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Originally published In Press as doi:10.1074/jbc.M409422200 on September 29, 2004 Originally published In Press as doi:10.1074/jbc.M409422200 on September 9, 2004

J. Biol. Chem., Vol. 279, Issue 45, 46787-46793, November 5, 2004
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Solution Structure of a Ubiquitin-like Domain from Tubulin-binding Cofactor B*

Betsy L. Lytle{ddagger}§, Francis C. Peterson{ddagger}§, Shi-Hong Qiu¶, Ming Luo¶, Qin Zhao{ddagger}||, John L. Markley{ddagger}||, and Brian F. Volkman{ddagger}§**

From the {ddagger}Center for Eukaryotic Structural Genomics, Madison, Wisconsin 53706-1549, §Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, Center for Biophysical Sciences and Engineering, Southeast Collaboratory for Structural Genomics, University of Alabama at Birmingham, Birmingham, Alabama 35294, and ||Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706

Received for publication, August 17, 2004 , and in revised form, September 2, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Proper folding and assembly of tubulin {alpha}{beta}-heterodimers involves a stepwise progression mediated by a group of protein cofactors A through E. Upon release of the tubulin monomers from the chaperonin CCT, they are acted upon by each cofactor in the folding pathway through a unique combination of protein interaction domains. Three-dimensional structures have previously been reported for cofactor A and the C-terminal CAP-Gly domain of cofactor B (CoB). Here we report the NMR structure of the N-terminal domain of Caenorhabditis elegans CoB and show that it closely resembles ubiquitin as was recently postulated on the basis of bioinformatic analysis (Grynberg, M., Jaroszewski, L., and Godzik, A. (2003) BMC Bioinformatics 4, 46). CoB binds partially folded {alpha}-tubulin monomers, and a putative tubulin-binding motif within the N-terminal domain is identified from sequence and structure comparisons. Based on modeling of the homologous cofactor E ubiquitin-like domain, we hypothesize that cofactors B and E may associate via their {beta}-grasp domains in a manner analogous to the PB1 and caspase-activated deoxyribonuclease superfamily of protein interaction domains.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
The cytoskeleton of eukaryotic cells is a network of microfilaments, intermediate filaments, and microtubules. The microtubules, hollow tubes made up of {alpha}{beta}-tubulin heterodimers, provide structural support for the cell and play a role in cell division, cell movement, and intracellular transport. The actin and tubulin subunits from which microfilaments and microtubules are comprised require the cytosolic chaperonin CCT (also known as c-cpn or TriC) for proper folding (1). However, in contrast to the folding of actin and {gamma}-tubulin, which require only chaperonin and ATP, the folding and dimerization of {alpha}- and {beta}-tubulin is a complex process involving the additional presence of GTP and five tubulin folding cofactors (2–4). After release from CCT, quasi-native folding intermediates are captured and stabilized by cofactors A or D (in the case of {beta}-tubulin) or by cofactors B and E (in the case of {alpha}-tubulin) (5, 6). A supercomplex is then formed consisting of cofactors C–E and {alpha}- and {beta}-tubulin. Finally, the {alpha}{beta}-tubulin heterodimer is released upon hydrolysis of GTP (7, 8). In addition, the interaction of cofactor D with native tubulin is regulated by the small G protein Arl2 (9).

Of the five cofactors, structures have been determined only for cofactor A (10, 11) and the C-terminal glycine-rich cytoskeleton-associated protein (CAP-Gly) domain of cofactor B (12). However, a recent study combining modeling and fold prediction tools identified previously unknown domains of cofactors B–E (13). These include a spectrin-like domain in cofactor C, HEAT and Armadillo domains in cofactor D, and leucine-rich repeats in cofactor E. In addition, the N and C termini of cofactors B and E, respectively, were predicted to adopt a ubiquitin-like {beta}-grasp fold. Here we report the structure of the Caenorhabditis elegans cofactor B ubiquitin-like (CoB1 Ubl) domain, which was solved as part of a structural genomics effort directed at novel protein domains from eukaryotic organisms. While its fold closely resembles that of ubiquitin, it is unlikely to function as a ubiquitin-like covalent modifier of other target proteins. Instead, we hypothesize that the domain has separate interaction sites for binding both {alpha}-tubulin and cofactor E.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Cloning—We developed an Escherichia coli-based expression system to produce the N-terminal half of tubulin folding cofactor B from C. elegans, residues 1–120. PCR was used to amplify a gene fragment coding for the N-terminal 120 amino acids with BamHI and HindIII sites at the 5' and 3' ends, respectively, to facilitate insertion into a modified pQE30 vector (Qiagen, Valencia, CA). The coding sequence of the insert was verified by DNA sequencing.

