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Protein Science (2006), 15:1248-1259. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society
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Solution structure of the ubiquitin-associated domain of human BMSC-UbP and its complex with ubiquitin

Yong-Gang Chang1,2, Ai-Xin Song1, Yong-Guang Gao1,2, Yan-Hong Shi2,3, Xiao-Jing Lin1, Xue-Tao Cao4, Dong-Hai Lin2 and Hong-Yu Hu1

1 Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Shanghai 200031, China
2 Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, China
3 Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Shanghai 201203, China
4 Institute of Immunology, Secondary Military Medical University, Shanghai 200433, China

(RECEIVED November 22, 2005; FINAL REVISION February 15, 2006; ACCEPTED February 19, 2006)


   Abstract
TOP
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 References
 
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 ~17 µM. 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.

Keywords: ubiquitin-associated domain; solution structure; complex; BMSC-UbP


   Introduction
TOP
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 References
 
Ubiquitination is a post-translational process that covalently modifies the substrate proteins with ubiquitin in all eukaryotic organisms (Hochstrasser 1996). The modification process requires in general the sequential action of three enzymes, including ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3) (Hershko and Ciechanover 1998). The great majority of proteins in eukaryotic cells are degraded by the 26S proteasome following the polyubiquitin attachment. In addition to marking proteins for breakdown by the proteasome, ubiquitination has been implicated in a variety of cellular processes, including cell cycle progression, signal transduction, transcriptional regulation, DNA repair, receptor down-regulation, endocytosis, ribosomal function, and quality control in endoplasmic reticulum (Hochstrasser 1996; Ciechanover 1998; Hershko and Ciechanover 1998; Conaway et al. 2002).

Ubiquitination signals could be divided into monoubiquitination, multiple monoubiquitination, and polyubiquitination (Haglund et al. 2003). Among these cellular processes, ubiquitin-binding motifs hold a key to the answer as to how the signals are transmitted and recognized. Several such motifs have been characterized, including UBA (ubiquitin-associated) (Chen et al. 2001; Mueller and Feigon 2002; Mueller et al. 2004), UIM (ubiquitin-interacting motif) (Raiborg et al. 2002; Shih et al. 2002), CUE (coupling of ubiquitin to ER degradation) (Davies et al. 2003; Shih et al. 2003), NZF (Npl4 zinc finger) (Meyer et al. 2002; Wang et al. 2003; Alam et al. 2004), UEV (ubiquitin E2 variant), (Garrus et al. 2001; Pornillos et al. 2002; Sundquist et al. 2004), and GLUE (GRAM-like ubiquitin-binding in Eap45) (Slagsvold et al. 2005).

The UBA domain, ~45 residues, was initially identified in subsets of E2, E3s, and USP (ubiquitin-specific protease) superfamilies, and other proteins of functions other than ubiquitination and deubiquitination (Hofmann and Bucher 1996). It usually locates at the C terminus of UBA-containing proteins, although the N-terminal UBA domains have also been characterized. Proteins containing the UBA domains include, for example, HHR23A (the human homolog of yeast RAD23), a protein involved in DNA repair (Watkins et al. 1993); p62, a protein that mediates diverse cellular functions including control of NF-{kappa}B signaling and transcriptional activation (Geetha and Wooten 2002); p47, a major adaptor molecule of the cytosolic AAA ATPase p97 (Yuan et al. 2004); and ubiquilin-2, a protein linking integrin-associated protein and cytoskeleton (Kleijnen et al. 2000).

Human BMSC-UbP, a novel ubiquitin-like protein identified from the cDNA library of human bone marrow stromal cells (BMSC), is distributed in a variety of tissues and enriched in heart, skeletal muscle, testis, thyroid and adrenal gland (Liu et al. 2003). Sequence alignment shows that it contains an N-terminal ubiquitin-like domain (UBQ) and a C-terminal UBA domain. Notably, a large number of proteins share the same domain distribution as in BMSC-UbP, for example, yeast Dsk2, human HHR23A, ubiquilin-1 (also named hPLIC1), ubiquilin-2 (hPLIC2) and A1Up. The UBA domains of yeast Dsk2 and its human homologs ubiquilin-1, ubiquilin-2, and A1Up share ~50% sequence identity with BMSC-UbP UBA domain. In addition, the N-terminal UBQ domains of ubiquilin-2 and yeast Dsk2 have been found binding to the S5a subunit of the proteasome (Walters et al. 2002).

To gain clues for biological functions of human BMSC-UbP, we solved the solution structure of the UBA domain by heteronuclear multidimensional NMR techniques and studied the interaction between the UBA domain and ubiquitin by chemical shift perturbation and mutagenesis experiments. Based on the reciprocal titration results, we elucidated the structure of the BMSC-UbP UBA–ubiquitin complex by the program HADDOCK (Dominguez et al. 2003). The structure-based interaction of the UBA domain for monoubiquitin may provide important hints for understanding of the polyubiquitin recognition.


