C2H2 zinc finger and the acetyltransferase domain
By making use of the natively bound zinc ion, the crystal structure was determined by single-wavelength anomalous dispersion (SAD) from a dataset collected at the zinc peak wavelength (Supplementary Table S1)
Summary
Cohesin is a protein complex whose core subunits, Smc[1], Smc[3], Scc[1], and SA1/SA2 form a ring-like structure encircling the DNA. A notable structural feature of this basic patch is the presence of two neighboring conserved lysines[17,18] Acetylation of these residues by yeast acetyltransferase Eco[1] or its mammalian orthologues Esco[1] and Esco[2] (establishment of cohesion) decreases the positive charge of the patch, which weakens DNA binding and lessens ATPase activity[17]. A subsequent study by Chao et al has observed that in the Xenopus xEco2/Smc[3] peptide structure, the Smc[3] D107 does not point towards the ε-amino group of the substrate lysines but interacts with two conserved R621 and W623 residues of xEco[2] This suggests that D107 of Smc[3] plays a role tethering the enzyme to the substrate rather than acting as a general base[37]. Both studies noted that this particular aspartic acid is not strictly conserved among Esco homologs, suggesting that other residues at the active site may contribute to catalysis
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