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Nadine Merkley

Publications and source records attributed to Nadine Merkley.

4 recordsLinked to original sources
PubMed2005

Ubiquitin manipulation by an E2 conjugating enzyme using a novel covalent intermediate.

Degradation of misfolded and damaged proteins by the 26 S proteasome requires the substrate to be tagged with a polyubiquitin chain. Assembly of polyubiquitin chains and subsequent substrate labeling potentially involves three enzymes, an E1, E2, and E3. E2 proteins are key enzymes and form a thioester intermediate through their catalytic cysteine with the C-terminal glycine (Gly76) of ubiquitin. This thioester intermediate is easily hydrolyzed in vitro and has eluded structural characterization. To overcome this, we have engineered a novel ubiquitin-E2 disulfide-linked complex by mutating Gly76 to Cys76 in ubiquitin. Reaction of Ubc1, an E2 from Saccharomyces cerevisiae, with this mutant ubiquitin resulted in an ubiquitin-E2 disulfide that could be purified and was stable for several weeks. Chemical shift perturbation analysis of the disulfide ubiquitin-Ubc1 complex by NMR spectroscopy reveals an ubiquitin-Ubc1 interface similar to that for the ubiquitin-E2 thioester. In addition to the typical E2 catalytic domain, Ubc1 contains an ubiquitin-associated (UBA) domain, and we have utilized NMR spectroscopy to demonstrate that in this disulfide complex the UBA domain is freely accessible to non-covalently bind a second molecule of ubiquitin. The ability of the Ubc1 to bind two ubiquitin molecules suggests that the UBA domain does not interact with the thioester-bound ubiquitin during polyubiquitin chain formation. Thus, construction of this novel ubiquitin-E2 disulfide provides a method to characterize structurally the first step in polyubiquitin chain assembly by Ubc1 and its related class II enzymes.

Catalytic Domain↗
PubMed2004

Solution structure of the flexible class II ubiquitin-conjugating enzyme Ubc1 provides insights for polyubiquitin chain assembly.

E2 conjugating enzymes form a thiol ester intermediate with ubiquitin, which is subsequently transferred to a substrate protein targeted for degradation. While all E2 proteins comprise a catalytic domain where the thiol ester is formed, several E2s (class II) have C-terminal extensions proposed to control substrate recognition, dimerization, or polyubiquitin chain formation. Here we present the novel solution structure of the class II E2 conjugating enzyme Ubc1 from Saccharomyces cerevisiae. The structure shows the N-terminal catalytic domain adopts an alpha/beta fold typical of other E2 proteins. This domain is physically separated from its C-terminal domain by a 22-residue flexible tether. The C-terminal domain adopts a three-helix bundle that we have identified as an ubiquitin-associated domain (UBA). NMR chemical shift perturbation experiments show this UBA domain interacts in a regioselective manner with ubiquitin. This two-domain structure of Ubc1 was used to identify other UBA-containing class II E2 proteins, including human E2-25K, that likely have a similar architecture and to determine the role of the UBA domain in facilitating polyubiquitin chain formation.

Amino Acid Sequence↗
PubMed2003

Interaction of the tail with the catalytic region of a class II E2 conjugating enzyme.

Ubiquitination plays an important role in many biological processes, including DNA repair, cell cycle regulation, and protein degradation. In the latter pathway the ubiquitin-conjugating enzymes or E2 enzymes are important proteins forming a key E2-ubiquitin thiolester prior to substrate labelling. While the structure of the 150-residue catalytic domain has been well characterized, a subset of E2 enzymes (class II) carry a variable length C-terminal "tail" where structural detail is not available. The presence of this C-terminal extension plays an important role in target recognition, ubiquitin chain assembly and oligomerization. In this work NMR spectroscopy was used to determine the secondary structure of the 215-residue yeast E2 protein Ubc1 and the interactions of its C-terminus with the catalytic domain. The C-terminal tail of Ubc1 was found to contain three alpha-helices between residues D169-S176, K183-L193 and N203-L213 providing the first evidence for a well-defined secondary structure in this region. Chemical shift mapping indicated that residues in the L2 loop of the catalytic domain were most affected indicating the C-terminus of Ubc1 likely interacts with this region. This site of interaction is distinct from that observed in the E2-ubiquitin thiolester and may act to protect the catalytic C88 residue and direct the interaction of ubiquitin in the thiolester intermediate.

Binding Sites↗