Gali Prag

Ubiquitin-binding domains.

The covalent modification of proteins by ubiquitination is a major regulatory mechanism of protein degradation and quality control, endocytosis, vesicular trafficking, cell-cycle control, stress response, DNA repair, growth-factor signalling, transcription, gene silencing and other areas of biology. A class of specific ubiquitin-binding domains mediates most of the effects of protein ubiquitination. The known membership of this group has expanded rapidly and now includes at least sixteen domains: UBA, UIM, MIU, DUIM, CUE, GAT, NZF, A20 ZnF, UBP ZnF, UBZ, Ubc, UEV, UBM, GLUE, Jab1/MPN and PFU. The structures of many of the complexes with mono-ubiquitin have been determined, revealing interactions with multiple surfaces on ubiquitin. Inroads into understanding polyubiquitin specificity have been made for two UBA domains, whose structures have been characterized in complex with Lys48-linked di-ubiquitin. Several ubiquitin-binding domains, including the UIM, CUE and A20 ZnF (zinc finger) domains, promote auto-ubiquitination, which regulates the activity of proteins that contain them. At least one of these domains, the A20 ZnF, acts as a ubiquitin ligase by recruiting a ubiquitin-ubiquitin-conjugating enzyme thiolester adduct in a process that depends on the ubiquitin-binding activity of the A20 ZnF. The affinities of the mono-ubiquitin-binding interactions of these domains span a wide range, but are most commonly weak, with Kd>100 microM. The weak interactions between individual domains and mono-ubiquitin are leveraged into physiologically relevant high-affinity interactions via several mechanisms: ubiquitin polymerization, modification multiplicity, oligomerization of ubiquitinated proteins and binding domain proteins, tandem-binding domains, binding domains with multiple ubiquitin-binding sites and co-operativity between ubiquitin binding and binding through other domains to phospholipids and small G-proteins.

A ubiquitin‐binding motif required for intramolecular monoubiquitylation, the CUE domain

Monoubiquitylation is a regulatory signal, like phosphorylation, that can alter the activity, location or structure of a protein. Monoubiquitin signals are likely to be recognized by ubiquitin‐binding proteins that transmit the regulatory information conferred by monoubiquitylation. To identify monoubiquitin‐binding proteins, we used a mutant ubiquitin that lacks the primary site of polyubiquitin chain formation as bait in a two‐hybrid screen. The C‐terminus of Vps9, a protein required in the yeast endocytic pathway, interacted specifically with monoubiquitin. The region required for monoubiquitin binding mapped to the Vps9 CUE domain, a sequence previously identified by database searches as similar to parts of the yeast Cue1 and mammalian Tollip proteins. We demonstrate that CUE domains bind directly to monoubiquitin and we have defined crucial interaction surfaces on both binding partners. The Vps9 CUE domain is required to promote monoubiquitylation of Vps9 by the Rsp5 hect domain ubiquitin ligase. Thus, we conclude that the CUE motif is an evolutionarily conserved monoubiquitin‐binding domain that mediates intramolecular monoubiquitylation.

Mechanism of ubiquitin recognition by the CUE domain of Vps9p.

Coupling of ubiquitin conjugation to ER degradation (CUE) domains are approximately 50 amino acid monoubiquitin binding motifs found in proteins of trafficking and ubiquitination pathways. The 2.3 A structure of the Vps9p-CUE domain is a dimeric domain-swapped variant of the ubiquitin binding UBA domain. The 1.7 A structure of the CUE:ubiquitin complex shows that one CUE dimer binds one ubiquitin molecule. The bound CUE dimer is kinked relative to the unbound CUE dimer and wraps around ubiquitin. The CUE monomer contains two ubiquitin binding surfaces on opposite faces of the molecule that cannot bind simultaneously to a single ubiquitin molecule. Dimerization of the CUE domain allows both surfaces to contact a single ubiquitin molecule, providing a mechanism for high-affinity binding to monoubiquitin.

