The yeast Epsin Ent1 is recruited to membranes through multiple independent interactions

In addition to its well known role in targeting proteins for proteasomal degradation, ubiquitin (Ub) is also involved in promoting internalization of cell surface proteins into the endocytic pathway. Moreover, putative Ub interaction motifs (UIMs) as well as Ub-associated (UBA) domains have been identified in key yeast endocytic proteins (the epsins Ent1 and Ent2, and the Eps15 homolog Ede1). In this study, we characterized the interaction of Ub with the Ede1 UBA domain and with the UIMs of Ent1. Our data suggest that the UIMs and the UBA are involved in binding these proteins to biological membranes. We also show that the Ent1 ENTH domain binds to phosphoinositides in vitro and that Ent1 NPF motifs interact with the EH domain-containing proteins Ede1 and Pan1. Our findings indicate that the ENTH domain interaction with membrane lipids cooperates with the binding of membrane-associated Ub moieties. These events may in turn favor the occurrence of other interactions, for instance EH-NPF recognition, thus stabilizing networks of low affinity binding partners at endocytic sites.     

Solution structure of Vps27 UIM-ubiquitin complex important for endosomal sorting and receptor downregulation

Monoubiquitylation is a well-characterized signal for the internalization and sorting of integral membrane proteins to distinct cellular organelles. Recognition and transmission of monoubiquitin signals is mediated by a variety of ubiquitin-binding motifs such as UIM, UBA, UEV, VHS and CUE in endocytic proteins. The yeast Vps27 protein requires two UIMs for efficient interactions with ubiquitin and for sorting cargo into multivesicular bodies. Here we show that the individual UIMs of Vps27 exist as autonomously folded alpha-helices that bind ubiquitin independently, non-cooperatively and with modest affinity. The Vps27 N-terminal UIM engages the Leu8-Ile44-Val70 hydrophobic patch of ubiquitin through a helical surface conserved in UIMs of diverse proteins, including that of the S5a proteasomal regulatory subunit. The Leu8-Ile44-Val70 ubiquitin surface is also the site of interaction for CUE and UBA domains in endocytic proteins, consistent with the view that ubiquitin-binding endocytic proteins act serially on the same monoubiquitylated cargo during transport from cell surface to the lysosome.     

The Function of Yeast Epsin and Ede1 Ubiquitin-Binding Domains During Receptor Internalization

The formation of a primary endocytic vesicle is a dynamic process involving the transient organization of adaptor and scaffold proteins at the plasma membrane. Epsins and Eps15-like proteins are ubiquitin-binding proteins that act early in this process. The yeast epsins, Ent1 and Ent2, carry functional ubiquitin-interacting motifs (UIMs), whereas the yeast Eps15-like protein, Ede1, has a C-terminal ubiquitin-associated (UBA) domain. Analysis of mutants lacking early endocytic adaptors reveals that the ubiquitin-binding domains (UBDs) of Ent2 and Ede1 are likely to function primarily to mediate protein–protein interactions between components of the early endocytic machinery. Cells that lack epsin and Ede1 UBDs are able to internalize activated, ubiquitinated receptors. Furthermore, under conditions in which epsin UIMs are important for receptor internalization, receptors internalized via both ubiquitin-dependent and ubiquitin-independent signals require the UIMs, indicating that UIM function is not restricted to ubiquitinated receptors. Epsin UIMs share function with non-UBD protein–protein interaction motifs in Ent2 and Ede1, and the Ede1 UBA domain appears to negatively regulate interactions between endocytic proteins. Together, our results suggest that the ubiquitin-binding domains within the yeast epsin Ent2 and Ede1 are involved in the formation and regulation of the endocytic network.     

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.     

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.     

Atypical binding of the Swa2p UBA domain to ubiquitin

Swa2p is an auxilin-like yeast protein that is involved in vesicular transport and required for uncoating of clathrin-coated vesicles. Swa2p contains a ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin (Ub)-mediated processes. We have determined a structural model of the Swa2p UBA domain in complex with Ub using NMR spectroscopy and molecular docking. Ub recognition occurs predominantly through an atypical interaction in which UBA helix α1 and the N-terminal part of helix α2 bind to Ub. Mutation of Ala148, a key residue in helix α1, to polar residues greatly reduced the affinity of the UBA domain for Ub and revealed a second low-affinity Ub-binding site located on the surface formed by helices α1 and α3. Surface plasmon resonance showed that the Swa2p UBA domain binds K48- and K63-linked di-Ub in a non-linkage-specific manner. These results reveal convergent evolution of a Ub-binding site on helix α1 of UBA domains involved in membrane protein trafficking.     

