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1.
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.  相似文献   

2.
Cbl proteins are E3 ubiquitin ligases that are negative regulators of many receptor tyrosine kinases. Cbl-b and c-Cbl contain a ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin-mediated processes. Despite high sequence identity, Cbl UBA domains display remarkably different ubiquitin-binding properties. Here, we report the crystal structure of the UBA domain of Cbl-b in complex with ubiquitin at 1.9 A resolution. The structure reveals an atypical mechanism of ubiquitin recognition by the first helix of the UBA. Helices 2 and 3 of the UBA domain form a second binding surface, which mediates UBA dimerization in the crystal and in solution. Site-directed mutagenesis demonstrates that Cbl-b dimerization is regulated by ubiquitin binding and required for tyrosine phosphorylation of Cbl-b and ubiquitination of Cbl-b substrates. These studies demonstrate a role for ubiquitin in regulating biological activity by promoting protein dimerization.  相似文献   

3.
The cellular inhibitor of apoptosis (cIAP) proteins are essential RING E3 ubiquitin ligases that regulate apoptosis and inflammatory responses. cIAPs contain a ubiquitin-associated (UBA) domain that binds ubiquitin and is implicated in the regulation of cell survival and proteasomal degradation. Here we show that mutation of the MGF and LL motifs in the UBA domain of cIAP1 caused unfolding and increased cIAP1 multimonoubiquitylation. By developing a UBA mutant that disrupted ubiquitin binding but not the structure of the UBA domain, we found that the UBA domain enhances cIAP1 and cIAP2 ubiquitylation. We demonstrate that the UBA domain binds to the UbcH5b∼Ub conjugate, and this promotes RING domain-dependent monoubiquitylation. This study establishes ubiquitin-binding modules, such as the UBA domain, as important regulatory modules that can fine tune the activity of E3 ligases.  相似文献   

4.
E2 conjugating enzymes form a thiol ester intermediate with ubiquitin, which is subsequently transferred to a substrate protein targeted for degradation. While all E2 proteins comprise a catalytic domain where the thiol ester is formed, several E2s (class II) have C-terminal extensions proposed to control substrate recognition, dimerization, or polyubiquitin chain formation. Here we present the novel solution structure of the class II E2 conjugating enzyme Ubc1 from Saccharomyces cerevisiae. The structure shows the N-terminal catalytic domain adopts an alpha/beta fold typical of other E2 proteins. This domain is physically separated from its C-terminal domain by a 22-residue flexible tether. The C-terminal domain adopts a three-helix bundle that we have identified as an ubiquitin-associated domain (UBA). NMR chemical shift perturbation experiments show this UBA domain interacts in a regioselective manner with ubiquitin. This two-domain structure of Ubc1 was used to identify other UBA-containing class II E2 proteins, including human E2-25K, that likely have a similar architecture and to determine the role of the UBA domain in facilitating polyubiquitin chain formation.  相似文献   

5.
The conjugation of polyubiquitin to target proteins acts as a signal that regulates target stability, localization, and function. Several ubiquitin binding domains have been described, and while much is known about ubiquitin binding to the isolated domains, little is known with regard to how the domains interact with polyubiquitin in the context of full-length proteins. Isopeptidase T (IsoT/USP5) is a deubiquitinating enzyme that is largely responsible for the disassembly of unanchored polyubiquitin in the cell. IsoT has four ubiquitin binding domains: a zinc finger domain (ZnF UBP), which binds the proximal ubiquitin, a UBP domain that forms the active site, and two ubiquitin-associated (UBA) domains whose roles are unknown. Here, we show that the UBA domains are involved in binding two different polyubiquitin isoforms, linear and K48-linked. Using isothermal titration calorimetry, we show that IsoT has at least four ubiquitin binding sites for both polyubiquitin isoforms. The thermodynamics of the interactions reveal that the binding is enthalpy-driven. Mutation of the UBA domains suggests that UBA1 and UBA2 domains of IsoT interact with the third and fourth ubiquitins in both polyubiquitin isoforms, respectively. These data suggest that recognition of the polyubiquitin isoforms by IsoT involves considerable conformational mobility in the polyubiquitin ligand, in the enzyme, or in both.  相似文献   

6.
The ubiquitin-associated (UBA) domain is one of the most frequently occurring motifs that recognize ubiquitin tags. Dsk2p, a UBA-containing protein from Saccharomyces cerevisiae, is involved in the ubiquitin-proteasome proteolytic pathway and has been implicated in spindle pole duplication. Here we present the solution structure of the UBA domain of Dsk2p (Dsk2(UBA)) in complex with ubiquitin. The structure reveals that the UBA domain uses a mode of ubiquitin recognition that is similar to that of the CUE domain, another ubiquitin binding motif that shares low sequence homology but high structural similarity with UBA domains. These two domains, as well as the structurally unrelated ubiquitin binding motif UIM, provide a common, crucial recognition site for ubiquitin, comprising a hydrogen-bonding acceptor for the amide group of Gly-47, and a methyl group that packs against the hydrophobic pocket of ubiquitin formed by Leu-8, Ile-44, His-68, and Val-70.  相似文献   