   Results
TOP
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 References
 
GB1 fusion improves the structure determination of BMSC-UbP UBA domain
As shown in Figure 1A, UBA domains usually locate at the C terminus of UBA-containing proteins and UBQ domains are frequently accompanying at the N terminus. The UBA domains share low sequence homology though adopting a similar fold as evident for the proteins with structures determined (Fig. 1B). However, BMSC-UbP UBA domain shares ~50% sequence identity with yeast Dsk2 and its homologs ubiquilin and A1Up (Fig. 1C), which have been shown to bind polyubiquitin (Funakoshi et al. 2002; Ko et al. 2004; Riley et al. 2004). To determine what fold BMSC-UbP UBA domain might take and whether it could bind with ubiquitin, we first cloned the corresponding sequence of BMSC-UbP UBA domain into pGEX-4T-3 plasmid, yielding a GST–UBA fusion protein. Unfortunately, the free UBA domain readily precipitates after removing GST tag by thrombin cleavage. Then, a solubility-enhancement tag (SET) GB1 (Zhou et al. 2001) was used as a fusion partner to enhance the solubility of BMSC-UbP UBA domain. 15N-labeled HGB1-UBA was prepared for recording the 1H-15N HSQC spectra. Figure 2A shows the wide chemical-shift dispersion of HGB1-UBA on the HSQC spectrum, indicating the well-folded structures of both GB1 and BMSC-UbP UBA domain. Obviously, little overlay of the resonance peaks between GB1 and BMSC-UbP UBA makes it easy to assign two sets of the peaks. We assigned most of the backbone and side-chain resonances and inspected the NOEs of the fusion protein. There is no interdomain NOE between GB1 and the UBA domain except for the sequential NOEs of the linker residues. From chemical shift index (Fig. 2B), the secondary structure of GB1 was assigned to be beta1beta2{alpha}beta3beta4, characteristic of the GB1 domain (Gronenborn et al. 1991), and that of BMSC-UbP UBA was identified as three {alpha}-helices, reminiscent of the typical three-helix fold of UBA domains.


Figure 1
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  		Figure 1. Multiple alignments of selected UBA domains. (A) Schematic representation of several UBA-containing proteins showing that UBA domains usually locate at the C terminus, whereas the accompanying UBQ domains occur at the N terminus. UBA domains are in black, and UBQ domains are in gray. Residue numbers are shown to the right of the diagram of each protein. Hu, Homo sapiens. (B) Sequence alignment of BMSC-UbP_UBA (338–380), p62_UBA (387–440), HHR23A_UBA(1) (163–200), and HHR23A_UBA(2) (320–363) whose structures have been determined by NMR. Amino acid residues in gray indicate identity or similarity. (C) Sequence alignment of the UBA domains of ubiquilin-1 (also called hPLIC1), ubiquilin-2 (hPLIC2), A1Up and yeast Dsk2, showing that they share high sequence homology. The SwissProt entry codes for the sequences are Q96S82 (BMSC-UbP), P48510 (Dsk2), Q9UMX0 (ubiquilin-1), Q9UHD9 (ubiquilin-2), Q9NRR5 (A1Up), P54725 (HHR23A), and Q13501 (p62).

 


Figure 2
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  		Figure 2. Sequence-specific assignment and secondary structure identification for the HGB1-UBA fusion protein. (A) 1H-15N HSQC spectrum of HGB1-UBA. Both GB1 and the UBA domain have wide chemical-shift dispersions in the spectrum. Almost all the 1H-15N correlation peaks have been assigned by heteronuclear multidimensional NMR techniques. Only the peaks attributed to the backbone amides of BMSC-UbP UBA domain (residues 65–108) are labeled in the figure. (B) Chemical shift index (CSI) reflecting the secondary structures of HGB1-UBA. CSI consensus shows the identification of secondary structures using the chemical shifts of 1H{alpha}, 13C{alpha}, 13Cbeta, and 13CO of HGB1-UBA. The indices indicate residues residing in {alpha}-helix (–1) and beta-sheet (+1), indicative of three {alpha}-helix composition of the UBA domain under study. The GB1 (residues 8–62) and UBA (65–108) domains are indicated, respectively, while the N-terminal His6-tag and two additional residues (Gly63 and Ser64) from the BamHI restriction site are omitted.

 
BMSC-UbP UBA domain adopts a highly compact three-helix bundle
Ten out of 200 calculated structures were selected on the basis of their lowest overall energies. Figure 3A shows the backbone C{alpha} traces of the 10-structure ensemble. Ramachandran plot reveals that the distributions of the backbone dihedral angles ({varphi}, {phi}) within the structured region of the ensemble (residues 65–108) are 92.4% of the residues in favored regions, 6.8% in additionally allowed regions, 0.8% in generously allowed regions, and none in disallowed regions (Table 1). The average RMSD for the 10 structures (residues 67–104) is 0.47 Å for backbone and 1.14 Å for overall heavy atoms.


Figure 3
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  		Figure 3. Solution structure of BMSC-UbP UBA domain. (A) Superimposition of the backbone C{alpha} traces representing a bundle of 10 refined structures. (B) Ribbon representation of the structure of BMSC-UbP UBA domain. The three helices from the N to the C terminus are labeled as {alpha}1, {alpha}2, and {alpha}3. (C) Structure of BMSC-UbP UBA domain showing the characteristic hydrophobic core. The side chains of the residues that contribute to the hydrophobic core are depicted in neon style.

 


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  		Table 1. Experimental restraints used in structure calculation and statistics for the BMSC-UbP UBA domain

 
Figure 3B shows BMSC-UbP UBA domain comprising mainly three tightly packed helices. Helix-1 consists of residues 67–76, helix-2 of residues 81–91, and helix-3 of residues 95–104. Loop-1 and loop-2 include residues 77–80 and 92–94, respectively. The three-helix bundle is stabilized by a highly hydrophobic core, which is formed by the side chains of Trp66, Leu70, Leu73, Leu83, Leu85, Leu88, Ile95, and Ile102 (Fig. 3C). The side chain of Trp66 in helix-1 makes intimate contact with those of Leu85 and Leu88 in helix-2, burying Leu85 deeply in the core. The side chain of Ala87 in helix-2 is in close contact with those of Ala98 and Ile102 in helix-3. Notably, Ala87, Leu88, and Ala98 (corresponding to the numbering in Fig. 1B as residues 30, 31, and 41) are conserved for the UBA domains (Fig. 1B). For loop-1, the side chain of Ile78 contacts closely with that of Met76 (the C-terminal residue in helix-1); Gly77 between these two residues provides the necessary conformation for the specific interaction. This glycine residue is conserved in almost all the UBA domains. In addition, hydrogen bond formation between the carbonyl oxygen of Leu73 and the amide hydrogen of Ile78 contributes to the packing of helix-1 and helix-2. Loop-2 is critical for determining the angle between helix-2 and helix-3. Four residues, Thr91, Gly92, Gly93, and Asp94 form a beta-turn, among which the gamma oxygen of Thr91 hydrogen bonds with the amide hydrogen of Asp94. The C terminus folds back onto helix-3, being similar to that of HHR23A UBA (1) (Mueller and Feigon 2002).

Comparison of BMSC-UbPUBA domain with those of p62 and HHR23A
Figure 4 shows the structure representatives of the UBA domains of BMSC-UbP, HHR23A(2) (Withers-Ward et al. 2000), p62 (Ciani et al. 2003), and HHR23A(1) (Mueller and Feigon 2002). These domains adopt the similar fold of three tightly packed helices stabilized by a hydrophobic core. As in the UBA domain structures already solved, BMSC-UbP UBA domain possesses a characteristic MGI motif instead of the common M/L-G-F/Y motifs in the essential loop-1 (Fig. 4). Helices for the four UBA domains are delimited according to the results from secondary structure calculation of their respective mean structures. The angles between the axes of {alpha}1 and {alpha}2 for the four domains are 134°, 127°, 131°, and 123°, respectively, whereas those of {alpha}2 and {alpha}3 are 109°, 125°, 132°, and 104°. The relatively rigid orientation of {alpha}1 and {alpha}2 may be due to the conservation of the M/L-G-F/Y/I motifs, which have been shown to be critical for ubiquitin binding. All domains show the close contact of M/L and F/Y/I that may dictate the overall orientation of {alpha}1 and {alpha}2. By contrast, the angles between {alpha}2 and {alpha}3 show relatively high variation, which could be explained by the presence of relatively flexible residues in the beta-turn region. Interestingly, the {alpha}1/{alpha}3 angles are 67°, 36°, 32°, and 47°, respectively, making BMSC-UbP UBA the most compact helix bundle of the four domains. Another structural feature shared by BMSC-UbP UBA and HHR23A UBA(1) is the folding of the C terminus back onto the compact three-helix bundle, which is evidenced by the NOEs between Ile102 and Ala107 of BMSC-UbP UBA and those between Ile202 and Tyr197 of HHR23A UBA(1) (Mueller and Feigon 2002). In all, the four domains take a three-helix-bundle fold that is stabilized by a hydrophobic core. The MGI motif in BMSC-UbP UBA extends the characteristic M/L-G-F/Y as M/L-G-F/Y/I motif, indicative of a common role of such motifs in ubiquitin recognition by the UBA domains.


Figure 4
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  		Figure 4. Comparison of the UBA domain structures. Shown are the ribbon representations of BMSC-UbP UBA (A), HHR23A UBA(2) (B), p62 UBA (C), and HHR23A UBA(1) (D). The side chains of the residues in the conserved M/L-G-F/Y/I motifs are highlighted in neon style. The PDB codes for the structures are 2CWB (BMSC-UbP UBA), 1DV0 [HHR23A UBA(2)], 1QO2 (p62 UBA), and 1IFY [HHR23A UBA(1)], respectively.

