38 research outputs found
The nucleotide-binding site of Aquifex aeolicus LpxC
The structure of UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) in complex with UDP is reported. The complex allows for a description of how the enzyme recognizes and binds a nucleotide moiety and enables the construction of an LpxC-substrate model
Casitas B-lineage lymphoma linker helix mutations found in myeloproliferative neoplasms affect conformation
Background: Casitas B-lineage lymphoma (Cbl or c-Cbl) is a RING ubiquitin ligase that negatively regulates protein
tyrosine kinase (PTK) signalling. Phosphorylation of a conserved residue (Tyr371) on the linker helix region (LHR)
between the substrate-binding and RING domains is required to ubiquitinate PTKs, thereby flagging them for
degradation. This conserved Tyr is a mutational hotspot in myeloproliferative neoplasms. Previous studies have
revealed that select point mutations in Tyr371 can potentiate transformation in cells and mice but not all possible
mutations do so. To trigger oncogenic potential, Cbl Tyr371 mutants must perturb the LHR-substrate-binding
domain interaction and eliminate PTK ubiquitination. Although structures of native and pTyr371-Cbl are available,
they do not reveal how Tyr371 mutations affect Cblās conformation. Here, we investigate how Tyr371 mutations
affect Cblās conformation in solution and how this relates to Cblās ability to potentiate transformation in cells.
Results: To explore how Tyr371 mutations affect Cblās properties, we used surface plasmon resonance to measure
Cbl mutant binding affinities for E2 conjugated with ubiquitin (E2āUb), small angle X-ray scattering studies to
investigate Cbl mutant conformation in solution and focus formation assays to assay Cbl mutant transformation
potential in cells. Cbl Tyr371 mutants enhance E2āUb binding and cause Cbl to adopt extended conformations
in solution. LHR flexibility, RING domain accessibility and transformation potential are associated with the extent
of LHR-substrate-binding domain perturbation affected by the chemical nature of the mutation. More disruptive
mutants like Cbl Y371D or Y371S are more extended and the RING domain is more accessible, whereas Cbl Y371F
mimics native Cbl in solution. Correspondingly, the only Tyr371 mutants that potentiate transformation in cells are
those that perturb the LHR-substrate-binding domain interaction.
Conclusions: c-Cblās LHR mutations are only oncogenic when they disrupt the native state and fail to ubiquitinate
PTKs. These findings provide new insights into how LHR mutations deregulate c-Cbl
A general strategy for discovery of inhibitors and activators of RING and U-box E3 ligases with ubiquitin variants
RING and U-box E3 ubiquitin ligases regulate diverse eukaryotic processes and have been implicated in numerous diseases, but targeting these enzymes remains a major challenge. We report the development of three ubiquitin variants (UbVs), each binding selectively to the RING or U-box domain of a distinct E3 ligase: monomeric UBE4B, phosphorylated active CBL, or dimeric XIAP. Structural and biochemical analyses revealed that UbVs specifically inhibited the activity of UBE4B or phosphorylated CBL by blocking the E2ā¼Ub binding site. Surprisingly, the UbV selective for dimeric XIAP formed a dimer to stimulate E3 activity by stabilizing the closed E2ā¼Ub conformation. We further verified the inhibitory and stimulatory functions of UbVs in cells. Our work provides a general strategy to inhibit or activate RING/U-box E3 ligases and provides a resource for the research community to modulate these enzymes
Identification and characterization of mutations in ubiquitin required for non-covalent dimer formation
Ubiquitin (Ub) is a small protein that post-translationally modifies a variety of substrates in eukaryotic cells to modulate substrate function. The ability of Ub to interact with numerous protein domains makes Ub an attractive scaffold for engineering ubiquitin variants (UbVs) with high target specificity. Previously, we identified a UbV that formed a non-covalent stable dimer via a Ī²-strand exchange, and in the current work we identified and characterized the minimal substitutions in the primary sequence of Ub required to form a higher ordered complex. Using solution angle scattering and X-ray crystallography, we show that a single substitution of residue Gly10 to either Ala or Val is sufficient to convert Ub from a monomer to a dimer. We also investigate contributions to dimer formation by the residues in the surrounding sequence. These results can be used to develop next-generation phage-display libraries of UbVs to engineer new interfaces for protein recognition
DELTEX2 C-terminal domain recognizes and recruits ADP-ribosylated proteins for ubiquitination
Cross-talk between ubiquitination and ADP-ribosylation regulates spatiotemporal recruitment of key players in many signaling pathways. The DELTEX family ubiquitin ligases (DTX1 to DTX4 and DTX3L) are characterized by a RING domain followed by a C-terminal domain (DTC) of hitherto unknown function. Here, we use two label-free mass spectrometry techniques to investigate the interactome and ubiquitinated substrates of human DTX2 and identify a large proportion of proteins associated with the DNA damage repair pathway. We show that DTX2-catalyzed ubiquitination of these interacting proteins requires PARP1/2-mediated ADP-ribosylation and depends on the DTC domain. Using a combination of structural, biochemical, and cell-based techniques, we show that the DTX2 DTC domain harbors an ADP-riboseābinding pocket and recruits poly-ADP-ribose (PAR)āmodified proteins for ubiquitination. This PAR-binding property of DTC domain is conserved across the DELTEX family E3s. These findings uncover a new ADP-riboseābinding domain that facilitates PAR-dependent ubiquitination
Structural insights into the catalysis and regulation of E3 ubiquitin ligases
Covalent attachment (conjugation) of one or more ubiquitin molecules to protein substrates governs numerous eukaryotic cellular processes, including apoptosis, cell division and immune responses. Ubiquitylation was originally associated with protein degradation, but it is now clear that ubiquitylation also mediates processes such as proteināprotein interactions and cell signalling depending on the type of ubiquitin conjugation. Ubiquitin ligases (E3s) catalyse the final step of ubiquitin conjugation by transferring ubiquitin from ubiquitin-conjugating enzymes (E2s) to substrates. In humans, more than 600 E3s contribute to determining the fates of thousands of substrates; hence, E3s need to be tightly regulated to ensure accurate substrate ubiquitylation. Recent findings illustrate how E3s function on a structural level and how they coordinate with E2s and substrates to meticulously conjugate ubiquitin. Insights regarding the mechanisms of E3 regulation, including structural aspects of their autoinhibition and activation are also emerging
Building a Model of the Active Site of Rubisco
(Statement of Responsibility) by Lori Buetow(Thesis) Thesis (B.A.) -- New College of Florida, 1996(Electronic Access) RESTRICTED TO NCF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE(Bibliography) Includes bibliographical references.(Source of Description) This bibliographic record is available under the Creative Commons CC0 public domain dedication. The New College of Florida, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.(Local) Faculty Sponsor: Scudder, Pau