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The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR‐mediated protein–protein interactions

The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for... The EMBO Journal Vol.17 No.5 pp.1192–1199, 1998 The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein–protein interactions although not exclusively, nuclear in location (Chen et al., Amit K.Das, PatriciaT.W.Cohen and 1994). In mammals, PP5 has a widespread tissue distribu- David Barford tion. Elevated levels of PP5 in rapidly growing cells Laboratory of Molecular Biophysics, University of Oxford, compared with serum-deprived cells suggest a role in cell Rex Richards Building, South Parks Road, Oxford, OX1 3QU and growth (Cohen et al., 1996). MRC Protein Phosphorylation Unit, Department of Biochemistry, The primary structure of PP5 reveals a C-terminal University of Dundee, Dundee, DD1 4HN, UK catalytic domain and an N-terminal tetratricopeptide repeat Corresponding author (TPR) domain containing three tandem TPR motifs e-mail: [email protected] (Becker et al., 1994; Chen et al., 1994; Chinkers, 1994) (Figure 1). TPRs occur in over 25 proteins of diverse The tetratricopeptide repeat (TPR) is a degenerate 34 biological functions and are known to mediate protein– amino acid sequence identified in a wide variety of protein interactions (Goebl and Yanagida, 1991; Sikorski proteins, present in tandem arrays of 3–16 motifs, et al., 1991; Lamb et al., 1995) (Figure 1). