Investigation of structure and function of Esco1 and Esco2 acetyltransferases.

Cohesin is a protein complex, whose core subunits are assembled into a ring-like structure encircling the DNA. In this manner, cohesin traps DNA molecules and plays key roles in expression, repair, and segregation of eukaryotic genomes. Esco1 and Esco2 regulate the function of cohesin by acetylation of Smc3, a subunit of the complex. Esco1 and Esco2 show distinct patterns of expression during the cell cycle. Esco1 is present constantly during the cell cycle; however, Esco2 is highly abundant during the S-phase. Both enzymes have important implications in human diseases. Mutations of Esco1 have been linked with bladder and endometrial cancer while mutations in Esco2 have been associated to Roberts syndrome (RBS), a developmental disorder with defective sister chromatid cohesion. Esco1 and Esco2 belong to the GCN5-related N-acetyltransferases (GNAT) family. While the N-terminal parts of Esco1 and Esco2 are highly divergent and likely account for the functional differences, the C-terminal GNAT acetyltransferase domains of these enzymes are conserved. Comprehensive investigation of the catalytic mechanism of the Esco1 and Esco2 acetyltransferases is essential to understand the role of cohesin acetylation in different cellular functions. Here, we combined the Mus musculus Esco2 (MmEsco2) structure, in vitro biochemistry, and cell-based studies to identify the catalytic residues of Esco1 and Esco2 and to gain insights into the functions of these residues in catalysis. We determined the structure of the acetyltransferase domain of MmEsco2, natively in com-plex with coenzyme A (CoA) at 1.8 A resolution. To characterize the active site of Esco2, a number of potential catalytic residues were chosen considering their proximity and side chain orientation toward the CoA in the MmEsco2/CoA complex structure. Next, the functional role of these potential catalytic residues, S566, D567, E491, and S527 was investigated. For this purpose, in vitro mutational analysis using incubation of human recombinant cohesin complex with different variants of full-length human ESCO1 and assessment of SMC3 acetylation was performed (sufficient amounts of either mouse or human Esco2 could not be purified due to low expression and solubility). To complement the in vitro assay, in vivo mutational analysis was performed by transfection of Esco1- and Esco2-deficient mouse embryonic fibroblasts (MEFs) with different mutants of Esco1 and Esco2. Smc3 acetylation for various mutants of MmEsco1 and Smc3 acetylation along with sister chromatid cohesion for various mutants of 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 ab-sence 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