Structure of the ESCRT-II endosomal trafficking complex

The multivesicular-body (MVB) pathway delivers transmembrane proteins and lipids to the lumen of the endosome. The multivesicular-body sorting pathway has crucial roles in growth-factor-receptor downregulation, developmental signalling, regulation of the immune response and the budding of certain enveloped viruses such as human immunodeficiency virus. Ubiquitination is a signal for sorting into the MVB pathway, which also requires the functions of three protein complexes, termed ESCRT-I, -II and -III (endosomal sorting complex required for transport). Here we report the crystal structure of the core of the yeast ESCRT-II complex, which contains one molecule of the Vps protein Vps22, the carboxy-terminal domain of Vps36 and two molecules of Vps25, and has the shape of a capital letter ‘Y’. The amino-terminal coiled coil of Vps22 and the flexible linker leading to the ubiquitin-binding NZF domain of Vps36 both protrude from the tip of one branch of the ‘Y’. Vps22 and Vps36 form nearly equivalent interactions with the two Vps25 molecules at the centre of the ‘Y’. The structure suggests how ubiquitinated cargo could be passed between ESCRT components of the MVB pathway through the sequential transfer of ubiquitinated cargo from one complex to the next.

Participation of IHF and a distant UP element in the stimulation of the phage lambda PL promoter.

We have previously identified a UP element in the phage λ PL promoter, centred at position −90 from the transcription start site. Integration host factor (IHF), a heterodimeric DNA‐binding and ‐bending protein, binds upstream of the λ PL promoter in a region overlapping the UP element. Stimulation of transcription by IHF requires an intact αCTD and affects the initial binding of RNA polymerase to the promoter. We propose a model for the stimulation of PL by IHF in which IHF bends the DNA to bring the distal UP sequence in closer proximity to the promoter core sequences to allow the docking of the αCTD of RNA polymerase. Furthermore, IHF may also participate in protein–protein interactions with the αCTD. In support of this model, we found that alanine substitutions in αCTD at positions 265, 268, 270 and 275 reduced PL promoter activity. Mutations in the IHF DNA binding site, as well as IHF mutant proteins exhibiting a decreased ability to bend the DNA, were both defective in stimulating the PL promoter. In addition, some of the mutated IHF residues are clustered at a protein surface that interacts with the UP DNA sequence. These residues may also participate in protein–protein interactions with the αCTD.

Structure-based in silico identification of ubiquitin-binding domains provides insights into the ALIX-V:ubiquitin complex and retrovirus budding

The ubiquitylation signal promotes trafficking of endogenous and retroviral transmembrane proteins. The signal is decoded by a large set of ubiquitin (Ub) receptors that tether Ub‐binding domains (UBDs) to the trafficking machinery. We developed a structure‐based procedure to scan the protein data bank for hidden UBDs. The screen retrieved many of the known UBDs. Intriguingly, new potential UBDs were identified, including the ALIX‐V domain. Pull‐down, cross‐linking and E3‐independent ubiquitylation assays biochemically corroborated the in silico findings. Guided by the output model, we designed mutations at the postulated ALIX‐V:Ub interface. Biophysical affinity measurements using microscale‐thermophoresis of wild‐type and mutant proteins revealed some of the interacting residues of the complex. ALIX‐V binds mono‐Ub with a Kd of 119 μM. We show that ALIX‐V oligomerizes with a Hill coefficient of 5.4 and IC50 of 27.6 μM and that mono‐Ub induces ALIX‐V oligomerization. Moreover, we show that ALIX‐V preferentially binds K63 di‐Ub compared with mono‐Ub and K48 di‐Ub. Finally, an in vivo functionality assay demonstrates the significance of ALIX‐V:Ub interaction in equine infectious anaemia virus budding. These results not only validate the new procedure, but also demonstrate that ALIX‐V directly interacts with Ub in vivo and that this interaction can influence retroviral budding.

