The 1.1-Å Structure of the Spindle Checkpoint Protein Bub3p Reveals Functional Regions
2005; Elsevier BV; Volume: 280; Issue: 14 Linguagem: Inglês
10.1074/jbc.m412919200
ISSN1083-351X
AutoresDavid K. Wilson, David Cerna, Erin R. Chew,
Tópico(s)Genomics and Chromatin Dynamics
ResumoBub3p is a protein that mediates the spindle checkpoint, a signaling pathway that ensures correct chromosome segregation in organisms ranging from yeast to mammals. It is known to function by co-localizing at least two other proteins, Mad3p and the protein kinase Bub1p, to the kinetochore of chromosomes that are not properly attached to mitotic spindles, ultimately resulting in cell cycle arrest. Prior sequence analysis suggested that Bub3p was composed of three or four WD repeats (also known as WD40 and β-transducin repeats), short sequence motifs appearing in clusters of 4–16 found in many hundreds of eukaryotic proteins that fold into four-stranded blade-like sheets. We have determined the crystal structure of Bub3p from Saccharomyces cerevisiae at 1.1 Å and a crystallographic R-factor of 15.3%, revealing seven authentic repeats. In light of this, it appears that many of these repeats therefore remain hidden in sequences of other proteins. Analysis of random and site-directed mutants identifies the surface of Bub3p involved in checkpoint function through binding of Bub1p and Mad3p. Sequence alignments indicate that these surfaces are mostly conserved across Bub3 proteins from diverse species. A structural comparison with other proteins containing WD repeats suggests that these folds may bind partner proteins using similar surface areas on the top and sides of the propeller. The sequences composing these regions are the most divergent within the repeat across all WD repeat proteins and could potentially be modulated to provide specificity in partner protein binding without perturbation of the core structure. Bub3p is a protein that mediates the spindle checkpoint, a signaling pathway that ensures correct chromosome segregation in organisms ranging from yeast to mammals. It is known to function by co-localizing at least two other proteins, Mad3p and the protein kinase Bub1p, to the kinetochore of chromosomes that are not properly attached to mitotic spindles, ultimately resulting in cell cycle arrest. Prior sequence analysis suggested that Bub3p was composed of three or four WD repeats (also known as WD40 and β-transducin repeats), short sequence motifs appearing in clusters of 4–16 found in many hundreds of eukaryotic proteins that fold into four-stranded blade-like sheets. We have determined the crystal structure of Bub3p from Saccharomyces cerevisiae at 1.1 Å and a crystallographic R-factor of 15.3%, revealing seven authentic repeats. In light of this, it appears that many of these repeats therefore remain hidden in sequences of other proteins. Analysis of random and site-directed mutants identifies the surface of Bub3p involved in checkpoint function through binding of Bub1p and Mad3p. Sequence alignments indicate that these surfaces are mostly conserved across Bub3 proteins from diverse species. A structural comparison with other proteins containing WD repeats suggests that these folds may bind partner proteins using similar surface areas on the top and sides of the propeller. The sequences composing these regions are the most divergent within the repeat across all WD repeat proteins and could potentially be modulated to provide specificity in partner protein binding without perturbation of the core structure. The spindle checkpoint is a signal transduction pathway that blocks the progress of the cell cycle through mitosis in response to kinetochores that are not attached to the spindles responsible for chromosomal segregation (1.Hardwick K.G. Trends Genet. 1998; 14: 1-4Abstract Full Text PDF PubMed Scopus (103) Google Scholar). A thorough functional understanding of the proteins that mediate this signal transduction mechanism is key in elucidating the mechanism by which cells maintain the proper number of chromosomes after cell division. Errors in the segregation of chromosomes have been shown to result in aneuploidy and have also been associated with tumorigenesis (2.Lengauer C. Kinzler K.W. Vogelstein B. Nature. 