Structural and functional characterization of a ubiquitin variant engineered for tight and specific binding to an alpha‐helical ubiquitin interacting motif

Ubiquitin interacting motifs (UIMs) are short α‐helices found in a number of eukaryotic proteins. UIMs interact weakly but specifically with ubiquitin conjugated to other proteins, and in so doing, mediate specific cellular signals. Here we used phage display to generate ubiquitin variants (UbVs) targeting the N‐terminal UIM of the yeast Vps27 protein. Selections yielded UbV.v27.1, which recognized the cognate UIM with high specificity relative to other yeast UIMs and bound with an affinity more than two orders of magnitude higher than that of ubiquitin. Structural and mutational studies of the UbV.v27.1‐UIM complex revealed the molecular details for the enhanced affinity and specificity of UbV.v27.1, and underscored the importance of changes at the binding interface as well as at positions that do not contact the UIM. Our study highlights the power of the phage display approach for selecting UbVs with unprecedented affinity and high selectivity for particular α‐helical UIM domains within proteomes, and it establishes a general approach for the development of inhibitors targeting interactions of this type.     

Yeast Two-Hybrid Analysis for Ubiquitin Variant Inhibitors of Human Deubiquitinases

We applied a yeast-two-hybrid (Y2H) analysis to screen for ubiquitin variant (UbV) inhibitors of a human deubiquitinase (DUB), ubiquitin-specific protease 2 (USP2). The Y2H screen used USP2 as the bait and a prey library consisting of UbVs randomized at four specific positions, which were known to interact with USP2 from phage display analysis. The screen yielded numerous UbVs that bound to USP2 both as a Y2H interaction in vivo and as purified proteins in vitro. The Y2H-derived UbVs inhibited the catalytic activity of USP2 in vitro with nanomolar-range potencies, and they bound and inhibited USP2 in human cells. Mutational and structural analysis showed that potent and selective inhibition could be achieved by just two substitutions in a UbV, which exhibited improved hydrophobic and hydrophilic contacts compared to the wild-type ubiquitin interaction with USP2. Our results establish Y2H as an effective platform for the development of UbV inhibitors of DUBs in vivo, providing an alternative strategy for the analysis of DUBs that are recalcitrant to phage display and other in vitro methods.     

Structural and functional characterization of ubiquitin variant inhibitors for the JAMM-family deubiquitinases STAMBP and STAMBPL1

Ubiquitination is a crucial posttranslational protein modification involved in a myriad of biological pathways. This modification is reversed by deubiquitinases (DUBs) that deconjugate the single ubiquitin (Ub) moiety or poly-Ub chains from substrates. In the past decade, tremendous efforts have been focused on targeting DUBs for drug discovery. However, most chemical compounds with inhibitory activity for DUBs suffer from mild potency and low selectivity. To overcome these obstacles, we developed a phage display-based protein engineering strategy for generating Ub variant (UbV) inhibitors, which was previously successfully applied to the Ub-specific protease (USP) family of cysteine proteases. In this work, we leveraged the UbV platform to selectively target STAMBP, a member of the JAB1/MPN/MOV34 (JAMM) metalloprotease family of DUB enzymes. We identified two UbVs (UbV^SP.1 and UbV^SP.3) that bind to STAMBP with high affinity but differ in their selectivity for the closely related paralog STAMBPL1. We determined the STAMBPL1-UbV^SP.1 complex structure by X-ray crystallography, revealing hotspots of the JAMM-UbV interaction. Finally, we show that UbV^SP.1 and UbV^SP.3 are potent inhibitors of STAMBP isopeptidase activity, far exceeding the reported small-molecule inhibitor BC-1471. This work demonstrates that UbV technology is suitable to develop molecules as tools to target metalloproteases, which can be used to further understand the cellular function of JAMM family DUBs.     

A Panel of Engineered Ubiquitin Variants Targeting the Family of Domains Found in Ubiquitin Specific Proteases (DUSPs)

Domains found in ubiquitin specific proteases (DUSPs) occur in seven members of the ubiquitin specific protease (USP) family. DUSPs are defined by a distinct structural fold but their functions remain largely unknown, although studies with USP4 suggest that its DUSP enhances deubiquitination activity. We used phage-displayed libraries of ubiquitin variants (UbVs) to derive protein-based tools to target DUSP family members with high affinity and specificity. We designed a UbV library based on insights from the structure of a previously identified UbV bound to the DUSP of USP15. The new library yielded 33 unique UbVs that bound to DUSPs from five different USPs (USP4, USP11, USP15, USP20 and USP33). For each USP, we were able to identify at least one DUSP that bound with high affinity and absolute specificity relative to the other DUSPs. We showed that UbVs targeting the DUSPs of USP15, USP11 and USP20 inhibited the catalytic activity of the enzyme, despite the fact that the DUSP is located outside of the catalytic domain. These findings provide an alternative means of inhibiting USP activity by targeting DUSPs, and this mechanism could be potentially extended other DUSP-containing USPs.     

Solution structure of UIM and interaction of tandem ubiquitin binding domains in STAM1 with ubiquitin

STAM1 and Hrs are the components of ESCRT-0 complex for lysosomal degradation of membrane proteins is composed of STAM1 Hrs and has multiple ubiquitin binding domains. Here, the solution structure of STAM1 UIM, one of the ubiquitin binding motif, was determined by NMR spectroscopy. The structure of UIM adopts an α-helix with amphipathic nature. The central hydrophobic residues in UIM provides the binding surface for ubiquitin binding and are flanked with positively and negatively charged residues on both sides. The docking model of STAM1 UIM-ubiquitin complex is suggested. In NMR and ITC experiments with the specifically designed mutant proteins, we investigated the ubiquitin interaction of tandem ubiquitin binding domains from STAM1. The ubiquitin binding affinity of the VHS domain and UIM in STAM1 was 52.4 and 94.9 μM, and 1.5 and 2.2 fold increased, respectively, than the value obtained from the isolated domain or peptide. The binding affinities here would be more physiologically relevant and provide more precise understanding in ESCRT pathway of lysosomal degradation.     

Structure and ubiquitin binding of the ubiquitin-interacting motif

Ubiquitylation is used to target proteins into a large number of different biological processes including proteasomal degradation, endocytosis, virus budding, and vacuolar protein sorting (Vps). Ubiquitylated proteins are typically recognized using one of several different conserved ubiquitin binding modules. Here, we report the crystal structure and ubiquitin binding properties of one such module, the ubiquitin-interacting motif (UIM). We found that UIM peptides from several proteins involved in endocytosis and vacuolar protein sorting including Hrs, Vps27p, Stam1, and Eps15 bound specifically, but with modest affinity (Kd = 0.1-1 mm), to free ubiquitin. Full affinity ubiquitin binding required the presence of conserved acidic patches at the N and C terminus of the UIM, as well as highly conserved central alanine and serine residues. NMR chemical shift perturbation mapping experiments demonstrated that all of these UIM peptides bind to the I44 surface of ubiquitin. The 1.45 A resolution crystal structure of the second yeast Vps27p UIM (Vps27p-2) revealed that the ubiquitin-interacting motif forms an amphipathic helix. Although Vps27p-2 is monomeric in solution, the motif unexpectedly crystallized as an antiparallel four-helix bundle, and the potential biological implications of UIM oligomerization are therefore discussed.     

Mapping the interactions between Lys48 and Lys63-linked di-ubiquitins and a ubiquitin-interacting motif of S5a