Synthetic biology approach to reconstituting the ubiquitylation cascade in bacteria

Covalent modification of proteins with ubiquitin (Ub) is widely implicated in the control of protein function and fate. Over 100 deubiquitylating enzymes rapidly reverse this modification, posing challenges to the biochemical and biophysical characterization of ubiquitylated proteins. We circumvented this limitation with a synthetic biology approach of reconstructing the entire eukaryotic Ub cascade in bacteria. Co‐expression of affinity‐tagged substrates and Ub with E1, E2 and E3 enzymes allows efficient purification of ubiquitylated proteins in milligram quantity. Contrary to in‐vitro assays that lead to spurious modification of several lysine residues of Rpn10 (regulatory proteasomal non‐ATPase subunit), the reconstituted system faithfully recapitulates its monoubiquitylation on lysine 84 that is observed in vivo. Mass spectrometry revealed the ubiquitylation sites on the Mind bomb E3 ligase and the Ub receptors Rpn10 and Vps9. Förster resonance energy transfer (FRET) analyses of ubiquitylated Vps9 purified from bacteria revealed that although ubiquitylation occurs on the Vps9‐GEF domain, it does not affect the guanine nucleotide exchanging factor (GEF) activity in vitro. Finally, we demonstrated that ubiquitylated Vps9 assumes a closed structure, which blocks additional Ub binding. Characterization of several ubiquitylated proteins demonstrated the integrity, specificity and fidelity of the system, and revealed new biological findings.

The Hetero-Hexameric Nature of a Chloroplast AAA+ FtsH Protease Contributes to Its Thermodynamic Stability

FtsH is an evolutionary conserved membrane-bound metalloprotease complex. While in most prokaryotes FtsH is encoded by a single gene, multiple FtsH genes are found in eukaryotes. Genetic and biochemical data suggest that the Arabidopsis chloroplast FtsH is a hetero-hexamer. This raises the question why photosynthetic organisms require a heteromeric complex, whereas in most bacteria a homomeric one is sufficient. To gain structural information of the possible complexes, the Arabidopsis FtsH2 (type B) and FtsH5 (type A) were modeled. An in silico study with mixed models of FtsH2/5 suggests that heteromeric hexamer structure with ratio of 4∶2 is more likely to exists. Specifically, calculation of the buried surface area at the interfaces between neighboring subunits revealed that a hetero-complex should be thermodynamically more stable than a homo-hexamer, due to the presence of additional hydrophobic and hydrophilic interactions. To biochemically assess this model, we generated Arabidopsis transgenic plants, expressing epitope-tagged FtsH2 and immuno-purified the protein. Mass-spectrometry analysis showed that FtsH2 is associated with FtsH1, FtsH5 and FtsH8. Interestingly, we found that ‘type B’ subunits (FtsH2 and FtsH8) were 2–3 fold more abundant than ‘type A’ (FtsH1 and FtsH5). The biochemical data corroborate the in silico model and suggest that the thylakoid FtsH hexamer is composed of two ‘type A’ and four ‘type B’ subunits.

Ubiquitylation‐dependent oligomerization regulates activity of Nedd4 ligases

Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac IKS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1‐helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1‐conjugated ubiquitin and the HECT ubiquitin‐binding patch pulls the α1‐helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the IKS channel, thus confirming the functional importance of E3‐ligase autoinhibition.

A phage display system designed to detect and study protein–protein interactions

Analysing protein–protein interactions is critical in proteomics and drug discovery. The usage of 2‐Hybrid (2λ) systems is limited to an in vivo environment. We describe a bacteriophage 2‐Hybrid system for studying protein interactions in vitro. Bait and prey are displayed as fusions to the surface of phage λ that are marked with different selectable drug‐resistant markers. An interaction of phages in vitro through displayed proteins allows bacterial infection by two phages resulting in double drug‐resistant bacterial colonies at very low multiplicity of infections. We demonstrate interaction of the protein sorting signal Ubiquitin with the Vps9‐CUE, a Ubiquitin binding domain, and by the interaction of (Gly‐Glu)4 and (Gly‐Arg)4 peptides. Interruptions of the phage interactions by non‐fused (free) bait or prey molecules show how robust and unique our approach is. We also demonstrate the use of Ubiquitin and CUE display phages to find binding partners in a λ‐display library. The unique usefulness to 2λ is also described.