 
BMSC-UbP UBA binds to the five-stranded beta-sheet surface of ubiquitin via a hydrophobic patch
Chemical shift perturbation experiments were carried out to map the contact surfaces on UBA and ubiquitin. Figure 5A shows the chemical shift change of ubiquitin upon titrating with HGB1-UBA at a molar ratio of four. By fitting the titration curves (Fig. 5B), the dissociation constant (KD) for BMSC-UbP UBA binding to ubiquitin is ~17 µM, similar to that for Dsk2 UBA–ubiquitin binding (14.8 µM). Titration of 15N-labeled HGB1-UBA with unlabeled ubiquitin shows that ubiquitin binds specifically to BMSC-UbP UBA, whereas the GB1 tag, experiencing little chemical shift change, serves as a negative control for ubiquitin binding (Fig. 6A). Residues on UBA exhibiting very significant chemical shift changes (>mean+SD) were mapped onto the structure of the UBA domain (Fig. 6B). Such residues include Met76 at the C terminus of helix-1, Gly77 and Ile78 in loop-1, and Ala98, Leu99, and Ile102 in helix-3, being defined as a hydrophobic patch involved in ubiquitin binding. Other UBA domains possess the similar patches for contacting ubiquitin. The significant chemical shift change of Trp66 may be due to a slight conformational change of the flexible region preceding helix-1. The UBA binding surface on ubiquitin was also mapped onto the five-stranded beta-sheet with significant chemical shift changes of residues Leu8, Ile13, Leu43, Ala46, Lys48, Gln49, Leu71, and Arg72 (Fig. 6C,D). These potential contacting residues identified by chemical shift perturbation will provide starting restraints for constructing the structural model of the complex.


Figure 5
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  		Figure 5. Determination of the binding affinity of BMSC-UbP UBA for ubiquitin. (A) Overlay of the 1H-15N HSQC spectra of ubiquitin free (red) and bound (black) to BMSC-UbP UBA. The molar ratio of ubiquitin to HGB1-UBA is 1:4. The three lines indicate the typical chemical-shift changes of residues Ile13, Lys48, and Leu71 of 15N-labeled ubiquitin upon titrating with HGB1-UBA. (B) Plot of weighted average chemical shift changes ({Delta}{delta}ave) for Ile13 ({blacksquare}), Lys48 (bullet), and Leu71 ({blacktriangleup}) against the molar ratio of HGB1-UBA:ubiquitin. The curves indicate the most favorable fitting using the equation described in the Materials and Methods section.

 


Figure 6
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  		Figure 6. Mapping the contact surface of BMSC-UbP UBA and ubiquitin by chemical shift perturbation. (A) Chemical shift changes of HGB1-UBA vs. its residue number. 15N-labeled HGB1-UBA was titrated stepwise with unlabeled ubiquitin to a final molar ratio of 1:4. The average chemical shift change ({Delta}{delta}ave) for every amide is plotted against residue number of HGB1-UBA. The solid and the dashed lines indicate the threshold values of mean and mean + SD for the chemical shift changes. Residues with the chemical shift changes above the mean + SD (dashed line) are considered involved in significant contact with ubiquitin as shown in B. (B) Mapping the significant contact residues on the BMSC-UbP UBA structure, including Trp66 (helix1); Met76, Gly77 and Ile78 (loop 1); Ala98, Leu99, Glu100 and L101 (helix 3). The side chains of these residues are highlighted in neon style. (C) As in A, except for the titration of 15N-labeled ubiquitin with HGB1-UBA. (D) As in B. The residues with significant chemical shift change are Leu8 and Ile13 (loop between beta1 and beta2), Leu43 (beta3), Ala46 (loop between beta3 and beta4), Lys48 and Gln49 (beta4), Leu71 (beta5), and Arg72 (C terminus). The ubiquitin structure is referenced from the crystal structure (PDB code 1UBQ).

 
Structure of the BMSC-UbP UBA–ubiquitin complex
Figure 7A and B, shows the C{alpha} traces of a bundle of the 10 refined BMSC-UbP UBA–ubiquitin complex and a ribbon representation as constructed by using the HADDOCK program. The average RMSD for the 10 structures for UBA (residues 67–104) is 0.63 Å for backbone and 1.20 Å for heavy atoms, and those for ubiquitin are 0.45 and 0.73 Å, respectively. BMSC-UbP UBA binds to the relatively hydrophobic concave surface defined by Leu8, Ile44, His68, and Val70 of ubiquitin (Fig. 7C,D). The side chain of Met76 pointing toward the center of the interacting surface of ubiquitin is in close contact with the side chains of Leu8 and His68 of ubiquitin. The {alpha}-hydrogen of Gly77 makes intimate contact with Gly47 and the side chain of Ile44 of ubiquitin, providing a possible explanation for the large chemical shift change of Lys48 that is in close proximity to Gly47 and Ile44. Contribution to the stability of the complex also comes from the hydrophobic interaction of Ile78 methyls with the side chains of Leu44 and Val70 of ubiquitin. Notably, the side chain of Leu99 makes very intimate contact with that of Leu8 of ubiquitin, further stabilizing the complex. Thus, we describe the three residues Met76, Ile78, and Leu99 as a "trident," which anchors the UBA domain to the hydrophobic concave surface of ubiquitin that is mainly comprised of Leu8, Ile44, His68, and Val70 residues (Fig. 7D). Similar recognition surface of ubiquitin was also reported for binding with other domains such as CUE (Kang et al. 2003) and NZF (Alam et al. 2004). K48- and K63-linked polyubiquitin has been found implicated in different cellular functions. The side chains of Lys48 and Lys63 of ubiquitin are shown in no contact with BMSC-UbP UBA domain despite large chemical shift change of Lys48 upon titration, leaving the possibility that one polyubiquitin string can bind with several UBA-containing proteins. The importance of the conservative M/L-G-F/Y/I motif is also evident in the structure of the Dsk2 UBA-ubiquitin complex constructed by intermolecular NOE restraints (Ohno et al. 2005). The overall structure of the BMSC-UbP UBA-ubiquitin complex is similar to that of the Dsk2 UBA–ubiquitin complex (Fig. 7F). Nevertheless, the orientation of both UBA domains toward ubiquitin is slightly different, which might be due to the difference in packing of the helical bundle (the backbone RMSD for the two UBA domains is 1.2 Å) (Fig. 7E). In addition, the hydrogen bond between the carbonyl group of Met342 in Dsk2-UBA and the amide group of Gly47 in ubiquitin is absent in the BMSC-UbP UBA-ubiquitin complex. In all, the conservative residues in loop-1 connecting {alpha}1 and {alpha}2 and the residues at the C terminus of {alpha}3 are determinant for the binding of UBA domains to the five-stranded beta-sheet surface of ubiquitin.