Numerous cellular processes are regulated by (poly)ubiquitin-mediated signaling events, which involve a covalent modification of the substrate protein by a single ubiquitin or a chain of ubiquitin molecules linked via a specific lysine. Remarkably, the outcome of polyubiquitination is linkage-dependent. For example, Lys48-linked chains are the principal signal for proteasomal degradation, while Lys63-linked chains act as nonproteolytic signals. Despite significant progress in characterization of various cellular pathways involving ubiquitin, understanding of the structural details of polyubiquitin chain recognition by downstream cellular effectors is missing. Here we use NMR to study the interaction of a ubiquitin-interacting motif (UIM) of the proteasomal subunit S5a with di-ubiquitin, the simplest model for polyubiquitin chain, to gain insights into the mechanism of polyubiquitin recognition by the proteasome. We have mapped the binding interface and characterized the stoichiometry and the process of UIM binding to Lys48- and Lys63-linked di-ubiquitin chains. Our data provide the first direct evidence that UIM binding involves a conformational transition in Lys48-linked di-ubiquitin, which opens the hydrophobic interdomain interface. This allows UIM to enter the interface and bind directly to the same ubiquitin hydrophobic-patch surface as utilized in UIM:monoubiquitin complexes. The results indicate that up to two UIM molecules can bind di-ubiquitin, and the binding interface between UIM and ubiquitin units in di-ubiquitin is essentially the same for both Lys48- and Lys63-linked chains. Our data suggest possible structural models for the binding of UIM and of full-length S5a to di-ubiquitin.     

Structural basis for specific recognition of Lys 63‐linked polyubiquitin chains by tandem UIMs of RAP80

RAP80 has a key role in the recruitment of the Abraxas–BRCC36–BRCA1–BARD1 complex to DNA‐damage foci for DNA repair through specific recognition of Lys 63‐linked polyubiquitinated proteins by its tandem ubiquitin‐interacting motifs (UIMs). Here, we report the crystal structure of the RAP80 tandem UIMs (RAP80‐UIM1‐UIM2) in complex with Lys 63‐linked di‐ubiquitin at 2.2 Å resolution. The two UIMs, UIM1 and UIM2, and the α‐helical inter‐UIM region together form a continuous 60 Å‐long α‐helix. UIM1 and UIM2 bind to the proximal and distal ubiquitin moieties, respectively. Both UIM1 and UIM2 of RAP80 recognize an Ile 44‐centered hydrophobic patch on ubiquitin but neither UIM interacts with the Lys 63‐linked isopeptide bond. Our structure suggests that the inter‐UIM region forms a 12 Å‐long α‐helix that ensures that the UIMs are arranged to enable specific binding of Lys 63‐linked di‐ubiquitin. This was confirmed by pull‐down analyses using RAP80‐UIM1‐UIM2 mutants of various length inter‐UIM regions. Further, we show that the Epsin1 tandem UIM, which has an inter‐UIM region similar to that of RAP80‐UIM1‐UIM2, also selectively binds Lys 63‐linked di‐ubiquitin.     

Double-sided ubiquitin binding of Hrs-UIM in endosomal protein sorting

Hrs has an essential role in sorting of monoubiquitinated receptors to multivesicular bodies for lysosomal degradation, through recognition of ubiquitinated receptors by its ubiquitin-interacting motif (UIM). Here, we present the structure of a complex of Hrs-UIM and ubiquitin at 1.7-A resolution. Hrs-UIM forms a single alpha-helix, which binds two ubiquitin molecules, one on either side. These two ubiquitin molecules are related by pseudo two-fold screw symmetry along the helical axis of the UIM, corresponding to a shift by two residues on the UIM helix. Both ubiquitin molecules interact with the UIM in the same manner, using the Ile44 surface, with equal binding affinities. Mutational experiments show that both binding sites of Hrs-UIM are required for efficient degradative protein sorting. Hrs-UIM belongs to a new subclass of double-sided UIMs, in contrast to its yeast homolog Vps27p, which has two tandem single-sided UIMs.     

Defining how ubiquitin receptors hHR23a and S5a bind polyubiquitin

Ubiquitin receptors connect substrate ubiquitylation to proteasomal degradation. HHR23a binds proteasome subunit 5a (S5a) through a surface that also binds ubiquitin. We report that UIM2 of S5a binds preferentially to hHR23a over polyubiquitin, and we provide a model for the ternary complex that we expect represents one of the mechanisms used by the proteasome to capture ubiquitylated substrates. Furthermore, we demonstrate that hHR23a is surprisingly adept at sequestering the ubiquitin moieties of a polyubiquitin chain, and provide evidence that it and the ubiquitylated substrate are committed to each other after binding.     

