Surface plasmon resonance to measure interactions of UbFs with their binding partners.

Ubiquitin family modifiers (UbFs) are protein-protein interaction modules acting within a variety of cellular processes. In combination with other techniques, surface plasmon resonance (SPR)-based technology has been used to characterize the interactions of UbFs with their binding partners. SPR binding assays allow the real-time detection of binding events with unlabeled analytes, yet are often hindered by the requirement for careful sample preparation and optimized assay conditions. This chapter aims to share our experience in SPR analysis of UbFs and provide helpful hints in sample preparation, experimental design, evaluation, and data interpretation.

The yeast E4 ubiquitin ligase Ufd2 interacts with the ubiquitin-like domains of Rad23 and Dsk2 via a novel and distinct ubiquitin-like binding domain.

Proteins containing ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains interact with various binding partners and function as hubs during ubiquitin-mediated protein degradation. A common interaction of the budding yeast UBL-UBA proteins Rad23 and Dsk2 with the E4 ubiquitin ligase Ufd2 has been described in endoplasmic reticulum-associated degradation among other pathways. The UBL domains of Rad23 and Dsk2 play a prominent role in this process by interacting with Ufd2 and different subunits of the 26 S proteasome. Here, we report crystal structures of Ufd2 in complex with the UBL domains of Rad23 and Dsk2. The N-terminal UBL-interacting region of Ufd2 exhibits a unique sequence pattern, which is distinct from any known ubiquitin- or UBL-binding domain identified so far. Residue-specific differences exist in the interactions of these UBL domains with Ufd2, which are coupled to subtle differences in their binding affinities. The molecular details of their differential interactions point to a role for adaptive evolution in shaping these interfaces.

Affinity makes the difference: nonselective interaction of the UBA domain of Ubiquilin-1 with monomeric ubiquitin and polyubiquitin chains.

Ubiquilin/PLIC proteins belong to the family of UBL-UBA proteins implicated in the regulation of the ubiquitin-dependent proteasomal degradation of cellular proteins. A human presenilin-interacting protein, ubiquilin-1, has been suggested as potential therapeutic target for treating Huntington's disease. Ubiquilin's interactions with mono- and polyubiquitins are mediated by its UBA domain, which is one of the tightest ubiquitin binders among known ubiquitin-binding domains. Here we report the three-dimensional structure of the UBA domain of ubiquilin-1 (UQ1-UBA) free in solution and in complex with ubiquitin. UQ1-UBA forms a compact three-helix bundle structurally similar to other known UBAs, and binds to the hydrophobic patch on ubiquitin with a K(d) of 20 microM. To gain structural insights into UQ1-UBA's interactions with polyubiquitin chains, we have mapped the binding interface between UQ1-UBA and Lys48- and Lys63-linked di-ubiquitins and characterized the strength of UQ1-UBA binding to these chains. Our NMR data show that UQ1-UBA interacts with the individual ubiquitin units in both chains in a mode similar to its interaction with mono-ubiquitin, although with an improved binding affinity for the chains. Our results indicate that, in contrast to UBA2 of hHR23A that has strong binding preference for Lys48-linked chains, UQ1-UBA shows little or no binding selectivity toward a particular chain linkage or between the two ubiquitin moieties in the same chain. The structural data obtained in this study provide insights into the possible structural reasons for the diversity of polyubiquitin chain recognition by UBA domains.

Ubiquitin receptors and ERAD: a network of pathways to the proteasome.

The elimination of misfolded proteins, known as protein quality control, is an essential cellular process. Removal of misfolded proteins from the secretory pathway depends on their recognition in the endoplasmic reticulum (ER) followed by their retrograde transport into the cytosol for degradation. The AAA-ATPase Cdc48/p97 facilitates the translocation of misfolded ER-proteins into the cytosol. Cdc48/p97 can dock onto the ER-membrane via direct interaction with ER-membrane proteins and/or indirectly via its substrate-recruiting cofactors, which interact with the ubiquitylated substrates at the membrane. This tight interaction in conjunction with the conformational changes induced upon ATP hydrolysis within Cdc48/p97 is thought to provide the driving force for the translocation reaction. Subsequently, a series of protein-protein interactions between the Cdc48/p97 complex, its cofactors, and the ubiquitylated substrates is instrumental for the proper delivery of the ER substrates to the proteasome. These protein-protein interactions are governed mainly by ubiquitin-fold and ubiquitin-binding domains.

