The Role of the Conserved SUMO-2/3 Cysteine Residueon Domain Structure Investigated Using Protein Chemical Synthesis

While the semi or total synthesis of ubiquitin or polyubiquitin conjugates has attracted a lot of attention the past decade, the preparation of small ubiquitin-like modifier (SUMO) conjugates is much less developed. We describe hereinafter some important molecular features to consider when preparing SUMO-2/3 conjugates by chemical synthesis using the native chemical ligation and extended methods. In particular, we clarify the role of the conserved cysteine residue on SUMO-2/3 domain stability and properties. Our data reveal that SUMO-2 and -3 proteins behave differently from the Cys → Ala modification with SUMO-2 being less impacted than SUMO-3, likely due to a stabilizing interaction occurring in SUMO-2 between its tail and the SUMO core domain. While the Cys → Ala modification has no effect on the enzyme-catalyzed conjugation, it shows a deleterious effect on the enzyme-catalyzed deconjugation process, especially with the SUMO-3 conjugate. Whereas it is often stated that SUMO-2 and SUMO-3 are structural...