1998; 396: 643-649Crossref PubMed Scopus (3390) Google Scholar). Defects in the spindle checkpoint provide one plausible explanation for this. Indeed, mutations have been found in at least one spindle checkpoint protein (Bub1 1The abbreviations used are: Bub, budding uninhibited by benomyl; HA, hemagglutinin; r.m.s., root mean square. , budding uninhibited by benomyl) in human cancers (3.Cahill D.P. Lengauer C. Yu J. Riggins G.J. Willson J.K.V. Markowitz S.D. Kinzler K.W. Vogelstein B. Nature. 1998; 392: 300-303Crossref PubMed Scopus (1314) Google Scholar). Moreover, expression levels of these proteins have been found to be altered in cancer tissue and cell lines (4.Grabsch H. Takeno S. Parsons W.J. Pomjanski N. Boecking A. Gabbert H.E. Mueller W. J. Pathol. 2003; 200: 16-22Crossref PubMed Scopus (142) Google Scholar). Other cancers associated with checkpoint defects have also been discussed previously (5.Musacchio A. Hardwick K.G. Nat. Rev. Mol. Cell Biol. 2002; 3: 731-741Crossref PubMed Scopus (471) Google Scholar). Bub3p is a 38-kDa WD repeat-containing protein and is conserved in organisms ranging from yeast to humans. Bub3p was initially discovered in a screen, searching for proteins involved in arresting the cell cycle in response to microtubule damage in budding yeast caused by the microtubule inhibitor benomyl (6.Hoyt M.A. Totis L. Roberts B.T. Cell. 1991; 66: 507-517Abstract Full Text PDF PubMed Scopus (899) Google Scholar). It functions in concert with several other proteins in yeast including Bub1p, Mad1p, Mad2p, and Mad3p, as well as Mps1p to mediate this pathway (7.Li R. Murray A.W. Cell. 1991; 66: 519-531Abstract Full Text PDF PubMed Scopus (933) Google Scholar). The function of the protein is to bind to kinetochores that are not attached to opposing microtubules, thereby activating a cascade of incompletely understood biochemical events. The end effect of this pathway is the inhibition of the anaphase-promoting complex (APC), which is a ubiquitin ligase complex responsible for the degradation of protein, maintaining the cell in metaphase (8.Morgan D.O. Nat. Cell Biol. 1999; 1: E47-E53Crossref PubMed Scopus (306) Google Scholar). WD repeats, also known as β-transducin repeats and WD40 repeats, occur in tandem clusters, contain ∼40 amino acids, and are widely distributed in many hundreds of eukaryotic proteins comprising a significant percentage of the genome. Approximately 2% of the proteins in Saccharomyces cerevisiae contain them, and it appears that similar values are likely in other organisms with sequenced genomes that are not as well annotated. The name is derived from a loosely conserved Trp-Asp dipeptide found at the C-terminal end of the consensus sequence. In general, these proteins function by specifically binding to one or more partner proteins. This is often done to present a protein substrate molecule to an enzyme such as a kinase or ubiquitin ligase. They also function as scaffolding proteins to aid in forming large complexes involved in processes such as mRNA splicing and translation initiation. Other roles for WD repeat proteins have been found in mediating signal transduction, cell cycle regulation, transcriptional regulation, vesicular trafficking, developmental regulation, mRNA processing, and numerous other physiologically relevant processes involving protein-protein interactions (9.Neer E.J. Schmidt C.J. Nambudripad R. Smith T.F. Nature. 1994; 371: 297-300Crossref PubMed Scopus (1292) Google Scholar, 10.van Nocker S. Ludwig P. BMC Genomics. 2003; 4: 50Crossref PubMed Scopus (236) Google Scholar). The WD repeats in Bub3p have been shown to bind at least two other checkpoint proteins, Mad3p (11.Hardwick K.G. Johnston R.C. Smith D.L. Murray A.W. J. Cell Biol. 2000; 148: 871-882Crossref PubMed Scopus (212) Google Scholar) and Bub1p, a serine/threonine kinase (12.Roberts R.T. Farr K.A. Hoyt M.A. Mol. Cell. Biol. 1994; 14: 8282-8291Crossref PubMed Scopus (190) Google Scholar). In order to understand how Bub3p binds its partners to activate the checkpoint and to increase our general understanding of WD proteins, we have determined the crystal structure of the protein from S. cerevisiae at 1.1-Å resolution using multiple isomorphous replacement. Identification and analysis of site-directed and random mutants that fail to rescue BUB3-null mutants have allowed us to map residues on the surface of the protein that are responsible for checkpoint function. Cloning, Purification, and Crystallization—The gene encoding Bub3p was amplified from S. cerevisiae genomic DNA using the primers 5′-CCATATGCAGATAGTACAAATTGAG and 5′-CCCCGGGGTTCTCATAGTCAAATATTAT, which include extra sequences that enable cleavage by NdeI for the 5′-primer and SmaI for the 3′-primer (underlined). The resulting insert was ligated into the NdeI/SmaI site in the vector pTYB2 (New England Biolabs). The resulting construct was sequenced and used to transform the expression strain ER2566. Overexpressed protein was purified using a chitin affinity column. Secondary purification was carried out using an HQ anion exchange column mounted on a Perseptive Biosystems Perfusion Chromatography System at pH 6.0 using a 0–0.5 m NaCl gradient. Monodisperse protein was concentrated to 13 mg/ml, and the buffer was changed to 10 mm Hepes, pH 7.3. Crystals were grown using the hanging drop vapor diffusion method. Drops containing 1 μl of protein and 1 μl of well solution were suspended over a well containing 14% polyethylene glycol 8000, 200 mm calcium acetate, 0.1 m cacodylate, pH 6.5. Irregularly shaped crystals appeared in several days and were harvested into 25% (v/v) glycerol, 75% (v/v) well solution, and flash-cooled in a 100 K nitrogen stream for data collection. Data Collection, Structure Determination, and Refinement—Native data were collected at beamline 5.0.2 at the Advanced Light Source to 1.1-Å resolution. Derivative data were collected on an R-AXIS IV mounted on a rotating anode (Table I). In both cases, the data were reduced using DENZO and SCALEPACK (13.Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38570) Google Scholar). The space group was determined to be P212121 based on the unit cell parameters (a = 52.27 Å, b = 74.09 Å, c = 94.09 Å, α = β = γ = 90°) and extinctions of axial reflections. Heavy atom refinement, density modification, and phasing calculations were performed using the CCP4 package (14.Collaborative Computational Crystallography Project, NActa Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19768) Google Scholar). Figures of merit before and after solvent flattening were 0.66 and 0.78, respectively. Isomorphous and anomalous differences were used to construct phase maps, which were interpreted using O (15.Jones T.A. Zou J.-Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13011) Google Scholar) and CHAIN (16.Sack J.S. J. Mol. Graphics. 1988; 6: 224Crossref Google Scholar). This structure was initially refined at 1.37-Å resolution using CNS, which reduced Rcryst and Rfree to 21.8% and 24.1%, respectively. Subsequent refinement employed SHELX (17.Sheldrick G.M. Schneider T.R. Carter C.W. Sweet R.M. Methods in Enzymology. 277. Academic Press, San Diego, CA1997: 319-343Google Scholar) using 1.1-Å data and anisotropic temperature factors. Addition of riding hydrogen atoms reduced the Rcryst and Rfree to the final values shown in Table I.Table IData collection, phasing, and refinement information for Bub3pNativeKAu(CN)2EMTSaEMTS, ethylmercurithiosalicylateK2PtCl4Resolution (Å)30–1.1 (1.13–1.10)bStatistics concerning the high resolution shell are shown in parentheses30–2.3 (2.38–2.30)30–2.3 (2.38–2.30)30–2.2 (2.28–2.20)Completeness (%)97.8 (91.3)99.3 (100.0)99.9 (99.9)92.6 (89.0)Rmerge (%)4.2 (42.2)5.3 (11.0)4.2 (8.2)6.5 (18.2)Mean I/σ(I)18.7 (3.5)19.5 (8.5)51.7 (22.9)21.6 (8.0)Phasing power (to 2.5 Å)1.451.681.15Rcryst (%)15.2Rfree (%)18.6R.m.s. deviation bonds (Å)0.022R.m.s. deviation angles (°)2.7a EMTS, ethylmercurithiosalicylateb Statistics concerning the high resolution shell are shown in parentheses Open table in a new tab Yeast Strains and Media—All haploid yeast strains used in this work were derived from the CRY1 1401 strain. Plasmids containing the random mutants were transformed using the lithium acetate method. Yeast media, growth conditions, stock solutions, and molecular techniques were as previously described (18.Guthrie C. Fink G.R. Methods Enzymol. 1991; 194: 1-933PubMed Google Scholar, 19.Hardwick K.G. Murray A.W. J. Cell Biol. 1995; 131: 709-720Crossref PubMed Scopus (199) Google Scholar). To construct the bub1::bub1-(HA)3::HIS3, bub3-Δ::kan,ura3-, can1-100,ade2-1,trp1-1,leu2-3,-112, his3-11, -15, ura3-1 strain used for rescue experiments, three HA tags were introduced at the endogenous locus of the Bub1 gene as an in-frame C-terminal fusion protein with the coding sequence as described (20.Puig O. Rutz B. Luukkonen B.G. Kandels-Lewis S. Bragado-Nilsson E. Seraphin B. Yeast. 1998; 14: 1139-1146Crossref PubMed Scopus (83) Google Scholar). Disruption of the BUB3 gene was performed as described (20.Puig O. Rutz B. Luukkonen B.G. Kandels-Lewis S. Bragado-Nilsson E. Seraphin B. Yeast. 1998; 14: 1139-1146Crossref PubMed Scopus (83) Google Scholar). Correct modifications were confirmed by PCR and Western blot analysis. The BUB3 construct containing the promoter and termination sequence used in rescue experiments was made using the primers 5′-ATCTAGAGGCAGACACCCCTTTGCATAATATGTCATC and 5′-AGTCGACGTGAGGAATTTGGGAGCAGACTTTTGGC. These primers amplified approximately 200 base pairs flanking the BUB3 open reading frame from the CRY1 strain and allowed cloning of this product into a yeast single copy plasmid, the Escherichia coli/yeast vector YCplac33 (21.Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: 527-534Crossref PubMed Scopus (2521) Google Scholar). PCR modification of the nucleotides directly upstream and downstream of the BUB3 open reading frame were used to remove the BUB3 coding region and replaced with an EcoRI and SmaI restriction sites and also incorporated thirteen copies of the Myc epitope (EQKLISEEDLN) to yield pDC, the empty plasmid used in rescue experiments. The BUB3 gene was amplified and inserted into pDC to make the wild-type Myc-tagged expression construct (pBUB3) for immunoprecipitation. The pBUB3 was tested for its ability to rescue the ΔBUB3 strain phenotype and then sequenced. Generation of Random and Site-directed Mutants—Random mutants were generated using the Diversify PCR random mutagenesis kit according to the manufacturer's instructions (Clontech). Primers 5′-CTATCTGATATCTGCAACACGAAAACACAACAG and 5′-GGAGGAGAAGCGAAGAGAGAGCGATGAATCTG were used to amplify the BUB3 open reading frame in pBUB3. The arginine to glutamate and serine/threonine to alanine site-directed mutants were generated using the mega primer PCR method (22.Colosimo A. Xu Z. Novelli G. Dallapiccola B. Gruenert D.C. BioTechniques. 1999; 5: 870-873Crossref Scopus (33) Google Scholar). The primer sequences were designed to frame the mutation site upstream and downstream by 20 bp to yield an in-frame codon mutation. In all cases, the mutated inserts were placed into the vector pDC. These plasmids were used to transform a ΔBUB3 strain of CRY1 (Bub1-HA3::HIS3, bub3Δ::kan, can1-100, ade2–1, trp1-1, leu2-3, -112, his3-11, -15,ura3-1). Transformants were plated on yeast synthetic drop out medium without uracil at 30 °C. Colonies were replica plated on to media supplemented with either 7.5 or 10 μg/ml of benzimidazole. After 2 days at 30 °C, colonies with inhibited growth were taken from the original plates and grown overnight in liquid medium. The plasmid associated with each colony was frozen for further use. Plasmids were analyzed by PCR for the BUB3 open reading frame. Positive clones were serially diluted onto plates with 7.5 or 10 μg/ml of benzimidazole. After 2 days, clones that again failed to grow were identified and sequenced. Immunoprecipitations—For co-immunoprecipitation experiments, extracts from yeast cells were grown at 30 °C to a density of 0.8 OD600 nm. Whole cell extracts were prepared by centrifugation of the culture, washing with water, and another centrifugation. The pellet was then weighed and resuspended in the same weight of lysis buffer 50 mm Hepes, pH 7.6, 75 mm KCl, 1 mm MgCl2, 1 mm EGTA, Triton X-100 0.5 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and protease complete mixture (Roche Applied Science). The suspension was then dripped in a 50-ml Falcon tube filled with liquid nitrogen. The frozen pellets were ground in a porcelain mortar and pestle cooled in liquid nitrogen. Cell rupture was monitored by phase contrast microscopy. The powder was then thawed on ice and cell debris was removed by centrifugation