A ubiquitin-interacting motif protects polyubiquitinated Met4 from degradation by the 26S proteasome.

Covalent attachment of ubiquitin to proteins regulates a host of cellular events by proteolysis dependent and independent mechanisms. A variety of protein domains that bind non-covalently to ubiquitin have been described and functionally linked to diverse cellular processes. Overall, however, the understanding and knowledge of the mechanisms by which ubiquitin-binding domains (UBDs) regulate these processes is limited. Here, we describe identification of a UBD in the yeast transcription factor Met4. Met4 activity, but not its stability, is regulated by polyubiquitination. We found that the UBD restricts the length of the polyubiquitin chain that is assembled on Met4, and prevents proteasomal recognition and degradation of polyubiquitinated Met4. Inactivation of the UBD allowed synthesis of longer ubiquitin chains on Met4 and transformed the normally stable polyubiquitinated Met4 into a short-lived protein. Our results demonstrate a function for UBDs in ubiquitin-chain synthesis and regulation of protein degradation.

Controlled synthesis of polyubiquitin chains.

Many intracellular signaling processes depend on the modification of proteins with polymers of the conserved 76-residue protein ubiquitin. The ubiquitin units in such polyubiquitin chains are connected by isopeptide bonds between a specific lysine residue of one ubiquitin and the carboxyl group of G76 of the next ubiquitin. Chains linked through K48-G76 and K63-G76 bonds are the best characterized, signaling proteasome degradation and nonproteolytic outcomes, respectively. The molecular determinants of polyubiquitin chain recognition are under active investigation; both the chemical structure and the length of the chain can influence signaling outcomes. In this article, we describe the protein reagents necessary to produce K48- and K63-linked polyubiquitin chains and the use of these materials to produce milligram quantities of specific-length chains for biochemical and biophysical studies. The method involves reactions catalyzed by linkage-specific conjugating factors, in which proximally and distally blocked monoubiquitins (or chains) are joined to produce a particular chain product in high yield. Individual chains are then deblocked and joined in another round of reaction. Successive rounds of deblocking and synthesis give rise to a chain of the desired length.

Diverse polyubiquitin interaction properties of ubiquitin-associated domains.

The ubiquitin-associated (UBA) domain occurs frequently in proteins involved in ubiquitin-dependent signaling pathways. Although polyubiquitin chain binding is considered to be a defining feature of the UBA domain family, the generality of this property has not been established. Here we have surveyed the polyubiquitin interaction properties of 30 UBA domains, including 16 of 17 occurrences in budding yeast. The UBA domains sort into four classes that include linkage-selective polyubiquitin binders and domains that bind different chains (and monoubiquitin) in a nondiscriminatory manner; one notable class ( approximately 30%) did not bind any ubiquitin ligand surveyed. The properties of a given UBA domain are conserved from yeast to mammals. Their functional relevance is further suggested by the ability of an ectopic UBA domain to alter the specificity of a deubiquitylating enzyme in a predictable manner. Conversely, non-UBA sequences can modulate the interaction properties of a UBA domain.

Structural determinants for selective recognition of a Lys48-linked polyubiquitin chain by a UBA domain.

Although functional diversity in polyubiquitin chain signaling has been ascribed to the ability of differently linked chains to bind in a distinctive manner to effector proteins, structural models of such interactions have been lacking. Here, we use NMR to unveil the structural basis of selective recognition of Lys48-linked di- and tetraubiquitin chains by the UBA2 domain of hHR23A. Although the interaction of UBA2 with Lys48-linked diubiquitin involves the same hydrophobic surface on each ubiquitin unit as that utilized in monoubiquitin:UBA complexes, our results show how the "closed" conformation of Lys48-linked diubiquitin is crucial for high-affinity binding. Moreover, recognition of Lys48-linked diubiquitin involves a unique epitope on UBA, which allows the formation of a sandwich-like diubiqutin:UBA complex. Studies of the UBA-tetraubiquitin interaction suggest that this mode of UBA binding to diubiquitin is relevant for longer chains.