3个六肽的胰岛素受体结合和体内生物活性

为了研究胰岛素受体结合部位的结构和功能,设计并用固相方法合成了３个六肽．在浓度大于１×１０３ｎｍｏｌ／Ｌ时,ｃｙｃｌｏ（Ｐｈｅ－Ｐｈｅ－Ｖａｌ－Ｌｅｕ－Ｔｙｒ－Ｇｌｙ）具有明显的胰岛素受体结合活力;Ｈ－Ｐｈｅ－Ｐｈｅ－Ｖａｌ－Ｌｅｕ－Ｔｙｒ－Ｇｌｙ－ＯＨ的这一活力则不明显;而Ｈ－Ｇｌｙ－Ｇｌｕ－Ａｒｇ－Ｇｌｙ－Ｐｈｅ－Ｐｈｅ－ＯＨ则增强胰岛素和其受体的亲和性．然而,它们都没有体内生物活性．这表明：环六肽部分模拟了胰岛素受体结合部位的空间构象;胰岛素受体结合部位的疏水性和其中的Ｂ２３Ｇｌｙ－Ｂ２４Ｐｈｅ－Ｂ２５Ｐｈｅ对胰岛素和其受体的结合起重要作用．     

Dimeric analogs of immunosuppressive decapeptide fragment of ubiquitin

Our previous studies revealed that ubiquitin and its decapeptide fragment with the LEDGRTLSDY sequence, located on the exposed molecule loop, strongly suppressed the immune response. This suggested that the loop may serve as a functional epitope of ubiquitin molecule and that a possible mechanism of biological action of the synthesized peptides is associated with interfering in interactions of ubiquitin with other molecules. Ubiquitin is known to exist in oligomeric forms, which can interact with various oligomeric receptors. We designed and synthesized new dimeric analogs of the ubiquitin fragment, to probe whether dimeric peptides may have higher affinity towards the ubiquitin receptors responsible for immunosuppression, which are believed to form oligomeric structures. Three dimerization strategies, N‐terminus to N‐terminus, C‐terminus to C‐terminus, and N‐terminus to C‐terminus (head‐to‐tail) via PEG derivatives were used to synthesize the dimeric peptides on solid support. In the course of our research, we developed a new and straightforward procedure of dimerization where α‐amino groups of the C‐terminal lysine residues of two peptide fragments were linked by PEG spacer directly on solid support. The effect of dimeric analogs on the immunological response was tested in the AFC in vitro experiment. The immunological tests showed that the head‐to‐tail dimerization caused a more profound increase in the biological activity than other tested dimerization methods. Our results suggest that such orientation of peptide components may correspond to orientation of the hypothetic ubiquitin receptors responsible for the immunomodulatory activity. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Structure of the UBA domain of Dsk2p in complex with ubiquitin molecular determinants for ubiquitin recognition

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.     

Synthesis and AT2 receptor-binding properties of angiotensin II analogues.

The present study investigates the importance of the amino acid side chains in the octapeptide angiotensin II (Ang II) for binding to the AT2 receptor. A Gly scan was performed where each amino acid in Ang II was substituted one-by-one with glycine. The resulting set of peptides was tested for affinity to the AT2 receptor (porcine myometrial membranes). For a comparison, the peptides were also tested for affinity to the AT1 receptor (rat liver membranes). Only the substitution of Arg2 reduced affinity to the AT2 receptor considerably (92-fold when compared with Ang II). For the other Gly-substituted analogues the affinity to the AT2 receptor was only moderately affected. To further investigate the role of the Arg2 side chain for receptor binding, we synthesized some N-terminally modified Ang II analogues. According to these studies a positive charge in the N-terminal end of angiotensin III [Ang II (2-8)] is not required for high AT2 receptor affinity but seems to be more important in Ang II. With respect to the AT1 receptor, [Gly2]Ang II and [Gly8]Ang II lacked binding affinity (Ki > 10 microM). Replacement of the Val3 or Ile5 residues with Gly produced only a slight decrease in affinity. Interestingly, substitution of Tyr4 or His6, which are known to be very important for AT1 receptor binding, resulted in only 48 and 14 times reduction in affinity, respectively.     

