Abstract
SUMO belongs to the ubiquitin-like family (UbL) of protein modifiers. SUMO is conserved among eukaryotes and is essential for the regulation of processes such as DNA damage repair, transcription, DNA replication and mitosis. UbL modification of proteins occurs via a specific enzymatic cascade formed by the crosstalk between the E1-activating enzyme, the E2-conjugating enzyme and the E3-ligase. An essential discrimination step in all UbL modifiers corresponds to the interaction between E1 and E2 enzymes, which is mediated by the recruitment of the E2 to the UFD domain (Ubiquitin-Fold Domain) of the E1 enzyme. To gain insights in the properties of this interface, we have compared the structures of the complexes between E1 UFD domain and E2 in human and yeast, revealing two alternative UFD platforms that interact with a conserved E2. Comparative sequence analysis of the E1 UFD domain indicates that the E2 binding region has been conserved across phylogenetic closely related species, in which higher sequence conservation can be found in the E2 binding region than in the entire UFD domain. These distinctive strategies for E1-E2 interactions through the UFD domain might be the consequence of a high selective pressure to ensure specificity of each modifier conjugation system.
Highlights
The post-translational modification pathway of proteins by ubiquitin-like family (UbL) (Ubiquitin-like modifiers) is characterized by the presence of specific enzymatic cascades (E1, E2 and E3 enzymes), which results in the formation of an isopeptidic bond between the UbL and the protein target[1,2]
The interaction between the ubiquitin-fold domain (UFD) of the E1-activating enzyme and the E2-conjugating enzyme has been revealed as a crucial discrimination step in the conjugation pathway of UbL modifiers[14,18,23,24]
Within the same UbL family, evolutionary distant species display a low degree of sequence conservation between E1 UFD domains
Summary
The post-translational modification pathway of proteins by UbLs (Ubiquitin-like modifiers) is characterized by the presence of specific enzymatic cascades (E1, E2 and E3 enzymes), which results in the formation of an isopeptidic bond between the UbL and the protein target[1,2]. The crystal structure of the thio-ester transfer intermediate of ubiquitin E1-E2 complex[15] revealed a dual binding of E2 to the UFD domain and to the catalytic E1 Cys-domain, which occurs after an significant rotation of the UFD domain, providing the structural basis for the isoenergetic thio-ester transfer between the E1 and the E2 enzymes[15] This interaction between E2 and the Cys domain of the E1 was proposed previously in the SUMO pathway by NMR analyses, E1 UFD-E2 interactions display higher affinity (Kd = 1.2 μM)[16] than E1 Cys-E2 interactions (Kd = 87 μM)[17], supporting a major role of the E1 UFD domain in E2 recruitment. Phylogenetic and homology analysis revealed that the region involved in Ubc[9] binding displays a slightly higher conservation degree than the UFD domain between phylogenetically closely related organisms, it displays higher variability, highlighting the relevance of this interface in the protein-protein specificity for each type of UbL modification
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