Crystal Structure of the Human Ubiquitin-activating Enzyme 5 (UBA5) Bound to ATP

Bacik, J.P.; Walker, John R.; Ali, Mehboob; Schimmer, Aaron D.; Dhe‐Paganon, Sirano

doi:10.1074/jbc.m110.102921

Cited by 58 publications

(76 citation statements)

References 35 publications

(69 reference statements)

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“…Similar to ATG7 and Uba4, Uba5 is homodimeric and lacks a defined catalytic cysteine domain (42). However, unlike ATG7 and Uba4, Uba5 does contain a UFD domain that is probably involved in recruiting its E2 (Ufc1) for transthiolation by analogy to the role of the UFD domain in canonical E1s and structural similarities between Ufc1 and other E2 enzymes (16,18).…”

Section: Discussionmentioning

confidence: 99%

Mechanistic Study of Uba5 Enzyme and the Ufm1 Conjugation Pathway

Gavin

Hoar

et al. 2014

Journal of Biological Chemistry

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Background: Human ubiquitin-like protein Ufm1 and its activating enzyme, Uba5, are involved in endoplasmic reticulum (ER) stress response. Results: The Uba5-Ufm1 conjugation pathway is characterized in both reconstituted systems and cellular settings. Conclusion: Uba5 activates Ufm1 via a distinct two-step mechanism. Significance: This study represents the first mechanistic characterization of Uba5, a homodimeric member of the E1 enzyme family.

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Section: Discussionmentioning

confidence: 99%

Mechanistic Study of Uba5 Enzyme and the Ufm1 Conjugation Pathway

Gavin

Hoar

et al. 2014

Journal of Biological Chemistry

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“…In a transesterification reaction, the activated Ufm1 is then transferred from Uba5 to the Cys116 of Ufc1 [3,5]. Although Ufc1 does not share much sequence identity with other E2 enzymes, a catalytic E2 core domain consisting of 10 amino acids was identified.…”

Section: The Ufm1 Cascadementioning

confidence: 99%

The Ufm1 Cascade

Daniel

Liebau

2014

Cells

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The ubiquitin-fold modifier 1 (Ufm1) is a posttranslational modifier that belongs to the ubiquitin-like protein (UBL) family. Ufm1 is present in nearly all eukaryotic organisms, with the exception of fungi. It resembles ubiquitin in its ability to be ligated to other proteins, as well as in the mechanism of ligation. While the Ufm1 cascade has been implicated in endoplasmic reticulum functions and cell cycle control, its biological role still remains poorly understood. In this short review, we summarize the current state of Ufm1 research and its potential role in human diseases, like diabetes, ischemic heart disease and cancer.

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“…1 were cloned and purified with similar methods (17). Briefly, cDNA templates (MHS1768-98980929.pCR-XL-TOPO) from Open Biosystems were cloned into the pET28MHL or pNICCH vector using the In-Fusion CF Dry-Down PCR cloning kit (639605, Clontech), transformed into Escherichia coli BL21(DE3) cells, and grown in Terrific Broth in the presence of 50 g/ml kanamycin at 37°C.…”

Section: Methodsmentioning

confidence: 99%

The Replication Focus Targeting Sequence (RFTS) Domain Is a DNA-competitive Inhibitor of Dnmt1

Syeda

Fagan

Wean

et al. 2011

Journal of Biological Chemistry

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115

136

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Dnmt1 (DNA methyltransferase 1) is the principal enzyme responsible for maintenance of cytosine methylation at CpG dinucleotides in the mammalian genome. The N-terminal replication focus targeting sequence (RFTS) domain of Dnmt1 has been implicated in subcellular localization, protein association, and catalytic function. However, progress in understanding its function has been limited by the lack of assays for and a structure of this domain. Here, we show that the naked DNA-and polynucleosome-binding activities of Dnmt1 are inhibited by the RFTS domain, which functions by virtue of binding the catalytic domain to the exclusion of DNA. Kinetic analysis with a fluorogenic DNA substrate established the RFTS domain as a 600-fold inhibitor of Dnmt1 enzymatic activity. The crystal structure of the RFTS domain reveals a novel fold and supports a mechanism in which an RFTS-targeted Dnmt1-binding protein, such as Uhrf1, may activate Dnmt1 for DNA binding.DNA cytosine methylation, an epigenetic mark that occurs predominantly at CpG dinucleotides, is the principal eukaryotic DNA modification (1). DNA methylation is a key component of gene silencing, which makes significant contributions to cell phenotype. Establishment and maintenance of DNA methylation patterns are governed by three catalytically active DNA methyltransferases: Dnmt3a, Dnmt3b, and Dnmt1. The Dnmt3 isoforms are responsible for de novo methylation during germ cell and embryonic development and bind with high affinity to unmethylated DNA sequences (2). CpG methylation patterns are maintained in mammals by Dnmt1 with hemimethylated CpG dinucleotides serving as preferred substrates.Human Dnmt1 (1616 amino acids) consists of a conserved C-terminal catalytic core (amino acids 1140 -1616) and a large N-terminal region (amino acids 1-1139) harboring multiple globular conserved domains, including the DMAP1 (DNA methyltransferase-associated protein 1)-binding domain (3), the proliferating cell nuclear antigen-binding domain (4), the replication focus targeting sequence (RFTS) 4 domain (residues 351-600) (5), the CXXC domain (6), and two bromo-adjacent homology (BAH) domains (see Fig. 1 ) (7).The CXXC domain is understood to contribute to catalytic activity by interacting with unmethylated CpG DNA substrates (6,8). This was observed in the recently solved crystal structures of Dnmt1, which encompass sequences from the CXXC domain to the C terminus (9). In the structures, the CXXC domain binds and holds unmethylated duplex CpG-containing DNA away from the active site, whereas the acidic linker between the CXXC and BAH1 domains is bound in the active site between the DNA segment and the S-adenosylhomocysteine product (9). This observation helps to explain the relationship between the CXXC and catalytic domains. However, it does not address the role of domains N-terminal to the CXXC domain, which are not present in the structures.We set out to clarify the structure and function of the RFTS domain. The RFTS domain is conserved by sequence (supplemental Fig. S1) and cont...

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Crystal Structure of the Human Ubiquitin-activating Enzyme 5 (UBA5) Bound to ATP

Cited by 58 publications

References 35 publications

Mechanistic Study of Uba5 Enzyme and the Ufm1 Conjugation Pathway

Mechanistic Study of Uba5 Enzyme and the Ufm1 Conjugation Pathway

The Ufm1 Cascade

The Replication Focus Targeting Sequence (RFTS) Domain Is a DNA-competitive Inhibitor of Dnmt1

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