Protein Expression and Purification—The pQE30T-CoB (1–120) expression construct was transformed into E. coli strain SG13009[pRPEP4] (Qiagen). Cells were grown at 37 °C in LB media containing 150 µg/ml ampicillin and 50 µg/ml kanamycin until the cell density reached A600 = 0.7. Protein expression was then induced by the addition of isopropyl-{beta}-D-thiogalactopyranoside to a final concentration of 1 mM. Following induction, cells were grown for another 5 h, harvested, and stored at –80 °C until processed further. For uniform labeling with 15N, cells were grown in M9 media containing 15N-ammonium chloride as the sole nitrogen source.

The recombinant N-terminal domain of CoB was found exclusively in the soluble fraction of the harvested cells and purified by the following procedure. Cell paste from a 1-liter culture was resuspended in 10 ml of lysis buffer (50 mM sodium phosphate, pH 7.4, 300 mM NaCl, 10 mM imidizole, 0.1% (v/v) 2-mercaptoethanol, 1 mM phenylmethylsufonyl fluoride) and lysed by two passes through a French pressure cell. The cell lysate was clarified by centrifugation at 10,000 x g for 15 min. The resulting protein solution was incubated with Ni+2-NTA resin (Qiagen) in batch mode for 30 min at room temperature, packed into a 5-ml disposable column, and washed with 3 x 10 ml portions of lysis buffer. The bound protein was eluted with 3 x 10 ml portions of elution buffer (50 mM sodium phosphate, pH 7.4, 300 mM NaCl, 250 mM imidizole, 0.1% (v/v) 2-mercaptoethanol). Eluted fractions were pooled and dialyzed against 4 liters of 50 mM sodium phosphate, pH 7.4, 100 mM NaCl, 0.1% (v/v) 2-mercaptoethanol overnight at 4 °C. Separation of the fusion tag was performed concurrently with dialysis by addition of a catalytic amount of TEV protease (~1,000:1). The His8 affinity tag was separated from the recombinant protein using a second Ni+2-NTA column. The TEV cleavage product was incubated with Ni+2-NTA resin as described above and washed with 3 x 10 ml portions of lysis buffer. Target protein that has been separated from the affinity tag no longer interacts with the column and is obtained from the wash fractions. Fractions containing pure target protein were pooled and dialyzed into 2 x 4 liters of 20 mM sodium phosphate, pH 6.5, 50 mM NaCl. Dialyzed protein was concentrated, and purity and identity were verified by SDS-PAGE and mass spectrometry.

During the course of NMR data collection for structural analysis, signals corresponding to residues at the C terminus gradually became weaker and split into multiple species, suggesting that this portion of the protein may be susceptible to proteolytic degradation. Mass spectrometry revealed a series of species derived from the initial product by removal of varying numbers of amino acids from the C terminus. As described below, residues beyond Val84 are unstructured and highly mobile, making the C terminus more susceptible to degradation. However, chemical shifts of residues 1–115 were unperturbed by heterogeneity at the C terminus, indicating that the protein tertiary structure was unaffected.

NMR Spectroscopy—All NMR data were acquired at 25 °C on a Bruker 600 MHz spectrometer equipped with a triple-resonance Cryo-ProbeTM and processed with NMRPipe software (14). Heteronuclear 15N-1H NOE values were determined from an interleaved pair of two-dimensional gradient sensitivity-enhanced correlation spectra of [U-15N]CoB Ubl domain acquired with and without a 3-s proton saturation period (15). Backbone resonance assignments were obtained in a semi-automated manner using the program Garant (16) with peak lists from three-dimensional HNCO, HNCACO, HNCA, HNCOCA, HNCACB, and CCONH spectra generated manually with XEASY software (17). Sidechain assignments were determined manually using three-dimensional 15N-edited TOCSY-HSQC and HCCH-TOCSY data and a 13C-edited NOESY-HSQC spectrum optimized for aromatic groups. Distance constraints were obtained from three-dimensional 15N-edited NOESY-HSQC and 13C-edited NOESY-HSQC spectra ({tau}mix = 80 ms). The total acquisition time for all NMR spectra was 232 h.

Initial structures were generated using the CANDID module of the torsion angle dynamics program CYANA (18). NOE peak intensities were converted into upper distance bounds with the CALIBA function of CYANA. Backbone {phi} and {psi} dihedral angle constraints were generated from secondary shifts of the 1H{alpha}, 13C{alpha}, 13C{beta}, 13C', and 15N nuclei shifts by the program TALOS (19). Additional NOE constraints were added in each round of calculations, and structures were generated using a simulated annealing protocol (10,000 steps total). Assignments for consistently violated restraints were reevaluated. Of the final 100 structures calculated, the 20 conformers with the lowest target function values were selected for analysis. Final refinement in explicit solvent with experimental constraints and nonbonded energy terms was performed in XPLOR-NIH (20) using a recently described protocol (21) that improves the quality of NMR structures in terms of validation criteria such as Ramachandran statistics and Z-scores.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Structure Determination—The solution conditions chosen for NMR spectroscopy were 20 mM NaPO4, pH 6.5, 50 mM NaCl, 10% 2H2O. Under these conditions, the number, dispersion, and line widths of the observed signals from a 1H-15N HSQC spectrum (Fig. 1) were all consistent with a homogeneous folded protein. The translational self-diffusion coefficient of 1.46 x 10–6 cm2s–1 measured by NMR pulsed field gradient methods (22), which is consistent with molecular mass {approx} 14 kDa, indicated that the protein is monomeric. Using manually obtained peak lists from a complete set of standard triple-resonance 3D NMR spectra, greater than 90% of the backbone 1H, 15N and 13C resonance assignments were accurately assigned by the program Garant (as described under "Experimental Procedures"). Sidechain assignments were completed by manual analysis of the CCONH and HCCH-TOCSY spectra.



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  		FIG. 1.
Two-dimensional 15N-1H HSQC spectrum of CoB Ubl domain. Resonance assignments are indicated by residue number and one-letter amino acid code. The cross-peak for Gly25 (boxed) is shifted due to frequency aliasing from its true 15N chemical shift of 99.1 ppm.

 
An automated procedure for iterative NOE assignment (18) was used in generating the structure of the protein. The final structure was generated from 114 dihedral angle constraints and 1732 unique, NOE-derived distance restraints. Molecular dynamics calculations in explicit solvent (21) (Table I) were used in the final refinement. Because no long range NOEs were observed for residues 91–120, and 15N-1H heteronuclear NOE values (Fig. 2A) confirmed that those C-terminal residues are dynamically disordered on the picosecond to nanosecond time-scale (23), the final structure calculations included only CoB residues 1–90. Low 15N–1H NOE values for residues at the N terminus (1–4) and an internal loop (52–56) indicate that these regions of CoB are also highly flexible. These dynamic properties are reflected in variations of the local backbone r.m.s.d. values (Fig. 2B) for the ensemble of structures, which is otherwise highly ordered (Fig. 2C). Statistics on experimental constraints, coordinate precision, and stereochemical quality as determined by PROCHECK (24) and WHATCHECK (25) are summarized in Table I.


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  		TABLE I
Structural statistics for 20 NMR structures

 



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  		FIG. 2.
Solution structure and backbone mobility of CoB Ubl domain. A, 15N-1H heteronuclear NOE values plotted as a function of residue number. Values for residues 91–120 (not shown) ranged from –0.4 to +0.2 consistent with an absence of defined tertiary structure beyond the extent of the {beta}-grasp domain. Other missing NOE values correspond to overlapped 1H-15N cross-peaks and proline residues. B, backbone (N, C{alpha}, C') atomic r.m.s.d. values plotted as a function of residue. Locations of secondary structure elements are indicated above the amino acid sequence. C, stereoview of the ensemble of 20 NMR structures, with helical, sheet, and loop residues colored magenta, green, and gray, respectively. D, ribbon representation of a representative conformer, colored as in C. E, surface representations showing the electrostatic surface potentials. The surface on the left is oriented the same as D, and the surface on the right is rotated 180° about the vertical axis. Red and blue represent negative and positive potentials, respectively.

 
The CoB Ubl domain (Fig. 2C) consists of a mixed five-stranded {beta}-sheet in the order 21534, a single major {alpha}-helix (residues 28–39) that packs in the concave groove of the {beta}-sheet, and a short 310 helix (residues 68–71) in the loop between strands 4 and 5. These secondary structure elements are consistent with those of ubiquitin (26) and form a tertiary structure corresponding to the common {beta}-grasp fold, which is widespread among proteins having little sequence or functional similarity. The tertiary structure is stabilized by interactions between the buried sidechains of the hydrophobic residues Tyr5, Leu7, Ile9, Leu28, Leu31, Leu35, Met46, Ile48, Leu50, Leu68, Val73, Ile79, and Ala81. In addition, the sidechain of Lys32 is buried within the core. All of these residues except Val73 are located in secondary structure elements. The aromatic ring of Tyr77 is partially exposed, causing extreme upfield shifting of the nearby Arg74 1HN resonance to 4.9 ppm because of ring current effects. The surface of CoB Ubl is devoid of any large hydrophobic patches like typical chaperones and is instead characterized by charged and polar residues (Fig. 2D).

Disordered C-terminal residues of the construct used in this analysis may serve as a flexible linker between the N-terminal Ubl domain and the C-terminal CAP-Gly domain (12) of CoB. However, by dividing the protein in the middle of this intervening sequence (residues 85–135), additional structural elements may have been disrupted. Although chemical shifts and NOE patterns did not indicate any regular secondary structures for residues 85–120, the secondary structure prediction program PSIPRED (27) predicts helices from 104–107 and 112–122. Similar predictions are made for the CoB ortholog sequences. In addition, helical {phi} and {psi} angles are predicted for residues 105–107 by the chemical shift analysis program TALOS. A short helical segment at this position would be reminiscent of 310 helices observed at the C termini of elongin B and the DCX domain, both members of the ubiquitin superfamily. The longer putative helix is perhaps an extension of a coiled-coil region predicted for residues 134–157 of the Schizosaccharomyces pombe CoB homolog, alp11 (28), a portion of which in turn coincides with the short helix observed in the CAP-Gly domain of C. elegans CoB (12). Moreover, coiled-coil domains are often found proximal to CAP-Gly domains (29), so it seems feasible that residues linking the N-terminal Ubl and C-terminal CAP-Gly domains of CoB contain a helical or coiledcoil structure in the intact protein.