Crystal structure of the ubiquitin binding domains of rabex-5 reveals two modes of interaction with ubiquitin

The interaction between ubiquitinated proteins and intracellular proteins harboring ubiquitin binding domains (UBDs) is critical to a multitude of cellular processes. Here, we report that Rabex-5, a guanine nucleotide exchange factor for Rab5, binds to Ub through two independent UBDs. These UBDs determine a number of properties of Rabex-5, including its coupled monoubiquitination and interaction in vivo with ubiquitinated EGFRs. Structural and biochemical characterization of the UBDs of Rabex-5 revealed that one of them (MIU, motif interacting with ubiquitin) binds to Ub with modes superimposable to those of the UIM (ubiquitin-interacting motif):Ub interaction, although in the opposite orientation. The other UBD, RUZ (Rabex-5 ubiquitin binding zinc finger) binds to a surface of Ub centered on Asp58(Ub) and distinct from the "canonical" Ile44(Ub)-based surface. The two binding surfaces allow Ub to interact simultaneously with different UBDs, thus opening new perspectives in Ub-mediated signaling.     

Competitive binding of UBPY and ubiquitin to the STAM2 SH3 domain revealed by NMR

To date, the signal transducing adaptor molecule 2 (STAM2) was shown to harbour two ubiquitin binding domains (UBDs) known as the VHS and UIM domains, while the SH3 domain of STAM2 was reported to interact with deubiquitinating enzymes (DUBs) like UBPY and AMSH. In the present study, NMR evidences the interaction of the STAM2 SH3 domain with ubiquitin, demonstrating that SH3 constitutes the third UBD of STAM2. Furthermore, we show that a UBPY-derived peptide can outcompete ubiquitin for SH3 binding and vice versa. These results suggest that the SH3 domain of STAM2 plays versatile roles in the context of ubiquitin mediated receptor sorting.     

S5a promotes protein degradation by blocking synthesis of nondegradable forked ubiquitin chains

Ubiquitin (Ub)–protein conjugates formed by purified ring‐finger or U‐box E3s with the E2, UbcH5, resist degradation and disassembly by 26S proteasomes. These chains contain multiple types of Ub forks in which two Ub's are linked to adjacent lysines on the proximal Ub. We tested whether cells contain factors that prevent formation of nondegradable conjugates and whether the forked chains prevent proteasomal degradation. S5a is a ubiquitin interacting motif (UIM) protein present in the cytosol and in the 26S proteasome. Addition of S5a or a GST‐fusion of S5a's UIM domains to a ubiquitination reaction containing 26S proteasomes, UbcH5, an E3 (MuRF1 or CHIP), and a protein substrate, dramatically stimulated its degradation, provided S5a was present during ubiquitination. Mass spectrometry showed that S5a and GST–UIM prevented the formation of Ub forks without affecting synthesis of standard isopeptide linkages. The forked Ub chains bind poorly to 26S proteasomes unlike those synthesized with S5a present or linked to Lys63 or Lys48 chains. Thus, S5a (and presumably certain other UIM proteins) function with certain E3/E2 pairs to ensure synthesis of efficiently degraded non‐forked Ub conjugates.     

Arginine kinase from Nautilus pompilius, a living fossil. Site-directed mutagenesis studies on the role of amino acid residues in the Guanidino specificity region

Arginine kinases were isolated from the cephalopods Nautilus pompilius, Octopus vulgaris, and Sepioteuthis lessoniana, and the cDNA-derived amino acid sequences have been determined. Although the origin and evolution of cephalopods have long been obscure, this work provides the first molecular evidence for the phylogenetic position of Cephalopoda in molluscan evolution. A crystal structure for Limulus arginine kinase showed that four amino acid residues (Ser(63), Gly(64), Val(65), and Tyr(68)) are hydrogen-bonded with the substrate arginine. We introduced three independent mutations, Ser(63) --> Gly, Ser(63) --> Thr, and Tyr(68) --> Ser, in Nautilus arginine kinase. One of the mutants had a considerably reduced substrate affinity, accompanied by a decreased V(max). In other mutants, the activity was lost almost completely. It is known that substantial conformational changes take place upon substrate binding in arginine kinase. We hypothesize that the hydrogen bond between Asp(62) and Arg(193) stabilizes the closed, substrate-bound state. Site-directed mutagenesis studies strongly support this hypothesis. The mutant (Asp(62) --> Gly or Arg(193) --> Gly), which destabilizes the maintenance of the closed state and/or perhaps disrupts the unique topology of the catalytic pocket, showed only a very weak activity (0.6-1.5% to the wild-type).     