Figure 7
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  		Figure 7. Structure of BMSC-UbP UBA-ubiquitin complex. (A) Superimposition of the backbone C{alpha} traces representing 10 refined structures of BMSC-UbP UBA–ubiquitin complex. Ubiquitin is shown in blue; BMSC-UbP UBA, in green. (B) Ribbon representation of the complex structure. (C) Interaction surfaces of BMSC-UbP UBA and ubiquitin. Electrostatic potential maps are shown for ubiquitin. BMSC-UbP UBA is represented in round ribbon except for omission of {alpha}2 for clarity. Side chains of residues Met76, Ile78, and Leu99 are shown in green and in neon style as indicated by red arrows. (D) Side chains of the hydrophobic residues that are in close contact: Met76, Ile78, and Leu99 of BMSC-UbP UBA (green) and Leu8, Ile44, His68, and Val70 of ubiquitin (bright gray) as displayed in neon style. The side chains of residues Lys48 and Lys63 are shown in blue, which face away from the contact surface in the complex despite large chemical shift change of Lys48 upon titration. (E) Overlay of the UBA domains from BMSC-UbP (residues 68–104, green) and Dsk2 (334–370, red) in each UBA–ubiquitin complex. (F) Structure of yeast Dsk2 UBA–ubiquitin complex for comparison. The ubiquitin molecule is positioned in the same orientation as that in B.

 
Evidence for the binding specificity from mutagenesis study
To obtain direct evidence for the specific interaction of BMSC-UbP UBA and ubiquitin, we constructed several mutants for both HGB1-UBA and ubiquitin. These mutants include M76A, I78A, L99A and L101A of HGB1-UBA, and K48A of ubiquitin. By chemical shift perturbation experiments, we studied the binding abilities of these mutants and compared the data with those of each wild-type protein (Fig. 8). M76A and I78A bind ubiquitin extremely weakly with the dissociation constants (KD) >200 µM, while L99A presents a KD of ~51 µM (Fig. 8A), suggesting that Met76 and Ile78 are very important to ubiquitin binding and Leu99 is also responsible for constructing the trident surface for hydrophobic interaction. On the contrary, L101A gives a KD of 12 µM for ubiquitin binding, being very similar to wild-type HGB1-UBA. Since the side chain of Leu101 orients away from the contacting surfaces of the complex, mutation of this residue has no significant impact on the binding ability. This is in good agreement with the result from HADDOCK analysis that Met76, Ile78, and Leu99 of HGB1-UBA are located on the contacting surfaces. As shown in Figure 7D, the side chain of Lys48 points away from the binding surface of ubiquitin, though it undergoes significant chemical shift change upon HGB1-UBA titration (Fig. 6C). To further demonstrate that this residue is not involved in contacting with HGB1-UBA, we compared the binding abilities of ubiquitin and its K48A mutant. The mutant exhibits similar binding ability to wild-type ubiquitin under experimental uncertainty (Fig. 8B). This indicates that Lys48 does not make contact with HGB1-UBA in the complex. Similarly, Leu101, though experiencing large chemical shift change upon ubiquitin binding (Fig. 6A), is not involved in directly contacting ubiquitin either. Therefore, the large chemical shift change of Lys48 of ubiquitin and Leu101 of HGB1-UBA observed in the reciprocal chemical shift perturbation experiments might result from backbone conformational change or aromatic ring current effect upon binding. Taken together, the mutagenesis study further supports the structural model from HADDOCK analysis that three residues (Met76, Ile78, and Leu99) of BMSC-UbP UBA form a trident anchoring the domain to the hydrophobic concave surface of ubiquitin.


Figure 8
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  		Figure 8. Mutations reveal the specific binding surfaces on BMSC-UbP UBA and ubiquitin. (A) Plot of weighted average chemical shift changes ({Delta}{delta}ave) of Leu71 of 15N-labeled ubiquitin against the molar ratio of HGB1-UBA mutants (M76A, I78A, L99A, and L101A) to ubiquitin. By data analysis, the dissociation constants of M76A and I78A are >200 µM, while those of L99A and L101A are 51 and 12 µM, respectively. (B) The average chemical shift changes of Glu100 of 15N-labeled HGB1-UBA vs. the molar ratio of ubiquitin and K48A mutant to HGB1-UBA. The dissociation constant of K48A binding with HGB1-UBA is 9 µM, which is comparable with that of wild-type ubiquitin binding (~5 µM). The curves indicate the most favorable fitting using the equation described in the Materials and Methods section.