Cofactor B Orthologs Sequence Comparison—Cofactor B is conserved among eukaryotes as one element of a sophisticated system for the folding and assembly of tubulin heterodimers. Overall sequence conservation among vertebrates is high with 93% sequence identity between human and murine CoB Ubl orthologs. However, the CoB Ubl domain is more divergent than the C-terminal CAP-Gly domain (Fig. 3). Whereas the C. elegans CoB CAP-Gly domain shares 65% amino acid identity with that of Homo sapiens, the corresponding Ubl domains are only 33% identical with only four residues strictly conserved across other species: Lys32, Gly40, Leu68, and Asp83. Of these, all but Asp83 are also conserved in ubiquitin and serve a structural role. In addition to the low sequence identity, the {beta}1–{beta}2 and {beta}3–{beta}4 loops vary in length among CoB sequences. Most notable is an insertion of 24 residues between strands 3 and 4 in the Ustilago maydis sequence compared with the vertebrate and other higher eukaryotic sequences.



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  		FIG. 3.
Sequence and secondary structures of CoB Ubl and alignment with some of its orthologs and human ubiquitin. Identical and similar residues in at least 13 of 15 ortholog sequences are shaded black and gray, respectively. In the consensus, based on conservation in 11 or more sequences, # represents small hydrophobic (M, I, L, V); %, aromatic hydrophobic (F, Y, W); b, basic; and a, acidic residues.

 
Structural Comparison to Other Ubl Domains—A search for homologous structures using both VAST (30) and FATCAT (31) revealed a high level of structural similarity to ubiquitin and other ubiquitin-like modifier proteins such as Nedd8 and Rub-1. Significant similarity was also found between other members of the ubiquitin superfamily, including elongin B (32), the UBX domains of FAF1 (33) and p47 (34), the Ras-binding domain of RalGDS (Protein Data Bank accession code 1RAX [PDB] ), and the Ubl domain of Hhr23B (35) (Fig. 4). Sequence similarity between the CoB Ubl domain and these proteins is very low (only 4–16% identity) with a majority of the type-conserved residues belonging to the hydrophobic core. Low sequence similarity has hindered the identification of Ubl domains in CoB sequences from other species, leading to the suggestion that the human (tubulin-specific chaperone B) and S. cerevisiae (Alf1) orthologs lack this N-terminal domain (36). However, we verified the presence of the Ubl domain in all of the sequences shown by using the Fold and Function Assignment System server (37), which predicted significant similarity to ubiquitin for all cofactor B sequences in a search of the Pfam data base.



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  		FIG. 4.
Superposition of CoB Ubl domain (gray) with (A) elongin B (1LM8 [PDB] , 1.7 Å), (B) ubiquitin (1AAR [PDB] , 2.1 Å), (C) UBX domain of FAF1 (1H8C [PDB] , 2.3 Å), (D) Ras-binding domain of RalGDS (1RAX [PDB] , 2.6 Å), and (E) N-DCX domain of doublecortin-like kinase (1MG4 [PDB] , 3.1 Å). In parentheses, the Protein Data Bank accession code is followed by the r.m.s.d. between {alpha} carbons of the aligned residues as calculated by FATCAT.

 
A key feature of the backbone structure of the CoB Ubl domain is the flexible loop (residues 52–56) between strands {beta}3 and {beta}4. This loop contains two solvent-exposed Asp residues, which are conserved among the mammalian CoB Ubl domains. Absent in ubiquitin and ubiquitin-related modifier proteins, in the UBX, PB1, and caspase-activated deoxyribonuclease (CAD) domains this loop is important for interactions with binding partners (34, 38, 39). Because of its recurring functional role, a scheme proposed for classification of ubiquitin-like domains would take the length of the {beta}3–{beta}4 loop into account (34). The UBX domain is defined in part by a conserved Phe-Pro sequence motif in the 4-residue loop; thus, along with other differences, CoB Ubl cannot be categorized as a UBX domain. However, as discussed below, it displays striking similarities to the CAD domain of ICAD and the PB1 domain type I, both of which contain conserved acidic residues within a 5-residue loop (39, 40).

The DCX domain was recently reported to be the first known microtubule-binding module with a canonical ubiquitin fold (41). Both the solution structure of the N-terminal DCX domain (one of two homologous tandem domains) from human doublecortin (Protein Data Bank accession code 1MJD [PDB] ) and the crystal structure of the equivalent domain from human doublecortin-like kinase (Protein Data Bank accession codes 1MFW [PDB] and 1MG4 [PDB] ) were determined. Although the DCX and CoB Ubl sequences display only 20% similarity, they are structurally similar with an r.m.s.d. of 3.1 Å between the {alpha} carbons of 72 equivalent positions (Fig. 4E). Like CoB Ubl, the DCX domain contains a 4-residue {beta}3–{beta}4 loop, whereas it has longer loops on either end of the {alpha}-helix. In addition, a short 310 helix is found in an extended C-terminal sequence, and specific interactions with a Trp sidechain anchor the C-terminal helix to the DCX domain. Although the CoB N-terminal construct examined in this study contains a similar sequence extension, none of the analogous stabilizing interactions between the Ubl domain and C terminus appear to be present, as shown by the enhanced mobility detected by NMR.