at 12,000 rpm for 10 min at 4 °C. The extracts were frozen in liquid nitrogen and stored at -80 °C. A total of 2 mg of yeast extract in a volume of 1 ml in lysis buffer was incubated overnight with 20 μl of a 50/50 agarose-conjugated anti-Myc antibodies equilibrated in lysis buffer (sc-40; Santa Cruz Biotechnology Inc.). Beads were washed three times with lysis buffer and then transferred to new tubes and washed three times with phosphate-buffered saline (140 mm NaHPO4, 1.8 mm KH2PO4, 138 mm NaCl, 2.7 mm KCl, pH 7.2). Beads were then incubated in SDS sample buffer for 5 min at 95 °C (80 mm Tris, pH 6.8, 2% SDS, 10% glycerol, 10 mm EDTA, 0.0015% bromphenol blue, and working concentration of complete protease mixture). Standard methods were used for SDS-PAGE, protein transfer to nitrocellulose, and Western analysis (23.Harlow E. Lane D. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988Google Scholar). Anti-Myc and anti-HA monoclonal antibodies were used at 1.0 μg/ml and 0.1 μg/ml respectively (9E10,12CA5; Roche Applied Science). Affinity-purified anti-Mad3p rabbit antibodies were used at 0.5 μg/ml. All antibodies were diluted in 4% Blotto (10 mm Tris-HCl, pH 8.0, 150 mm NaCl, 0.1% Tween 20, 4% nonfat powdered milk). The anti-Mad3p antibody was kindly provided by Kevin Hardwick, University of Edinburgh, UK. Blots were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse, at 1:5000 (NA 931, Amersham Biosciences) and HRP-conjugated anti-rabbit at a 1:2000 dilution in Blotto (sc-2004; Santa Cruz Biotechnology Inc.). After the final wash, blots were incubated in 10 mm Tris-HCl, pH 8.0, 150 mm, for 5 min and then developed with the Santa Cruz Biotechnology Western blotting luminol reagent according to the manufacturer's instructions. Overall Structure—Bub3p is a member of a large superfamily of proteins that contains tandem sequence repeats termed WD repeats after the Trp-Asp dipeptide that is sometimes found at the C-terminal end of the repeat. Crystal structures of WD repeat proteins such as the transducin β-subunit (24.Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1051) Google Scholar, 25.Sondek J. Bohm A. Lambright D.G. Hamm H.E. Sigler P.B. Nature. 1996; 379: 369-374Crossref PubMed Scopus (707) Google Scholar), budding yeast Tup1p (26.Sprague E.R. Redd M.J. Johnson A.D. Wolberger C. EMBO J. 2000; 19: 3016-3027Crossref PubMed Scopus (104) Google Scholar), the p40 subunit of the Arp2/3 complex (27.Robinson R.C. Turbedsky K. Kaiser D.A. Marchand J.-B. Higgs H.N. Choe S. Pollard T.D. Science. 2001; 294: 1679-1684Crossref PubMed Scopus (413) Google Scholar), and others have shown that the WD repeat effectively yields a four-stranded β-sheet. In almost all cases, these blade-like secondary structural modules pack into a seven-bladed propeller-like structure. By convention, these strands are labeled A through D starting with the innermost strand. Rather than forming a single blade, each repeat consists of the outside (D) strand of one blade then crosses over to create the inner three strands (A–C) of the next blade. The D strand from the first repeat is the outside strand for the last blade, creating an interlocking structure. Based on sequence analysis, Bub3 was believed to have three or four WD repeats (28.Fraschini R. Beretta A. Sironi L. Musacchio A. Lucchini G. Piatti S. EMBO J. 2001; 20: 6648-6659Crossref PubMed Scopus (155) Google Scholar, 29.Taylor S.S. Ha E. McKeon F. J. Cell Biol. 1998; 142: 1-11Crossref PubMed Scopus (361) Google Scholar). Despite this, threading techniques have suggested the existence of a seven-bladed propeller (28.Fraschini R. Beretta A. Sironi L. Musacchio A. Lucchini G. Piatti S. EMBO J. 2001; 20: 6648-6659Crossref PubMed Scopus (155) Google Scholar). The refined model contains all Bub3p residues (1–341) and a C-terminal proline, which was appended from cloning. The high resolution structure clearly reveals that Bub3p possesses seven structural repeats (Fig. 1). These are arranged in a circular manner as seen in other WD repeat proteins with a completely hydrated central core. Using the conventional definition of the "top" of the protein as the side containing the loops connecting the D and A strands (the DA loop) and the loops connecting the