 

   Discussion
TOP
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 References
 
BMSC-UbP contains the same UBQ–UBA domain distribution as in human HHR23A, A1Up, ubiquilins, and yeast Dsk2, which have been found interacting with the 26S proteasome through their UBQ domains and with ubiquitin through the UBA domains. We previously solved the solution structure of the C-terminal ubiquitin-like domain of human DC-UbP, another ubiquitin-like protein from dendritic cells (Gao et al. 2005). To understand the biological function of BMSC-UbP, we explore the possibility of its UBA domain interacting with ubiquitin. We have elucidated the solution NMR structure of the UBA domain and studied in detail its interaction with monoubiquitin, suggesting that, as well as DC-UbP, BMSC-UbP possibly functions as a shuttle factor involved in the ubiquitin system.

UBA domain is small (44 residues) and is not easy to be expressed alone in Escherichia coli. We have never successfully purified the GST fusion protein and removed the GST tag by thrombin cleavage. The free UBA domain easily precipitates in solution (data not shown), possibly due to the high percentage of hydrophobic residues in the sequence. The GB1 fusion makes it possible to study the solution structure of BMSC-UbP UBA and its complex with ubiquitin by dramatically enhancing the solubility of the domain. In addition, ubiquitin binding to the fusion protein has no impact on the chemical shifts of the GB1 tag (Fig. 6A). Thus, the GB1 tag holds the promise of facilitating structure determination of other ubiquitin-binding domains with low expression and poor solubility and stability.

The reciprocal NMR titration experiments have provided abundant valuable information for mapping the interfaces of BMSC-UbP UBA–ubiquitin complex. To further delineate the complex structure in detail, we employed the HADDOCK program to calculate the structural coordinates based on the chemical shift perturbation results. An ensemble of 10 NMR structures of BMSC-UbP UBA and a single crystal structure of ubiquitin were used as the starting structures, which allows a better sampling of all conformational possibilities during the rigid-body docking step (Dominguez et al. 2004). This complex structure shows a clear view of the interaction surfaces of both subunits and provides insights into the structural specificity for the UBA-ubiquitin interaction as shown in Figure 7. The mutagenesis study demonstrates that this program is practical to mapping the interaction surfaces and constructing the structural model for the protein complex. Since developed by Dominguez et al. (2003), this program has been successfully applied to the structural determination of several complexes (van Dijk et al. 2005).

BMSC-UbP UBA domain binds to the five-stranded beta-sheet of ubiquitin via a hydrophobic patch defined by the C terminus of helix-1, loop-1, and helix-3. The UBA domains of BMSC-UbP, ubiquilin-1, ubiquilin-2, A1Up, and yeast Dsk2 share relatively high sequence similarities, particularly in loop-1 and helix-3 (Fig. 1). The M-G-I motif of BMSC-UbP UBA domain, a critical determinant of the hydrophobic patch interacting with ubiquitin, joins in the family of M/L-G-F/Y motifs conserved in the UBA domains of some ubiquitin-associated proteins (Fig. 4). The five-stranded beta-sheet in ubiquitin is a common surface for interacting with the UBA domains (Fig. 7). These UBA domains can be probably classified into another domain subfamily, as distinct from the well-characterized UBA domains of HHR23A-like proteins. In regard to the UBQ–UBA domain distribution and binding specificity, we hypothesize that BMSC-UbP is a potential shuttle protein of ubiquitinated substrates to the proteasome for degradation.

Monoubiquitination and polyubiquitination has distinct cellular roles. A variety of UBA-containing proteins known to bind ubiquitin show strong preference to K48-linked polyubiquitin chains (Raasi et al. 2005). As evident in the complex structure, Lys48 and Lys63, two major sites for polyubiquitin formation, are far away from the contacting surface. This allows the K48- or K63-linked polyubiquitins to retain their exposed surfaces for UBA binding. Compared with K63-linked type, K48-linked tetraubiquitin is a more compact molecule, of which subunit 1 and 3 contact with each other via the beta-sheet surfaces (Beal et al. 1996; Tenno et al. 2004). Whether UBA domains could bind each subunit of K48-linked polyubiquitin awaits further NMR study of polyubiquitin with only one subunit labeled.

The main function of the UBA domains is binding with ubiquitin, either for ubiquitin signal generation (like in some E2s and E3s) and termination (in some USPs) or transporting ubiquitinated proteins to the 26S proteasome. The specificity of the UBA-containing proteins may come from two aspects: One is the UBA domain that binds with specific partners other than ubiquitin; another is other domains of the UBA-containing proteins that might influence their localization by binding different partners. For example, XPC-binding domain mediates the interaction of HHR23A with XPC (Kamionka and Feigon 2004), and p47 UBX domain mediates the interaction of p47 and p97 (Yuan et al. 2004). In particular, the UBQ–UBA proteins may lead ubiquitinated substrates to the 26S proteasome, although DNA repair-related HHR23A could also recognize K63-linked polyubiquitin that is demonstrated not to be a signal for degradation by the proteasome. Interestingly, some proteins such as yeast Rad23 can form homodimers through their UBA domains. The dimerization of UBA domains may impede their binding to the polyubiquitinated target proteins that are regulated by the UBA-containing proteins (Bertolaet et al. 2001).