Putative Tubulin-binding Residues—It has been shown that CoB sequesters {alpha}-tubulin from the CCT chaperonin and maintains it in an activated quasi-native conformation before passing it on to CoE (6). CoB and CoE each contain a Ubl domain and a CAP-Gly domain, though the order of domains is reversed between the two proteins. Furthermore, it has been demonstrated in fission yeast, where the CoB ortholog, Alp11, is essential for viability, that the Ubl domain performs the necessary function of CoB (42). On the other hand, the CoB CAP-Gly domain, a known plus-end microtubule-binding domain, is not essential and appears to play a role in stabilizing binding to {alpha}-tubulin. Thus, it can be inferred that CoB Ubl contains an {alpha}-tubulin interaction surface, and by analogy the C-terminal Ubl domain of CoE may display a similar binding site.

A comparison of 15 CoB Ubl and 14 CoE Ubl sequences (data not shown) reveals only three solvent-exposed residues that are highly type-conserved in both cofactors: a pair of basic residues in strand 2 (Lys20, Lys21) and a hydrophobic residue (Val38) in the {alpha}-helix (Fig. 5). These residues all cluster on the same face, and therefore it is possible that binding of CoB and CoE to {alpha}-tubulin involves these conserved residues. Furthermore, each cofactor has an additional conserved basic residue on the same surface, but in different positions on the {alpha}-helix, that could also be involved in electrostatic interactions with {alpha}-tubulin: Lys34 in CoB and Arg442 in CoE. Because {alpha}-tubulin has a negatively charged surface, it is not surprising that a putative {alpha}-tubulin-binding surface would contain basic residues.



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  		FIG. 5.
Sequence alignment of C. elegans and human CoB Ubl and CoE Ubl. Residue numbering is shown for the C. elegans CoB Ubl (above) and CoE Ubl (below). The secondary structures are based on the CoB Ubl structure. The putative tubulin-binding residues are highlighted in gray, the conserved hydrophobic core residues are highlighted yellow, and residues belonging to the CoB Ubl acidic patch and CoE Ubl basic patch are highlighted red and blue, respectively.

 
The doublecortin protein is essential for normal development of the cerebral cortex, and a number of disease-causing missense mutations map to the surface of the tandem ubiquitin-like DCX domains that bind microtubules (41). Although there is little sequence conservation between CoB Ubl and DCX, many of the doublecortin mutations are likely to affect the electrostatic potential and cluster in the same region of the C-terminal DCX domain surface as the putative {alpha}-tubulin-binding residues of the CoB and CoE Ubl domains described above. Because C-DCX binds to tubulin heterodimers and microtubules rather than free tubulin monomers, the details of those interactions may differ from those of CoB with {alpha}-tubulin. However, one of the disease-causing doublecortin substitutions is an Arg residue (Arg196) that is found on strand {beta}2, in a location analogous to the CoB Ubl "KK" motif. Thus, the cluster of basic residues on the CoB surface may serve a common functional role with similar microtubule binding elements of the doublecortin DCX domains.

The only known structure of a protein that binds monomeric tubulin is that of cofactor A (10, 11). Although its structure, a three-helix bundle, is completely different from that of CoB, like CoB Ubl its surface is highly hydrophilic. Cofactor A binding to {beta}-tubulin, which is highly homologous to {alpha}-tubulin, was studied using cofactor A peptides, and three interaction sites were identified (11). These sites contain mostly polar residues, both positively and negatively charged, but also some hydrophobic residues. It is therefore likely that the {alpha}-tubulin binding sites of CoB and CoE involve additional charged and nonpolar residues. Also, variation in the specific elements responsible for cofactor-tubulin interactions is to be expected based on previous biochemical studies. The {alpha}-tubulin complex with CoE is not as stable as that with CoB, and unlike CoB, CoE binds both free {alpha}-tubulin and native tubulin heterodimers (6). These differences in tubulin binding may arise from sequence features that distinguish the CoB and CoE Ubl domains from each other. For example, residues Glu19 and Pro23 on either side of the "KK" motif common to both proteins are uniquely conserved in CoB and CoE Ubl, respectively. Further structural or biochemical analysis of tubulin-cofactor complexes is needed to confirm functional roles for specific residues.