B and C strands (the BC loops), the protein measures ∼45 Å from top to bottom and 45 Å across the diameter. The top of the protein is characterized by the alternate stacking of DA and BC loops as opposed to the bottom, which has the AB and CD loops positioned roughly adjacent along the radius. There are no major structural excursions from the central core structure although somewhat elongated DA loops are found between blades 3 and 4 and blades 5 and 6. An elongated BC loop is also found in blade 7 (Fig. 1A). In addition to 512 water molecules, four calcium ions were fit to regions of strong density with close (<2.5 Å) contacts to electronegative protein atoms or ordered water molecules. Because 100–150 mm calcium acetate was necessary for crystallization, these sites probably reflect the high concentration of calcium in the crystallization medium rather than any physiological role of calcium. The nature of the binding sites also suggests that they are low affinity. Each ion has only one, two, or three protein ligands. One of these is typically a carboxylate from a glutamate or aspartate. Two of these are present on the top of the protein, one associated with repeat 6 and the other facilitating a crystal packing contact near repeat 7. Another forms a packing contact on repeat 4, and the fourth is found in the central pore (Fig. 1). Interestingly, non-WD repeat β-propeller enzymes have been shown to bind cofactors, including calcium, in the central tunnel (30.Neer E.J. Smith T.F. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 960-962Crossref PubMed Scopus (27) Google Scholar). Bub3p Function—Various experiments have demonstrated that Bub1p and Mad3p bind directly to Bub3p during checkpoint function (11.Hardwick K.G. Johnston R.C. Smith D.L. Murray A.W. J. Cell Biol. 2000; 148: 871-882Crossref PubMed Scopus (212) Google Scholar, 12.Roberts R.T. Farr K.A. Hoyt M.A. Mol. Cell. Biol. 1994; 14: 8282-8291Crossref PubMed Scopus (190) Google Scholar, 31.Seeley T.W. Wang L. Zhen J.Y. Biochem. Biophys. Res. Commun. 1999; 257: 589-595Crossref PubMed Scopus (40) Google Scholar). Sequence alignments have shown that both of these proteins have two homologous segments dubbed region I and region II (11.Hardwick K.G. Johnston R.C. Smith D.L. Murray A.W. J. Cell Biol. 2000; 148: 871-882Crossref PubMed Scopus (212) Google Scholar). In both proteins, region I has been shown to be involved in the interaction with Cdc20p, an activator of the anaphase promoting complex. Likewise, region II is responsible for Mad3p binding (11.Hardwick K.G. Johnston R.C. Smith D.L. Murray A.W. J. Cell Biol. 2000; 148: 871-882Crossref PubMed Scopus (212) Google Scholar) and Bub1p binding to Bub3p (29.Taylor S.S. Ha E. McKeon F. J. Cell Biol. 1998; 142: 1-11Crossref PubMed Scopus (361) Google Scholar). Sequence comparison between Bub1p and Mad3p show that 21 of 42 of the residues composing region II in Bub1p are identical, including many charged side chains (29.Taylor S.S. Ha E. McKeon F. J. Cell Biol. 1998; 142: 1-11Crossref PubMed Scopus (361) Google Scholar). This high level of sequence conservation in the Bub3p binding domain would strongly suggest that they bind to the same surface of Bub3p. To determine the location of surface residues interacting with partner proteins in Bub3p, the location of previously known mutations was examined. A deletion of residues 218–221 has been reported that corresponds to the removal of a major portion of strand C in WD repeat number 5 (29.Taylor S.S. Ha E. McKeon F. J. Cell Biol. 1998; 142: 1-11Crossref PubMed Scopus (361) Google Scholar). Because these are internal residues, their deletion would severely destabilize or hamper the folding of the protein rather than specifically affect binding. Two surface mutants have also been constructed (W31G and W120G) yielding a protein that fails to bind to a complex of the checkpoint proteins Mad2p, Mad3p, and Cdc20p, possibly by disrupting a direct interaction with Mad3p (28.Fraschini R. Beretta A. Sironi L. Musacchio A. Lucchini G. Piatti S. EMBO J. 2001; 20: 6648-6659Crossref PubMed Scopus (155) Google Scholar). These are found at the C-terminal end of the B strands of repeats 1 and 3 which composes a part of the top of the propeller, a region