Ubiquitin chain synthesis.

Several important signaling processes depend on the tagging of cellular proteins with "polyubiquitin chains"-ubiquitin polymers whose building blocks are connected by isopeptide bonds between G76 of one ubiquitin and a specific lysine residue of the next one. Here we describe procedures for the synthesis of polyubiquitin chains of defined lengths that are linked through the K48 or K63 side chains. The method involves a series of enzymatic reactions in which proximally and distally blocked monoubiquitins (or chains) are conjugated to produce a particular chain in high yield. Individual chains are then deblocked and joined in another round of reaction. Successive rounds of deblocking and synthesis can give rise to a chain of any desired length.

FAT10, a ubiquitin-independent signal for proteasomal degradation.

FAT10 is a small ubiquitin-like modifier that is encoded in the major histocompatibility complex and is synergistically inducible by tumor necrosis factor alpha and gamma interferon. It is composed of two ubiquitin-like domains and possesses a free C-terminal diglycine motif that is required for the formation of FAT10 conjugates. Here we show that unconjugated FAT10 and a FAT10 conjugate were rapidly degraded by the proteasome at a similar rate. Fusion of FAT10 to the N terminus of very long-lived proteins enhanced their degradation rate as potently as fusion with ubiquitin did. FAT10-green fluorescent protein fusion proteins were not cleaved but entirely degraded, suggesting that FAT10-specific deconjugating enzymes were not present in the analyzed cell lines. Interestingly, the prevention of ubiquitylation of FAT10 by mutation of all lysines or by expression in ubiquitylation-deficient cells did not affect FAT10 degradation. Thus, conjugation with FAT10 is an alternative and ubiquitin-independent targeting mechanism for degradation by the proteasome, which, in contrast to polyubiquitylation, is cytokine inducible and irreversible.

Binding of polyubiquitin chains to ubiquitin-associated (UBA) domains of HHR23A.

Ubiquitin-associated (UBA) domains are small protein domains that occur in the context of larger proteins and are likely to function as inter- and intramolecular communication elements in ubiquitin/polyubiquitin signaling. Although monoubiquitin/UBA complexes are well characterized, much less is known about UBA/polyubiquitin complexes, even though polyubiquitin chains are believed to be biologically relevant ligands of many UBA domain proteins. Here, we report the results of a quantitative study of the interaction of K48-linked polyubiquitin chains with UBA domains of the DNA repair/proteolysis protein HHR23A, using surface plasmon resonance and other approaches. We present evidence that the UBL domain of HHR23A negatively regulates polyubiquitin/UBA interactions and identify leucine 8 of ubiquitin as an important determinant of chain recognition. A striking relationship between binding affinity and chain length suggests that maximum affinity is associated with a conformational feature that is fully formed in chains of n = 4-6 and can be recognized by a single UBA domain of HHR23A. Our findings provide new insights into polyubiquitin chain recognition and set the stage for future structural investigations of UBA/polyubiquitin complexes.

NEDD8 ultimate buster-1L interacts with the ubiquitin-like protein FAT10 and accelerates its degradation.