Binding of Cofactor B and Cofactor E—Although the binding of CoB and CoE has been reported in fission yeast (43), the interaction has not been mapped to specific domains within either protein. Analysis of the CoB and CoE Ubl sequences revealed numerous basic residues in CoE Ubl that are not conserved in CoB Ubl (Fig. 5). This observation led us to speculate about the potential role for the Ubl domains to mediate the binding of CoB and CoE via electrostatic interactions, in a manner analogous to that of the recently defined PB1 (Phox and Bem1) domains (40, 44) and CAD domains (39), both of which display the ubiquitin-like {beta}-grasp fold. PB1 domain heterodimerization is necessary for the function of a wide variety of protein complexes such as Bem1/Cdc24p, p40phox/p67phox, PKC{zeta}/Par6, and MEK5/MEKK2 that are involved in diverse signaling pathways. The PB1 heterodimer is formed via docking of two acidic clusters on the type I domain with two basic clusters on the type II domain (38, Fig. 6A). The type I PB1 domain is defined by a conserved sequence motif, the "OPCA motif," made up of acidic residues located in the {beta}3–{beta}4 loop and an {alpha}-helix following strand {beta}4 (45). The only strictly conserved residue in type II PB1 domains is a basic residue found in strand {beta}1. Similar to PB1 domain heterodimerization, the CAD domain of CAD contains two basic clusters that interact with two acidic clusters on the CAD domain of its inhibitor (ICAD-CAD domain) (Fig. 6A). The acidic residues in ICAD-CAD domain are similarly found in the {beta}3–{beta}4 loop and after strand {beta}4.



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  		FIG. 6.
A, ribbon diagrams of the p40phox/p67phox and CAD/ICAD heterodimers showing the charged residues at the interface. B, ribbon diagrams of the CoE Ubl homology model and the CoB Ubl structure with the putative interacting sidechains shown. In both panels the acidic and basic sidechains are colored red and blue, respectively.

 
A homology model of CoE Ubl, based on the CoB Ubl structure and a sequence alignment optimized manually on the basis of multiple sequence alignments, was created using the automated model routine of the program Modeller (46, 47). The CoE Ubl model confirmed the presence of a basic region made up of Lys413 and Arg415 in strand {beta}1, Arg425 and Arg426 in strand {beta}2, Lys435 and Arg442 in helix {alpha}1, and Lys445 in the {alpha}1–{alpha}2 loop (Fig. 6B). Except for Arg425 and Arg426, these residues are conserved only in CoE Ubl and appear to be analogous to the residues of the PB1 and CAD domain basic clusters. Likewise, CoB Ubl contains conserved acidic residues within the {beta}3–{beta}4 loop (Asp54 and Asp55) as previously discussed. Additional acidic residues between strands {beta}4 and {beta}5 (Asp63, Asp70, Asp75) are found on the same face. Although acidic residues are not strictly conserved at these positions across species, a higher proportion of acidic residues than basic residues is found in the {beta}3–{beta}4, {beta}4–{alpha}3, and {alpha}3–{beta}5 loops of all sequences. Therefore, we hypothesize that, as observed in the PB1 and CAD heterodimers, acidic CoB residues may participate in electrostatic interactions with the basic residues of CoE Ubl. Additional residues, such as the conserved aromatic residue at position 71 of CoB Ubl, may be involved in the binding interface. Because of sequence variations and minor differences in tertiary structure, it is expected that a CoB/CoE complex would necessarily diverge in some respects from the examples set by the PB1 and CAD complexes. The interacting regions of a possible CoB/CoE complex are depicted in Fig. 6B; however, confirmation of this interaction must await further study.

The {beta}-Grasp Fold as a Ubiquitous Protein Interaction Domain—The CoB Ubl domain represents yet another example of the ubiquitin-like {beta}-grasp fold as a scaffold for the presentation of residues involved in protein-protein interactions. Although the proposed interactions are electrostatic in nature, like those of the DCX, PB1, and CAD domains, ubiquitin and other ubiquitin-like domains, such as UBX (34) and elongin B (48), form complexes stabilized primarily by hydrophobic interactions. The versatility of the {beta}-grasp fold appears to be a direct result of its stable hydrophobic core that requires a minimal level of sequence conservation. Thus, it is likely that as structural genomics efforts continue, many more new Ubl domain protein interactions will be discovered.


   FOOTNOTES
 
* This work was supported by the National Institutes of Health Protein Structure Initiative through Grant 1 P50 GM64598. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1T0Y [PDB] ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

All time-domain NMR data and chemical shift assignments have been deposited in BioMagResBank (www.bmrb.wisc.edu/) under BMRB accession number 6176 [BMRB] . Back

** To whom correspondence should be addressed. Tel.: 414-456-8400; Fax: 414-456-6510; E-mail: bvolkman{at}mcw.edu.

1 The abbreviations used are: CoB, cofactor B; Ubl, ubiquitin-like; NTA, nitrilotriacetic acid; NOE, nuclear Overhauser effect; HSQC, heteronuclear single quantum coherence; r.m.s.d., root mean square deviation; CAP-Gly, glycine-rich cytoskeleton-associated protein; CAD, caspase-activated deoxyribonuclease; CoE, cofactor E; PB1, Phox and Bem1; DCX, doublecortin-like domain. Back


