Identification of SkpA-CulA-F-box E3 ligase complexes in pathogenic Aspergilli.

The ubiquitin proteasome system is critical for the regulation of protein turnover, which is implicated in the modulation of a wide array of biological processes in eukaryotes, ranging from cell senescence to virulence in plant and human hosts. Proteins to be marked for ubiquitination and subsequent degradation are bound by F-box proteins, which are interchangeable substrate-recognising receptors. These F-box proteins bind a wide range of substrates and associate with the adaptor protein Skp1 and the scaffold Cul1 to form Skp1-Cul1-F-box (SCF) complexes. SCF complex components are highly conserved in eukaryotes, ranging from yeast to humans. However, information regarding the composition of these complexes and the biological roles of F-box proteins is limited, specifically in filamentous fungal species like the genus Aspergillus. In this study, we have identified 51 and 55 fbx-encoding genes in the genomes of two pathogenic fungi, A. fumigatus and A. flavus, respectively. Immunoprecipitations of the HA-tagged SkpA adaptor protein revealed that 26 F-box proteins in A. fumigatus and 30 F-box proteins in A. flavus are involved in SCF complex formation during vegetative growth. These interactome data also revealed that a diverse array of SCF complex conformations exist in response to various exogenous stressors. Lastly, we have provided evidence that the F-box protein Fbx45 interacts with SkpA in both species in response to Amphotericin B. Orthologs of the fbx45 gene are highly conserved in Aspergillus species, but are not present within the genomes of organisms such as yeast, plants or humans. This suggests that Fbx45 could potentially be a novel F-box protein that is unique to specific filamentous fungi such as Aspergillus species.

Cullin-RING ubiquitin E3 ligase regulation by the COP9 signalosome.

The cullin-RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The CUL4A-RBX1-DDB1-DDB2 complex (CRL4A(DDB2)) monitors the genome for ultraviolet-light-induced DNA damage. CRL4A(DBB2) is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4A(DDB2) and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN.

Structural basis of high-order oligomerization of the cullin-3 adaptor SPOP.

Protein ubiquitination in eukaryotic cells is mediated by diverse E3 ligase enzymes that each target specific substrates. The cullin E3 ligase complexes are the most abundant class of E3 ligases; they contain various cullin components that serve as scaffolds for interaction with substrate-recruiting adaptor proteins. SPOP is a BTB-domain adaptor of the cullin-3 E3 ligase complexes; it selectively recruits substrates via its N-terminal MATH domain, whereas its BTB domain mediates dimerization and interactions with cullin-3. It has recently been recognized that the high-order oligomerization of SPOP enhances the ubiquitination of substrates. Here, a dimerization interface in the SPOP C-terminus is identified and it is shown that the dimerization interfaces of the BTB domain and of the C-terminus act independently and in tandem to generate high-order SPOP oligomers. The crystal structure of the dimeric SPOP C-terminal domain is reported at 1.5 Šresolution and it is shown that Tyr353 plays a critical role in high-order oligomerization. A model of the high-order SPOP oligomer is presented that depicts a helical organization that could enhance the efficiency of substrate ubiquitination.

Single-particle EM reveals extensive conformational variability of the Ltn1 E3 ligase.

Ltn1 is a 180-kDa E3 ubiquitin ligase that associates with ribosomes and marks certain aberrant, translationally arrested nascent polypeptide chains for proteasomal degradation. In addition to its evolutionarily conserved large size, Ltn1 is characterized by the presence of a conserved N terminus, HEAT/ARM repeats predicted to comprise the majority of the protein, and a C-terminal catalytic RING domain, although the protein's exact structure is unknown. We used numerous single-particle EM strategies to characterize Ltn1's structure based on negative stain and vitreous ice data. Two-dimensional classifications and subsequent 3D reconstructions of electron density maps show that Ltn1 has an elongated form and presents a continuum of conformational states about two flexible hinge regions, whereas its overall architecture is reminiscent of multisubunit cullin-RING ubiquitin ligase complexes. We propose a model of Ltn1 function based on its conformational variability and flexibility that describes how these features may play a role in cotranslational protein quality control.

The mechanism of ubiquitination in the cullin-RING E3 ligase machinery: conformational control of substrate orientation.

In cullin-RING E3 ubiquitin ligases, substrate binding proteins, such as VHL-box, SOCS-box or the F-box proteins, recruit substrates for ubiquitination, accurately positioning and orienting the substrates for ubiquitin transfer. Yet, how the E3 machinery precisely positions the substrate is unknown. Here, we simulated nine substrate binding proteins: Skp2, Fbw7, beta-TrCP1, Cdc4, Fbs1, TIR1, pVHL, SOCS2, and SOCS4, in the unbound form and bound to Skp1, ASK1 or Elongin C. All nine proteins have two domains: one binds to the substrate; the other to E3 ligase modules Skp1/ASK1/Elongin C. We discovered that in all cases the flexible inter-domain linker serves as a hinge, rotating the substrate binding domain, optimally and accurately positioning it for ubiquitin transfer. We observed a conserved proline in the linker of all nine proteins. In all cases, the prolines pucker substantially and the pucker is associated with the backbone rotation toward the E2/ubiquitin. We further observed that the linker flexibility could be regulated allosterically by binding events associated with either domain. We conclude that the flexible linker in the substrate binding proteins orients the substrate for the ubiquitin transfer. Our findings provide a mechanism for ubiquitination and polyubiquitination, illustrating that these processes are under conformational control.

Cullin-5 plays multiple roles in cell fate specification and synapse formation during Drosophila development.

We describe a developmental analysis of Drosophila Cullin-5 (Cul-5) identified from the genome sequence on the basis of its high degree of homology to vertebrate and worm sequences. The gene is expressed in a restricted manner in ectodermal cells throughout development suggesting pleiotropic functions. We decided to examine the phenotypes of Cul-5 aberrations in two well-studied developmental systems: the neuromuscular junction (NMJ) and the developing sensory organ. Alteration of Cul-5 levels in motoneurons results in an increase in bouton number at the NMJ. The cells of a sensory organ on the adult notum arise from a single progenitor cell by regulated cell division. Aberrations in Cul-5 affect different steps in the lineage consistent with a role in cell fate determination, proliferation, and death. Such phenotypes highlight the multiple cellular processes in which Cul-5 can participate.