In summary, we have corroborated the previous finding that UBA domains bind to the five-stranded beta-sheet of ubiquitin via the hydrophobic patches. We also favor the idea that the binding specificities of the UBA-containing proteins could be conferred not only by the specific UBA domains but also by other UBA-binding proteins or other domains within the UBA-containing proteins. Characterization of the binding partners and ubiquitinated substrates of the UBA-containing proteins will open avenues to unravel the link between cellular processes and the ubiquitin system. The structural basis of the UBA domains in complex with ubiquitin may enrich our knowledge of the UBA-containing proteins that may modulate cellular ubiquitination processes.


   Materials and methods
TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
References
 
HGB1-UBA expression and purification
The coding sequence for the UBA domain, corresponding to residues 337–380 of BMSC-UbP, was amplified via PCR and cloned in frame with the GB1 domain into the pHGB expression plasmid using the BamHI/XhoI cloning sites. The construct expresses a chimeric protein that we designated HGB1-UBA, which includes from the N to the C terminus a His6 tag, a GB1 domain and a UBA domain. The BMSC-UbP UBA segment was then renumbered as 65–108. Site-directed mutagenesis was performed on this vector via PCR method. The pHGB-UBA plasmid was transformed into E. coli BL21 (DE3) CodonPlus cells. Cells harboring the plasmid were grown at 37°C in M9 minimal media. When an OD600 reached ~0.6, HGB1-UBA expression was induced at 25°C by adding IPTG to a final concentration of 0.1 mM, and incubated and shaken for 10 h. After cell lysis, the protein was purified through a Ni2+-NTA column (QIAGEN) followed by FPLC gel-filtration on a 16/40 Superose 12 column (Amersham Biosciences). 15N- and 15N/13C-labeled HGB1-UBA proteins were prepared by using the M9 media containing 15NH4Cl and/or 13C6-D-glucose as the sole nitrogen and/or carbon resource, respectively. Purified HGB1-UBA was dialyzed exhaustively against water, lyophilized, and stored at –20°C.

Ubiquitin expression and purification
The ubiquitin gene was cloned into pET-3a plasmid using the NdeI/BamHI sites. The pET-3a-Ub plasmid was transformed into E. coli BL21 (DE3) cells. Cells containing the plasmid were grown at 37°C in M9 minimal media until an OD600 of ~0.6. Expression was induced at 25°C for 10 h by adding IPTG to a final concentration of 0.2 mM. The protein was purified sequentially by acetic acid denaturation, 16/10 SP XL column and 16/60 Superdex 75 column (Amersham Biosciences) chromatography. 15N-labeled ubiquitin was prepared with 15NH4Cl as the sole nitrogen in M9 media. Purified ubiquitin was dialyzed exhaustively against water, lyophilized and stored at –20°C.

NMR spectroscopy and structure determination
HGB1-UBA sample (~1 mM) for NMR experiments was dissolved in a buffer containing 20 mM phosphate, 100 mM NaCl (pH 6.5). All spectra were recorded at 25°C on a Varian INOVA 600 spectrometer equipped with three RF channels and a triple-resonance pulsed-field gradient probe. Sequence-specific assignments of backbone 1HN, 15N, 1H{alpha}, 13C{alpha}, 13Cbeta, and 13CO for HGB1-UBA were performed by analyzing 3D HNCACB, CBCA(CO)NH, HNCO HN(CA)CO, and HNHA spectra. Side-chain assignments were achieved by using 3D HCCH-TOCSY (recorded in D2O) combined with 15N-TOCSY-HSQC. All spectra were processed with NMRPipe (Delagio et al. 1995) and analyzed by Sparky (T.D. Goddard and D.G. Kneller, University of California, San Francisco). Secondary structures were identified from chemical shifts of 1H{alpha}, 13C{alpha}, 13Cbeta, and 13CO by using chemical shift index (CSI) approach (Wishart et al. 1992). Three-dimensional 15N- and 13C-edited NOESY spectra ({tau}m = 100 msec) were recorded for obtaining distance restraints for structure calculation. A total of 877 unambiguous and 15 ambiguous NOE restraints combined with 45 CSI-derived dihedral angle restraints, all exclusively for the UBA domain, were employed by ARIA 1.2 (Nilges et al. 1997) to calculate the UBA structure. A family of 200 structures was calculated using the simulated annealing protocol. Ten structures of the lowest energy were selected, which exhibit no NOE violation >0.5 Å and no dihedral angle violation >5°. Structure assessment was performed by PROCHECK (Laskowski et al. 1993). The ribbon and surface graphs were displayed by using the program MOLMOL (Koradi et al. 1996). The coordinates of BMSC-UbP UBA domain (residues 65–108) and the necessary supporting data have been deposited in the Protein Data Bank (PDB) (code 2CWB).