FAT10 is an interferon-gamma-inducible ubiquitin-like protein that consists of two ubiquitin-like domains. FAT10 bears a diglycine motif at its C terminus that can form isopeptide bonds to so far unidentified target proteins. Recently we found that FAT10 and its conjugates are rapidly degraded by the proteasome and that the N-terminal fusion of FAT10 to a long lived protein markedly reduces its half-life. FAT10 may hence direct target proteins to the proteasome for degradation. In this study we report a new interaction partner of FAT10 that may link FAT10 to the proteasome. A yeast two-hybrid screen identified NEDD8 ultimate buster-1L (NUB1L) as a non-covalent binding partner of FAT10, and this interaction was confirmed by coimmunoprecipitation and glutathione S-transferase pull-down experiments. NUB1L is also an interferon-inducible protein that has been reported to interact with the ubiquitin-like protein NEDD8, thus leading to accelerated NEDD8 degradation. Here we show that NUB1L binds to FAT10 much stronger than to NEDD8 and that NEDD8 cannot compete with FAT10 for NUB1L binding. The interaction of FAT10 and NUB1L is specific as green fluorescent fusion proteins containing ubiquitin or SUMO-1 do not bind to NUB1L. The coexpression of NUB1L enhanced the degradation rate of FAT10 8-fold, whereas NEDD8 degradation was only accelerated 2-fold. Because NUB1 was shown to bind to the proteasome subunit RPN10 in vitro and to be contained in 26 S proteasome preparations, it may function as a linker that targets FAT10 for degradation by the proteasome.

Solution conformation of Lys63-linked di-ubiquitin chain provides clues to functional diversity of polyubiquitin signaling.

Diverse cellular events are regulated by post-translational modification of substrate proteins via covalent attachment of one or a chain of ubiquitin molecules. The outcome of (poly)ubiquitination depends upon the specific lysine residues involved in the formation of polyubiquitin chains. Lys48-linked chains act as a universal signal for proteasomal degradation, whereas Lys63-linked chains act as a specific signal in several non-degradative processes. Although it has been anticipated that functional diversity between alternatively linked polyubiquitin chains relies on linkage-dependent differences in chain conformation/topology, direct structural evidence in support of this model has been lacking. Here we use NMR methods to determine the structure of a Lys63-linked di-ubiquitin chain. The structure is characterized by an extended conformation, with no direct contact between the hydrophobic residues Leu8, Ile44, and Val70 on the ubiquitin units. This structure contrasts with the closed conformation observed for Lys48-linked di-ubiquitin wherein these residues form the interdomain interface (Cook, W. J., Jeffrey, L. C., Carson, M., Zhijian, C., and Pickart, C. M. (1992) J. Biol. Chem. 267, 16467-16471; Varadan, R., Walker, O., Pickart, C., and Fushman, D. (2002) J. Mol. Biol. 324, 637-647). Consistent with the open conformation of the Lys(63)-linked di-ubiquitin, our binding studies show that both ubiquitin domains in this chain can bind a ubiquitin-associated domain from HHR23A independently and in a mode similar to that for mono-ubiquitin. In contrast, Lys48-linked di-ubiquitin binds in a different, higher affinity mode that has yet to be determined. This is the first experimental evidence that alternatively linked polyubiquitin chains adopt distinct conformations.

Rad23 ubiquitin-associated domains (UBA) inhibit 26 S proteasome-catalyzed proteolysis by sequestering lysine 48-linked polyubiquitin chains.

Most substrates of the 26 S proteasome are recognized only following conjugation to a Lys48-linked polyubiquitin chain. Rad23 is one member of a family of proteins that possesses an N-terminal ubiquitin-like domain (UbL) and a C-terminal ubiquitin-associated domain(s) (UBA). Recent studies have shown that UbLs interact with 26 S proteasomes, whereas UBAs bind polyubiquitin chains. These biochemical properties suggest that UbL-UBA proteins may shuttle polyubiquitinated substrates to proteasomes. Here we show that contrary to prediction from this model, the effect of human Rad23A on the degradation of polyubiquitinated substrates catalyzed by purified proteasomes is exclusively inhibitory. Strong inhibition is dependent on the presence of both UBAs, independent of the UbL, and can be explained by competition between the UBA domains and the proteasome for binding to substrate-linked polyubiquitin chains. The UBA domains bind Lys48-linked polyubiquitin chains in strong preference to Lys63 or Lys29-linked chains, leading to selective inhibition of the assembly and disassembly of Lys48-linked chains. These results place constraints on the mechanism(s) by which UbL-UBA proteins promote proteasome-catalyzed proteolysis and reveal new properties of UBA domains.