Structural basis of ubiquitin recognition by the ubiquitin-associated (UBA) domain of the ubiquitin ligase EDD

EDD (or HYD) is an E3 ubiquitin ligase in the family of HECT (homologous to E6-AP C terminus) ligases. EDD contains an N-terminal ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin-mediated processes. Here, we use isothermal titration calorimetry (ITC), NMR titrations, and pull-down assays to show that the EDD UBA domain binds ubiquitin. The 1.85 A crystal structure of the complex with ubiquitin reveals the structural basis of ubiquitin recognition by UBA helices alpha1 and alpha3. The structure shows a larger number of intermolecular hydrogen bonds than observed in previous UBA/ubiquitin complexes. Two of these involve ordered water molecules. The functional importance of residues at the UBA/ubiquitin interface was confirmed using site-directed mutagenesis. Surface plasmon resonance (SPR) measurements show that the EDD UBA domain does not have a strong preference for polyubiquitin chains over monoubiquitin. This suggests that EDD binds to monoubiquitinated proteins, which is consistent with its involvement in DNA damage repair pathways.     

Solution structures of UBA domains reveal a conserved hydrophobic surface for protein-protein interactions

UBA domains are a commonly occurring sequence motif of approximately 45 amino acid residues that are found in diverse proteins involved in the ubiquitin/proteasome pathway, DNA excision-repair, and cell signaling via protein kinases. The human homologue of yeast Rad23A (HHR23A) is one example of a nucleotide excision-repair protein that contains both an internal and a C-terminal UBA domain. The solution structure of HHR23A UBA(2) showed that the domain forms a compact three-helix bundle. We report the structure of the internal UBA(1) domain of HHR23A. Comparison of the structures of UBA(1) and UBA(2) reveals that both form very similar folds and have a conserved large hydrophobic surface patch. The structural similarity between UBA(1) and UBA(2), in spite of their low level of sequence conservation, leads us to conclude that the structural variability of UBA domains in general is likely to be rather small. On the basis of the structural similarities as well as analysis of sequence conservation, we predict that this hydrophobic surface patch is a common protein-interacting surface present in diverse UBA domains. Furthermore, accumulating evidence that ubiquitin binds to UBA domains leads us to the prediction that the hydrophobic surface patch of UBA domains interacts with the hydrophobic surface on the five-stranded beta-sheet of ubiquitin. Detailed comparison of the structures of the two UBA domains, combined with previous mutagenesis studies, indicates that the binding site of HIV-1 Vpr on UBA(2) does not completely overlap the ubiquitin binding site.     

Solution structure of the ubiquitin-associated domain of human BMSC-UbP and its complex with ubiquitin

Ubiquitin is an important cellular signal that targets proteins for degradation or regulates their functions. The previously identified BMSC-UbP protein derived from bone marrow stromal cells contains a ubiquitin-associated (UBA) domain at the C terminus that has been implicated in linking cellular processes and the ubiquitin system. Here, we report the solution NMR structure of the UBA domain of human BMSC-UbP protein and its complex with ubiquitin. The structure determination was facilitated by using a solubility-enhancement tag (SET) GB1, immunoglobulin G binding domain 1 of Streptococcal protein G. The results show that BMSC-UbP UBA domain is primarily comprised of three alpha-helices with a hydrophobic patch defined by residues within the C terminus of helix-1, loop-1, and helix-3. The M-G-I motif is similar to the M/L-G-F/Y motifs conserved in most UBA domains. Chemical shift perturbation study revealed that the UBA domain binds with the conserved five-stranded beta-sheet of ubiquitin via hydrophobic interactions with the dissociation constant (KD) of approximately 17 microM. The structural model of BMSC-UbP UBA domain complexed with ubiquitin was constructed by chemical shift mapping combined with the program HADDOCK, which is in agreement with the result from mutagenesis studies. In the complex structure, three residues (Met76, Ile78, and Leu99) of BMSC-UbP UBA form a trident anchoring the domain to the hydrophobic concave surface of ubiquitin defined by residues Leu8, Ile44, His68, and Val70. This complex structure may provide clues for BMSC-UbP functions and structural insights into the UBA domains of other ubiquitin-associated proteins that share high sequence homology with BMSC-UbP UBA domain.     