Chemical shift perturbation experiments
15N-labeled HGB1-UBA was dissolved in above-mentioned NMR buffer, and unlabeled ubiquitin was added stepwise up to a final molar ratio of 1:4, with each step monitored by acquiring 2D 1H-15N HSQC spectrum. The reciprocal titration of 15N-labeled ubiquitin with unlabeled HGB1-UBA was performed accordingly with the starting ubiquitin concentration of 0.5 mM. The sequence-specific backbone assignment for ubiquitin was achieved by comparing the almost identical resonance dispersion of ubiquitin on the 1H-15N HSQC spectra as reported previously. The changes of amide 1H and 15N chemical shifts were averaged by using the formula {Delta}{delta}ave = {[1/2[({Delta}{delta}H)2 + (0.2{Delta}{delta}N)2]]}1/2. The dissociation constant for the UBA–ubiquitin binding was fitted by using the following equation (Kang et al. 2003):


Formula 1

where {Delta}{delta}ave stands for the weighted average chemical shift changes (in ppm) of the amide nitrogen and hydrogen; x, for the molar ratio of HGB1-UBA to ubiquitin; P0, for the initial concentration of labeled protein; {Delta}{delta}max, for the maximum change of weighted average chemical shifts; and KD, for the dissociation constant value to be fitted.

Docking for the UBA–ubiquitin complex
Based on the reciprocal titration results, we calculated the structure of BMSC-UbP UBA in complex with ubiquitin by using the HADDOCK software (Dominguez et al. 2003). The docking followed the procedure described previously (Dominguez et al. 2004). Ten NMR structures of BMSC-UbP UBA domain and one crystal structure of human ubiquitin (PDB code 1UBQ [PDB] ; Vijay-Kumar et al. 1987) were used for mapping the interfaces and docking the structures. The solvent accessibilities were calculated by NACCESS. Residues with {Delta}{delta}ave values greater than the mean value and with high solvent accessibilities were selected as the active residues, and the solvent-accessible surface neighbors of these active residues were defined as passive residues. For the UBA domain, the active residues are Gln71, Met76, Gly77, Gln89, Gln96, Glu100, and Ala104, and the passive residues are Gln72, Asp75, Gln79, Phe103, and Pro108. For ubiquitin, seven active residues (Leu8, Lys11, Ala46, Gly47, Gln49, Glu51, and Leu73) and eight passive residues (Thr9, Gly10, Gly35, Ala46, Arg54, Arg74, Gly75, and Gly76) were selected. One thousand initial complex structures were generated by rigid body energy minimization, and 200 structures with the lowest energy were selected for torsion angle dynamics and subsequent Cartesian dynamics calculation in an explicit water solvent. There are two most populated clusters in all. The average RMSD values for the lowest-energy structure are 1.2 and 9.5 Å for clusters 1 and 2, respectively. In addition, cluster 1 has lower average intermolecular energy than cluster 2. Ten refined complex structures of cluster 1 were selected for analysis. The accession code for the BMSC-UbP UBA–ubiquitin complex in the PDB is 2DEN.


   Footnotes
 
Reprint requests to: Hong-Yu Hu, Institute of Biochemistry and Cell Biology, 320 Yue-yang Road, Shanghai 200031, China; e-mail: hyhu{at}sibs.ac.cn; fax: +86-021-54921011; or Dong-Hai Lin, Institute of Materia Medica, 555 Zuchongzhi Road, Shanghai 201203, China; e-mail: dhlin{at}mail.shcnc.ac.cn; fax: +86-021-50806036.

Abbreviations: BMSC, bone marrow stromal cells; CSI, chemical shift index; CUE, coupling of ubiquitin to ER degradation; GB1, immunoglobulin G binding domain 1 of Streptococcal protein G; GST, glutathione S-transferase; HADDOCK, high ambiguity driven protein–protein docking; HGB1, GB1 with N-terminal His6 tag; HSQC, heteronuclear single quantum coherence; IPTG: isopropyl-beta, D-thiogalactopyranoside; NOESY, nuclear Overhauser effect spectroscopy; PDB, Protein Data Bank; ppm, parts per million; RMSD, root-mean-square deviation; SET, solubility-enhancement tag; SD, standard deviation; A1Up, ataxin-1 ubiquitin-like interacting protein; Ub, ubiquitin; UBA, ubiquitin-associated domain; UbP, ubiquitin-like protein; UbL, UBQ, or UBX, ubiquitin-like domain; UIM, ubiquitin-interacting motif; USP, ubiquitin specific protease.

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051995006.


   Acknowledgments
 
We thank Drs. A.M. Bonvin, S.J. Hubbard, and M.J. Zhang for providing the usage of the HADDOCK and NACCESS software and the pHGB expression plasmid. We also thank Dr. Y.X. Wang for critical reading of the manuscript. This work was supported by grants from the 863 Hi-Tech program (2002BA711A13), the Chinese Academy of Sciences (KSCX1-SW-17, KSCX2-SW-209), Shanghai Commission of Science and Technology (03JC14081), and One Hundred Talents Program of the Chinese Academy of Sciences.


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