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Role of the Zn(2+) motif of E1 in SUMO adenylation.

ABSTRACT: Post-translational modifications by ubiquitin-like proteins are among the most important mechanisms for regulating a wide variety of cellular functions. In these modifications an E1 enzyme activates each ubiquitin-like protein (Ubl) by adenylation of the Ubl C-terminal COOH group and then forms a thioester bond with the adenylated C-terminal COOH group of the Ubl. Previous x-ray crystallography studies revealed a conserved zinc motif in the SUMO and NEDD8 E1; however, the function of this Zn(2+) motif is unclear. In this study, using quantitative ATP:PPi isotope exchange assays in combination with site-directed mutagenesis, we show that the conserved Zn(2+) motif in the SUMO E1 is important for SUMO adenylation and is critical for the E1 pseudo-ordered substrate binding mechanism. Furthermore, Zn(2+) motif mutants showed significantly reduced k(cat) values for ATP:PPi isotope exchange assays, suggesting that the Zn(2+) motif is important in binding and preventing SUMO adenylate from dissociating from E1 before formation of the thioester conjugate. Because the Zn(2+) motif is located in a cross-over loop that is known to have conformational flexibility, the results described here suggest that this cross-over loop interacts with Ubl in the multistep, dynamic process of Ubl activation by E1s.

SUBMITTER: Wang J

PROVIDER: S-EPMC2911299 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Role of the Zn(2+) motif of E1 in SUMO adenylation.

Wang Jianghai J Chen Yuan Y

The Journal of biological chemistry 20100525 31

Post-translational modifications by ubiquitin-like proteins are among the most important mechanisms for regulating a wide variety of cellular functions. In these modifications an E1 enzyme activates each ubiquitin-like protein (Ubl) by adenylation of the Ubl C-terminal COOH group and then forms a thioester bond with the adenylated C-terminal COOH group of the Ubl. Previous x-ray crystallography studies revealed a conserved zinc motif in the SUMO and NEDD8 E1; however, the function of this Zn(2+) ...[more]

PMID: 20501649

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Project description:The E1-like superfamily is central to ubiquitin (Ub) conjugation, biosynthesis of cysteine, thiamine, and MoCo, and several secondary metabolites. Yet, its functional diversity and evolutionary history is not well understood. We develop a natural classification of this superfamily and use it to decipher the major adaptive trends occurring in the evolution of the E1-like superfamily. Within the Rossmann fold, E1-like proteins are closest to NAD(P)/FAD-dependent dehydrogenases and S-AdoMet-dependent methyltransferases. Hence, their phosphotransfer activity is an independent catalytic "invention" with respect to such activities seen in other Rossmannoid folds. Sequence and structure analysis reveals a striking diversity of residues and structures involved in adenylation, sulfotransfer, and substrate binding between different E1-like families, allowing us to predict previously uncharacterized functional adaptations. E1-like proteins are fused to several previously undetected domains, such as a predicted sulfur transfer domain containing a novel superfamily of the TATA-binding protein fold, different types of catalytic domains, a novel winged helix-turn-helix domain and potential adaptor domains related to Ub conjugation. On the basis of these fusions, we develop a generalized model for the linking of E1 catalyzed adenylation/thiolation with further downstream reactions. This is likely to involve a dynamic interplay between the E1 active sites and diverse fused C-terminal domains. We also predict participation of E1-like domains in previously uncharacterized bacterial secondary metabolism pathways, new cysteine biosynthesis systems, such as those associated with archaeal O-phosphoseryl tRNA, metal-sulfur cluster assembly (e.g., in nitrogen fixation) and Ub-conjugation. Evolutionary reconstructions suggest that the last universal common ancestor contained a single E1-like domain possessing both phosphotransfer and thiolating activities and participating in multiple sulfotransfer reactions. The E1-like superfamily subsequently expanded to include 26 families clustering into three major radiations. These are broadly involved in Ub activation, cofactor and cysteine biosynthesis, and biosynthesis of secondary metabolites. In light of this, we present evidence that in eukaryotes other E1-like enzymes such as Urm1 were independently recruited for Ubl conjugation, probably functioning without conventional E2-like enzymes.

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Role of the Zn(2+) motif of E1 in SUMO adenylation.

Publications

Role of the Zn(2+) motif of E1 in SUMO adenylation.

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