Complex of Fas-associated Factor 1 (FAF1) with Valosin-containing Protein (VCP)-Npl4-Ufd1 and Polyubiquitinated Proteins Promotes Endoplasmic Reticulum-associated Degradation (ERAD)

Fas-associated factor 1 (FAF1) is a ubiquitin receptor containing multiple ubiquitin-related domains including ubiquitin-associated (UBA), ubiquitin-like (UBL) 1, UBL2, and ubiquitin regulatory X (UBX). We previously showed that N-terminal UBA domain recognizes Lys⁴⁸-ubiquitin linkage to recruit polyubiquitinated proteins and that a C-terminal UBX domain interacts with valosin-containing protein (VCP). This study shows that FAF1 interacts only with VCP complexed with Npl4-Ufd1 heterodimer, a requirement for the recruitment of polyubiquitinated proteins to UBA domain. Intriguingly, VCP association to C-terminal UBX domain regulates ubiquitin binding to N-terminal UBA domain without direct interaction between UBA and UBX domains. These interactions are well characterized by structural and biochemical analysis. VCP-Npl4-Ufd1 complex is known as the machinery required for endoplasmic reticulum-associated degradation. We demonstrate here that FAF1 binds to VCP-Npl4-Ufd1 complex via UBX domain and polyubiquitinated proteins via UBA domain to promote endoplasmic reticulum-associated degradation.     

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.     

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 μM. 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.     

UBA domains of DNA damage-inducible proteins interact with ubiquitin

Rad23 is a highly conserved protein involved in nucleotide excision repair (NER) that associates with the proteasome via its N-terminus. Its C-terminal ubiquitin-associated (UBA) domain is evolutionarily conserved from yeast to humans. However, the cellular function of UBA domains is not completely understood. Recently, RAD23 and DDI1, both DNA damage-inducible genes encoding proteins with UBA domains, were implicated genetically in Pds1-dependent mitotic control in yeast. The UBA domains of RAD23 and DDI1 are required for these interactions. Timely degradation of Pds1 via the ubiquitin/proteasome pathway allows anaphase onset and is crucial for chromosome maintenance. Here, we show that Rad23 and Ddi1 interact directly with ubiquitin and that this interaction is dependent on their UBA domains, providing a possible mechanism for UBA-dependent cell cycle control. Moreover, we show that a hydrophobic surface on the UBA domain, which from structural work had been predicted to be a protein-protein interaction interface, is indeed required for ubiquitin binding. By demonstrating that UBA domains interact with ubiquitin, we have provided the first indication of a cellular function for the UBA domain.     

Solution Structure of the Ubiquitin-associated (UBA) Domain of Human Autophagy Receptor NBR1 and Its Interaction with Ubiquitin and Polyubiquitin

NBR1 (neighbor of BRCA1 gene 1) is a protein commonly found in ubiquitin-positive inclusions in neurodegenerative diseases. Due to its high architectural similarity to the well studied autophagy receptor protein p62/SQSTM1, NBR1 has been thought to analogously bind to ubiquitin-marked autophagic substrates via its C-terminal ubiquitin-associated (UBA) domain and deliver them to autophagosomes for degradation. Unexpectedly, we find that NBR1 differs from p62 in its UBA structure and accordingly in its interaction with ubiquitin. Structural differences are observed on helix α-3, which is tilted farther from helix α-2 and extended by approximately one turn in NBR1. This results not only in inhibition of a p62-type self-dimerization of NBR1 UBA but also in a significantly higher affinity for monoubiquitin as compared with p62 UBA. Importantly, the NBR1 UBA-ubiquitin complex structure shows that the negative charge of the side chain in front of the conserved MGF motif in the UBA plays an integral role in the recognition of ubiquitin. In addition, NMR and isothermal titration calorimetry experiments show that NBR1 UBA binds to each monomeric unit of polyubiquitin with similar affinity and by the same surface used for binding to monoubiquitin. This indicates that NBR1 lacks polyubiquitin linkage-type specificity, in good agreement with the nonspecific linkages observed in intracellular ubiquitin-positive inclusions. Consequently, our results demonstrate that the structural differences between NBR1 UBA and p62 UBA result in a much higher affinity of NBR1 for ubiquitin, which in turn suggests that NBR1 may form intracellular inclusions with ubiquitylated autophagic substrates more efficiently than p62.     