7.
p62/SQSTM1/A170 is a multimodular protein that is found in ubiquitin-positive inclusions associated with neurodegenerative diseases. Recent findings indicate that p62 mediates the interaction between ubiquitinated proteins and autophagosomes, leading these proteins to be degraded via the autophagy-lysosomal pathway. This ubiquitin-mediated selective autophagy is thought to begin with recognition of the ubiquitinated proteins by the C-terminal ubiquitin-associated (UBA) domain of p62. We present here the crystal structure of the UBA domain of mouse p62 and the solution structure of its ubiquitin-bound form. The p62 UBA domain adopts a novel dimeric structure in crystals, which is distinctive from those of other UBA domains. NMR analyses reveal that in solution the domain exists in equilibrium between the dimer and monomer forms, and binding ubiquitin shifts the equilibrium toward the monomer to form a 1:1 complex between the UBA domain and ubiquitin. The dimer-to-monomer transition is associated with a structural change of the very C-terminal end of the p62 UBA domain, although the UBA fold itself is essentially maintained. Our data illustrate that dimerization and ubiquitin binding of the p62 UBA domain are incompatible with each other. These observations reveal an autoinhibitory mechanism in the p62 UBA domain and suggest that autoinhibition plays a role in the function of p62.  相似文献   

8.
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.  相似文献   

9.
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 Kd 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.  相似文献   

10.
Wnt/β-catenin signaling plays a central role in development and is also involved in a diverse array of diseases. β-Catenin activity is tightly regulated via a multiprotein complex that includes the kinase glycogen synthase kinase-3β (GSK-3β). GSK-3β phosphorylates β-catenin, marking it for ubiquitination and degradation via the proteasome. Thus in regulation of the Wnt pathway, the ubiquitin system is known to be involved mostly in mediating the turnover of β-catenin, resulting in reduced Wnt signaling levels. Here we report that an arm of the ubiquitin system increases β-catenin protein levels. We show that GSK-3β directly interacts with the E3 ubiquitin ligase identified by differential display (EDD) that also binds β-catenin. Expression of EDD leads to enhanced nuclear accumulation of both GSK-3β and β-catenin and results in up-regulation of β-catenin expression levels and activity. Importantly, EDD ubiquitinates β-catenin through Lys29- or Lys11-linked ubiquitin chains, leading to enhanced stability of β-catenin. Our results demonstrate a role for the ubiquitin system in up-regulation of the Wnt signaling pathway, suggesting that EDD could function as a colorectal oncogene.  相似文献   

11.
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.  相似文献   

12.
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.  相似文献   

13.
Mechanism of ubiquitin recognition by the CUE domain of Vps9p   总被引:9,自引:0,他引:9  
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.  相似文献   

14.
Garner TP  Long J  Layfield R  Searle MS 《Biochemistry》2011,50(21):4665-4674
The scaffold protein p62/SQSTM1 acts as a hub in regulating a diverse range of signaling pathways which are dependent upon a functional ubiquitin-binding C-terminal UBA domain. Mutations linked to Paget's disease of bone (PDB) commonly cluster within the UBA domain. The p62 UBA domain is unique in forming a highly stable dimer which regulates ubiquitin recognition by using overlapping surface patches in both dimerization and ubiquitin binding, making the two association events competitive. NMR structural analysis and biophysical methods show that some PDB mutations modulated the ubiquitin binding affinity by both direct and indirect mechanisms that affect UBA structural integrity, dimer stability, and contacts at the UBA-ubiquitin interface. In other cases, common PDB mutations (P392L in particular) result in no significant change in ubiquitin binding affinity for the UBA domain in isolation; however, all PDB UBA mutations lead to loss of function with respect to ubiquitin binding in the context of full-length p62, suggesting a more complex underlying mechanism.  相似文献   

15.
E2-25K is an ubiquitin-conjugating enzyme with the ability to synthesize Lys48-linked polyubiquitin chains. E2-25K and its homologs represent the only known E2 enzymes which contain a C-terminal ubiquitin-associated (UBA) domain as well as the conserved catalytic ubiquitin-conjugating (UBC) domain. As an additional non-covalent binding surface for ubiquitin, the UBA domain must provide some functional specialization. We mapped the protein–protein interface involved in the E2-25K UBA/ubiquitin complex by solution nuclear magnetic resonance (NMR) spectroscopy and subsequently modeled the structure of the complex. Domain–domain interactions between the E2-25K catalytic UBC domain and the UBA domain do not induce significant structural changes in the UBA domain or alter the affinity of the UBA domain for ubiquitin. We determined that one of the roles of the C-terminal UBA domain, in the context of E2-25K, is to increase processivity in Lys48-linked polyubiquitin chain synthesis, possibly through increased binding to the ubiquitinated substrate. Additionally, we see evidence that the UBA domain directs specificity in polyubiquitin chain linkage.  相似文献   