   ACKNOWLEDGMENTS
 
We thank Adam Godzik for assistance with the cofactor E model and Kelly Kjer for assistance with protein purification.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Melki, R., Vainberg, I., Chow, R., and Cowan, N. (1993) J. Cell Biol. 122, 1301–1310[Abstract/Free Full Text]
  2. Gao, Y., Vainberg, I. E., Chow, R. L., and Cowan, N. J. (1993) Mol. Cell. Biol. 13, 2478–2485[Abstract/Free Full Text]
  3. Lopez-Fanarraga, M., Avila, J., Guasch, A., Coll, M., and Zabala, J. C. (2001) J. Struct. Biol. 135, 219–229[CrossRef][Medline] [Order article via Infotrieve]
  4. Lewis, S. A., Tian, G., and Cowan, N. J. (1997) Trends Cell Biol. 7, 479–484[CrossRef][Medline] [Order article via Infotrieve]
  5. Tian, G., Huang, Y., Rommelaere, H., Vandekerckhove, J., Ampe, C., and Cowan, N. J. (1996) Cell 86, 287–296[CrossRef][Medline] [Order article via Infotrieve]
  6. Tian, G., Lewis, S. A., Feierbach, B., Stearns, T., Rommelaere, H., Ampe, C., and Cowan, N. J. (1997) J. Cell Biol. 138, 821–832[Abstract/Free Full Text]
  7. Tian, G., Bhamidipati, A., Cowan, N. J., and Lewis, S. A. (1999) J. Biol. Chem. 274, 24054–24058[Abstract/Free Full Text]
  8. Fontalba, A., Paciucci, R., Avila, J., and Zabala, J. (1993) J. Cell Sci. 106, 627–632[Abstract]
  9. Bhamidipati, A., Lewis, S. A., and Cowan, N. J. (2000) J. Cell Biol. 149, 1087–1096[Abstract/Free Full Text]
  10. Steinbacher, S. (1999) Nat. Struct. Biol. 6, 1029–1032[CrossRef][Medline] [Order article via Infotrieve]
  11. Guasch, A., Aloria, K., Perez, R., Avila, J., Zabala, J. C., and Coll, M. (2002) J. Mol. Biol. 318, 1139–1149[CrossRef][Medline] [Order article via Infotrieve]
  12. Li, S., Finley, J., Liu, Z. J., Qiu, S. H., Chen, H., Luan, C. H., Carson, M., Tsao, J., Johnson, D., Lin, G., Zhao, J., Thomas, W., Nagy, L. A., Sha, B., DeLucas, L. J., Wang, B. C., and Luo, M. (2002) J. Biol. Chem. 277, 48596–48601[Abstract/Free Full Text]
  13. Grynberg, M., Jaroszewski, L., and Godzik, A. (2003) BMC Bioinformatics 4, 46[CrossRef][Medline] [Order article via Infotrieve]
  14. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) J. Biomol. NMR 6, 277–293[Medline] [Order article via Infotrieve]
  15. Farrow, N. A., Muhandiram, R., Singer, A. U., Pascal, S. M., Kay, C. M., Gish, G., Shoelson, S. E., Pawson, T., Forman-Kay, J. D., and Kay, L. E. (1994) Biochemistry 33, 5984–6003[CrossRef][Medline] [Order article via Infotrieve]
  16. Bartels, C., Billeter, M., Güntert, P., and Wüthrich, K. (1996) J. Biomol. NMR 7, 207–213
  17. Bartels, C., Xia, T.-H., Billeter, M., Güntert, P., and Wüthrich, K. (1995) J. Biomol. NMR 5, 1–10[Medline] [Order article via Infotrieve]
  18. Herrmann, T., Guntert, P., and Wuthrich, K. (2002) J. Mol. Biol. 319, 209–227[CrossRef][Medline] [Order article via Infotrieve]
  19. Cornilescu, G., Delaglio, F., and Bax, A. (1999) J. Biomol. NMR 13, 289–302[CrossRef][Medline] [Order article via Infotrieve]
  20. Schwieters, C. D., Kuszewski, J. J., Tjandra, N., and Clore, G. M. (2003) J. Magn. Reson. 160, 65–73[CrossRef][Medline] [Order article via Infotrieve]
  21. Linge, J. P., Williams, M. A., Spronk, C. A., Bonvin, A. M., and Nilges, M. (2003) Proteins 50, 496–506[CrossRef][Medline] [Order article via Infotrieve]
  22. Altieri, A. S., Hinton, D. P., and Byrd, R. A. (1995) J. Am. Chem. Soc. 117, 7566–7567[CrossRef]
  23. Palmer, A. G., III (2001) Annu. Rev. Biophys. Biomol. Struct. 30, 129–155[CrossRef][Medline] [Order article via Infotrieve]
  24. Laskowski, R. A., Rullmannn, J. A., MacArthur, M. W., Kaptein, R., and Thornton, J. M. (1996) J. Biomol. NMR 8, 477–486[Medline] [Order article via Infotrieve]