Specificity of the interaction between ubiquitin-associated domains and ubiquitin

Ubiquitin-associated (UBA) domains are found in a large number of proteins with diverse functions involved in ubiquitination, DNA repair, and signaling pathways. Recent studies have shown that several UBA domain proteins interact with ubiquitin (Ub), specifically p62, the phosphotyrosine-independent ligand of the SH2 domain of p56(lck); HHR23A, a human nucleotide excision repair protein; and DDI1, another damage-inducible protein. NMR chemical shift mapping reveals that Ub binds specifically but weakly to a conserved hydrophobic epitope on HHR23A UBA(1) and UBA(2) and that the UBA domains bind on the hydrophobic patch on the surface of the five-stranded beta-sheet of Ub. Models of the UBA(1)-Ub and UBA(2)-Ub complexes obtained from de novo docking reveal different orientations of the UBA domains on the Ub surface compared with those obtained by homology modeling with the related CUE domains, which also bind Ub. Our results suggest that UBA domains may interact with Ub as well as other proteins in more than one way while utilizing the same binding surface.     

Structural basis for ubiquitin recognition by SH3 domains

The SH3 domain is a protein-protein interaction module commonly found in intracellular signaling and adaptor proteins. The SH3 domains of multiple endocytic proteins have been recently implicated in binding ubiquitin, which serves as a signal for diverse cellular processes including gene regulation, endosomal sorting, and protein destruction. Here we describe the solution NMR structure of ubiquitin in complex with an SH3 domain belonging to the yeast endocytic protein Sla1. The ubiquitin binding surface of the Sla1 SH3 domain overlaps substantially with the canonical binding surface for proline-rich ligands. Like many other ubiquitin-binding motifs, the SH3 domain engages the Ile44 hydrophobic patch of ubiquitin. A phenylalanine residue located at the heart of the ubiquitin-binding surface of the SH3 domain serves as a key specificity determinant. The structure of the SH3-ubiquitin complex explains how a subset of SH3 domains has acquired this non-traditional function.     

The specificity of ubiquitin binding to ubiquilin-1 is regulated by sequences besides its UBA domain

UBQLN proteins regulate proteostasis by facilitating clearance of misfolded proteins through the proteasome and autophagy degradation pathways. Consistent with its proteasomal function, UBQLN proteins contain both UBL and UBA domains, which bind subunits of the proteasome, including the S5a subunit, and ubiquitin chains, respectively. Conclusions regarding the binding properties of UBQLN proteins have been derived principally through studies of its individual domains, not the full-length (FL) proteins. Here we describe the in vitro binding properties of FL-UBQLN1 with the S5a subunit of the proteasome and two different lysine-linked (K48 or K63) ubiquitin chains. We show that in contrast to its isolated UBA domain, which binds almost equally well with both K48 and K63 ubiquitin chains, FL UBQLN1 binds preferentially with K63 chains. Furthermore, we show that deletion of the UBL domain from UBQLN1 abrogates ubiquitin binding. Taken together these results suggest that sequences outside of the UBA domain in UBQLN1 function to regulate the specificity and binding with different ubiquitin moieties. We also show that the UBL domain of UBQLN1 is required for S5a binding and that its binding to UBQLN1, in turn, enhances K48 ubiquitin chain binding to the complex. We discuss the implications of our findings with the known function of UBQLN proteins in protein degradation.     

NMR reveals a different mode of binding of the Stam2 VHS domain to ubiquitin and diubiquitin

The VHS domain of the Stam2 protein is a ubiquitin binding domain involved in the recognition of ubiquitinated proteins committed to lysosomal degradation. Among all VHS domains, the VHS domain of Stam proteins is the strongest binder to monoubiqiuitin and exhibits preferences for K63-linked chains. In the present paper, we report the solution NMR structure of the Stam2-VHS domain in complex with monoubiquitin by means of chemical shift perturbations, spin relaxation, and paramagnetic relaxation enhancements. We also characterize the interaction of Stam2-VHS with K48- and K63-linked diubiquitin chains and report the first evidence that VHS binds differently to these two chains. Our data reveal that VHS enters the hydrophobic pocket of K48-linked diubiquitin and binds the two ubiquitin subunits with different affinities. In contrast, VHS interacts with K63-linked diubiquitin in a mode similar to its interaction with monoubiquitin. We also suggest possible structural models for both K48- and K63-linked diubiquitin in interaction with VHS. Our results, which demonstrate a different mode of binding of VHS for K48- and K63-linked diubiquitin, may explain the preference of VHS for K63- over K48-linked diubiquitin chains and monoubiquitin.