implicated in partner protein binding in other WD repeat proteins (Fig. 2A). To further define the protein binding surface of Bub3p, we have used error-prone PCR to produce mutant proteins that fail to rescue a BUB3-null strain when plated on selective media containing benomyl (Fig. 3). Several hundred colonies that gave reproducible phenotypes were further characterized. After identifying and discarding those with empty vectors, multiple mutations, frameshifts, and stop codons, there remained eight clones with single point mutations at positions 2, 188, 191, 192, 193, 226, 276, and 278. Mapping of these mutants to the protein structure revealed that most occur on the top and side of the propeller (Fig. 2A), regions which have been implicated in protein binding in other WD proteins (Fig. 4) (24.Lambright D.G. Sondek J. Bohm A. Skiba N.P. Hamm H.E. Sigler P.B. Nature. 1996; 379: 311-319Crossref PubMed Scopus (1051) Google Scholar, 26.Sprague E.R. Redd M.J. Johnson A.D. Wolberger C. EMBO J. 2000; 19: 3016-3027Crossref PubMed Scopus (104) Google Scholar, 27.Robinson R.C. Turbedsky K. Kaiser D.A. Marchand J.-B. Higgs H.N. Choe S. Pollard T.D. Science. 2001; 294: 1679-1684Crossref PubMed Scopus (413) Google Scholar, 32.Mohri K. Vorobiev S. Fedorov A.A. Almo S.C. Ono S. J. Biol. Chem. 2004; 279: 31697-31707Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 33.Loew A. Ho Y.-K. Blundell T. Bax B. Structure. 1998; 6: 1007-1019Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar).Fig. 4Conservation of protein binding surfaces in known structures of WD repeat proteins complexed with partner proteins. Functional surfaces of the WD repeat proteins Gβ and p40 indicate regions involved in partner protein binding and are similar to the Bub3p surface shown in Fig. 2, A and B. All models have an orientation similar to Fig. 1. Interaction surfaces: A, Gα binding to Gβ (35.Wall M.A. Coleman D.E. Lee E. Iniguez-Lluhi J.A. Posner B.A. Gilman A.G. Sprang S.R. Cell. 1995; 83: 1047-1058Abstract Full Text PDF PubMed Scopus (1014) Google Scholar); B, phosducin binding to Gβ (33.Loew A. Ho Y.-K. Blundell T. Bax B. Structure. 1998; 6: 1007-1019Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar); C, p16 binding to p40 (27.Robinson R.C. Turbedsky K. Kaiser D.A. Marchand J.-B. Higgs H.N. Choe S. Pollard T.D. Science. 2001; 294: 1679-1684Crossref PubMed Scopus (413) Google Scholar); D, p20 binding to p40 (27.Robinson R.C. Turbedsky K. Kaiser D.A. Marchand J.-B. Higgs H.N. Choe S. Pollard T.D. Science. 2001; 294: 1679-1684Crossref PubMed Scopus (413) Google Scholar) are shown in red. These figures were prepared using the program GRASP (45.Nicholls A. Sharp K.A. Honig B. Proteins: Struct., Funct., Genet. 1991; 11: 281-296Crossref PubMed Scopus (5316) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The effects of several of the mutations can be rationalized based upon structural results. The Q2L mutation may cause the formation of an unstable N terminus of Bub3p. The hydrophobic properties of leucine could affect the stability in the solvent-exposed surface of the D strand. This mutation is at the site of the "molecular velcro" of Bub3p where the N terminus forms the first D strand of the last blade, thereby securing the head to the tail end of the protein (Fig. 1A). This is a region that has been noted by others as important for protein stability. Despite this, the mutant appears to be stable within the cell as shown by Western blots (Fig. 5A). The mutant E188V removes the hydrogen bond of Glu-188 to the Trp-174 side chain, putatively perturbing its interactions with Arg-176, located 4 Å away. This arginine is solvent-exposed, and its mutation to a glutamate destroys Bub3p checkpoint function (described below). The surface mutations G191R, L192E, and K193T also appear on the top side of the propeller and are also located within proximity of arginine at position 217. The mutation of this arginine to a glutamate also destroys checkpoint function. Residues Glu-188, Gly-191, Leu-192, and Lys-193 are found between the N terminus and the middle of the D strand of repeat 5. The Q226L mutant is located at the top of strand C and D of repeat 6. S276P and W278R a
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