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.
MmEsco2 were used as readouts. In vivo results from mutational analysis differed from the in vitro results. The single mutants were catalytically inactive in the in vitro assays, while the same mutants exhibited detectable activity in vivo. This inconsistency could be due to the absence of required cohesin regulatory factors in vitro. Such limitation makes it clear that an in vivo activity assessment of various Esco1 and Esco2 mutants is crucial in addition to in vitro analysis.Taken together, the results from in vitro and in vivo mutational analysis reveal that the four conserved catalytic residues S566, D567, E491, and S527 in the active site of MmEsco2 and corresponding residues in MmEsco1 cooperatively play a role in deprotonation of the lysine substrate. In this proposed mechanism, the general bases, namely aspartate and glutamate, abstract the proton of substrate lysine via serine residues.
Cohesin is a protein complex encircles the DNA and regulates the separation of sister chromatids during cell division. Following a catalytic mechanism that is insufficiently understood, Esco1 and Esco2 acetyltransferases acetylate Smc3 subunit of cohesin, thereby inducing a stabilization of cohesin on DNA. As a prerequisite for structureguided investigation of enzymatic activity, we determine here the crystal structure of the mouse Esco2/CoA complex at 1.8 Å resolution. We reconstitute the entire cohesin as a tetrameric assembly and use it as a physiologically-relevant substrate 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 MmEsco2 active site cleft, are critical for catalysis. Our experiments supports a catalytic mechanism of acetylation where residues D567 and E491 are general bases that deprotonate the εamino group of lysine substrate, via two nearby serine residues -S566 and S527-that possess a proton relay function.
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