A ubiquitin-interacting motif from Hrs binds to and occludes the ubiquitin surface necessary for polyubiquitination in monoubiquitinated proteins

The ubiquitin-interacting motif (UIM) is a short, approximately 20 residue, structural element, which is present in, but not limited to, the proteins involved in endocytotic and proteasomal degradation. UIMs facilitate endocytotic vesicular sorting of the monoubiquitinated proteins and may be important for the targeting of the polyubiquitinated proteins to the proteasome. Using heteronuclear NMR backbone and side-chain chemical shift mapping of the ubiquitin interaction surface, the UIM from the hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, specifically binds to the ubiquitin hydrophobic surface using UIM's well-conserved central helical LALAL motif. Molecular modeling of the ubiquitin:UIM_Hrs complex suggests that binding occurs through a specific interaction of Leu263 and Leu267 of the UIM_Hrs with two ubiquitin hydrophobic patches located in close proximity to the ubiquitin major polyubiquitination site, Lys48. Intramolecular binding of ubiquitin to a UIM in monoubiquitinated proteins would render Lys48 unavailable for further ubiquitination, thus, explaining the absolute requirement of UIMs for monoubiquitination. Two leucines, Leu265 and Leu269, located on the opposite face of UIM_Hrs can also interact, albeit less favorably than Leu263 and Leu267, with the ubiquitin hydrophobic patches, suggesting a possible mode for polyubiquitin:UIM binding and apparent preference of UIMs for polyubiquitins.     

Coarse-grained models for simulations of multiprotein complexes: application to ubiquitin binding

We develop coarse-grained models and effective energy functions for simulating thermodynamic and structural properties of multiprotein complexes with relatively low binding affinity (K_d > 1 μM) and apply them to binding of Vps27 to membrane-tethered ubiquitin. Folded protein domains are represented as rigid bodies. The interactions between the domains are treated at the residue level with amino-acid-dependent pair potentials and Debye-Hückel-type electrostatic interactions. Flexible linker peptides connecting rigid protein domains are represented as amino acid beads on a polymer with appropriate stretching, bending, and torsion-angle potentials. In simulations of membrane-attached protein complexes, interactions between amino acids and the membrane are described by residue-dependent short-range potentials and long-range electrostatics. We parameterize the energy functions by fitting the osmotic second virial coefficient of lysozyme and the binding affinity of the ubiquitin-CUE complex. For validation, extensive replica-exchange Monte Carlo simulations are performed of various protein complexes. Binding affinities for these complexes are in good agreement with the experimental data. The simulated structures are clustered on the basis of distance matrices between two proteins and ranked according to cluster population. In ∼ 70% of the complexes, the distance root-mean-square is less than 5 Å from the experimental structures. In ∼ 90% of the complexes, the binding interfaces on both proteins are predicted correctly, and in all other cases at least one interface is correct. Transient and nonspecifically bound structures are also observed. With the validated model, we simulate the interaction between the Vps27 multiprotein complex and a membrane-tethered ubiquitin. Ubiquitin is found to bind preferentially to the two UIM domains of Vps27, but transient interactions between ubiquitin and the VHS and FYVE domains are observed as well. These specific and nonspecific interactions are found to be positively cooperative, resulting in a substantial enhancement of the overall binding affinity beyond the ∼ 300 μM of the specific domains. We also find that the interactions between ubiquitin and Vps27 are highly dynamic, with conformational rearrangements enabling binding of Vps27 to diverse targets as part of the multivesicular-body protein-sorting pathway.