  25. Hooft, R. W., Vriend, G., Sander, C., and Abola, E. E. (1996) Nature 381, 272[Medline] [Order article via Infotrieve]
  26. Vijay-Kumar, S., Bugg, C. E., and Cook, W. J. (1987) J. Mol. Biol. 194, 531–544[CrossRef][Medline] [Order article via Infotrieve]
  27. McGuffin, L. J., Bryson, K., and Jones, D. T. (2000) Bioinformatics 16, 404–405[Abstract/Free Full Text]
  28. Radcliffe, P. A., and Toda, T. (2000) Mol. Gen. Genet. 263, 752–760[CrossRef][Medline] [Order article via Infotrieve]
  29. Rickard, J. E., and Kreis, T. E. (1996) Trends Cell Biol. 6, 178–183[CrossRef][Medline] [Order article via Infotrieve]
  30. Gibrat, J. F., Madej, T., and Bryant, S. H. (1996) Curr. Opin. Struct. Biol. 6, 377–385[CrossRef][Medline] [Order article via Infotrieve]
  31. Ye, Y., and Godzik, A. (2003) Bioinformatics 19, ii246–ii255[Abstract]
  32. Min, J. H., Yang, H., Ivan, M., Gertler, F., Kaelin, W. G., Jr., and Pavletich, N. P. (2002) Science 296, 1886–1889[Abstract/Free Full Text]
  33. Buchberger, A., Howard, M. J., Proctor, M., and Bycroft, M. (2001) J. Mol. Biol. 307, 17–24[CrossRef][Medline] [Order article via Infotrieve]
  34. Dreveny, I., Kondo, H., Uchiyama, K., Shaw, A., Zhang, X., and Freemont, P. S. (2004) EMBO J. 23, 1030–1039[CrossRef][Medline] [Order article via Infotrieve]
  35. Fujiwara, K., Tenno, T., Sugasawa, K., Jee, J. G., Ohki, I., Kojima, C., Tochio, H., Hiroaki, H., Hanaoka, F., and Shirakawa, M. (2004) J. Biol. Chem. 279, 4760–4767[Abstract/Free Full Text]
  36. Hartmann-Petersen, R., and Gordon, C. (2004) Semin. Cell Dev. Biol. 15, 247–259[CrossRef][Medline] [Order article via Infotrieve]
  37. Rychlewski, L., Jaroszewski, L., Li, W., and Godzik, A. (2000) Protein Sci. 9, 232–241[Medline] [Order article via Infotrieve]
  38. Wilson, M. I., Gill, D. J., Perisic, O., Quinn, M. T., and Williams, R. L. (2003) Mol. Cell 12, 39–50[CrossRef][Medline] [Order article via Infotrieve]
  39. Otomo, T., Sakahira, H., Uegaki, K., Nagata, S., and Yamazaki, T. (2000) Nat. Struct. Biol. 7, 658–662[CrossRef][Medline] [Order article via Infotrieve]
  40. Yoshinaga, S., Kohjima, M., Ogura, K., Yokochi, M., Takeya, R., Ito, T., Sumimoto, H., and Inagaki, F. (2003) EMBO J. 22, 4888–4897[CrossRef][Medline] [Order article via Infotrieve]
  41. Kim, M. H., Cierpicki, T., Derewenda, U., Krowarsch, D., Feng, Y., Devedjiev, Y., Dauter, Z., Walsh, C. A., Otlewski, J., Bushweller, J. H., and Derewenda, Z. S. (2003) Nat. Struct. Biol. 10, 324–333[CrossRef][Medline] [Order article via Infotrieve]
  42. Radcliffe, P. A., and Toda, T. (2000) in Mol. Gen. Genet. 263, 752–760[CrossRef][Medline] [Order article via Infotrieve]
  43. Radcliffe, P. A., Hirata, D., Vardy, L., and Toda, T. (1999) Mol. Biol. Cell 10, 2987–3001[Abstract/Free Full Text]
  44. Terasawa, H., Noda, Y., Ito, T., Hatanaka, H., Ichikawa, S., Ogura, K., Sumimoto, H., and Inagaki, F. (2001) EMBO J. 20, 3947–3956[CrossRef][Medline] [Order article via Infotrieve]
  45. Ponting, C. P., Ito, T., Moscat, J., Diaz-Meco, M. T., Inagaki, F., and Sumimoto, H. (2002) Trends Biochem. Sci. 27, 10[CrossRef][Medline] [Order article via Infotrieve]
  46. Sali, A., and Blundell, T. L. (1993) J. Mol. Biol. 234, 779–815[CrossRef][Medline] [Order article via Infotrieve]
  47. Fiser, A., and Sali, A. (2003) Methods Enzymol. 374, 461–491[Medline] [Order article via Infotrieve]
  48. Stebbins, C. E., Kaelin, W. G., and Pavletich, N. P. (1999) Science 284, 455–461[Abstract/Free Full Text]

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