16.
The BUZ/Znf-UBP domain is a distinct ubiquitin-binding module found in the cytoplasmic deacetylase HDAC6, the E3 ubiquitin ligase BRAP2/IMP, and a subfamily of deubiquitinating enzymes. Here, we report the solution structure of the BUZ domain of Ubp-M, a ubiquitin-specific protease, and its interaction with ubiquitin. Unlike the BUZ domain from isopeptidase T (isoT) that contains a single zinc finger, the Ubp-M BUZ domain features three zinc-binding sites consisting of 12 residues. These zinc ligands form a pair of cross-braced ring fingers encapsulated within a third zinc finger in the primary structure. In contrast to isoT, which can form an N-terminal loop swapped dimer in the crystal state, the formation of additional zinc fingers in the Ubp-M BUZ domain restricts its N-terminal loop to intra-domain interactions. The ubiquitin-binding site of the Ubp-M BUZ domain is mapped to the highly conserved, concave surface formed by the alpha 3 helix and the central beta-sheet. We further show that this site binds to the C-terminal tail of free ubiquitin, and corresponding peptides display essentially the same binding affinities as full-length ubiquitin does for the Ubp-M BUZ domain. However, modification of the G76(Ub) carboxylate group either by a peptide or isopeptide bond abolishes BUZ-domain interaction. The unique ubiquitin-recognition mode of the BUZ domain family suggests that they may function as "sensors" of free ubiquitin in cells to achieve regulatory roles in many aspects of ubiquitin-dependent processes.  相似文献   

17.
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.  相似文献   

18.
Lee PC  Sowa ME  Gygi SP  Harper JW 《Molecular cell》2011,43(3):392-405
Vertebrates express two enzymes for activation of ubiquitin-UBA1, which is responsible for activation of the vast majority of E2 conjugating enzymes, and UBA6, which uses the dedicated E2, USE1. However, targets and E3s for UBA6-USE1 are unknown. Here, we demonstrate that UBA6-USE1 functions with the UBR1-3 subfamily of N-recognin E3s to degrade the N-end rule substrates RGS4, RGS5, and Arg (R)-GFP. This pathway functions in the cytoplasm in parallel with the UBA1-UBE2A/B-UBR2 cascade, which promotes turnover of nuclear RGS4/5 proteins and an apparently phenotypically distinct pool of cytoplasmic RGS4/5. UBR2 promotes Lys48 (K48)-specific ubiquitin discharge from, and RGS4 ubiquitylation by, both USE1 and UBE2A in vitro. This work provides insight into the machinery employed by the UBA6-USE1 cascade to promote protein turnover and suggests that the UBA6 and UBA1 pathways can function in parallel with the same E3 to degrade the same targets in a spatially distinct manner.  相似文献   

19.
Meyer HH  Wang Y  Warren G 《The EMBO journal》2002,21(21):5645-5652
The multiple functions of the p97/Cdc48p ATPase can be explained largely by adaptors that link its activity to different cellular pathways, but how these adaptors recognize different substrates is unclear. Here we present evidence that the mammalian adaptors, p47 and Ufd1-Npl4, both bind ubiquitin conjugates directly and so link p97 to ubiquitylated substrates. In the case of Ufd1-Npl4, which is involved in endoplasmic reticulum (ER)-associated degradation and nuclear envelope reassembly, binding to ubiquitin is mediated through a putative zinc finger in Npl4. This novel domain (NZF) is conserved in metazoa and is both present and functional in other proteins. In the case of p47, which is involved in the reassembly of the ER, the nuclear envelope and the Golgi apparatus, binding is mediated by a UBA domain. Unlike Ufd1-Npl4, it binds ubiquitin only when complexed with p97, and binds mono- rather than polyubiquitin conjugates. The UBA domain is required for the function of p47 in mitotic Golgi reassembly. Together, these data suggest that ubiquitin recognition is a common feature of p97-mediated reactions.  相似文献   

20.
Interaction between the signal-transducing adapter molecule 1 (STAM1) Vps27/Hrs/Stam (VHS) domain and ubiquitin was investigated by nuclear magnetic resonance (NMR) spectroscopy. NMR evidence showed that the structure of STAM1 VHS domain resembles that of other VHS domains, especially the homologous domain of STAM2. We found that the VHS domain binds to ubiquitin via its hydrophobic patch consisting of N-terminus of helix 2 and C-terminus of helix 4 in which Trp26 on helix 2 plays a pivotal role in the binding. The binding between VHS and ubiquitin seems to be very similar to that between ubiquitin associated domain (UBA) and ubiquitin, however, the direction of α-helices involved in the ubiquitin binding is opposite. Here, we propose a novel ubiquitin binding site and the manner of ubiquitin recognition of the STAM1 VHS domain.

Structured summary

MINT-6804185:STAM1 (uniprotkb:Q92783) binds (MI:0407) to ubiquitin (uniprotkb:P62988) by nuclear magnetic resonance (MI:0077)  相似文献   

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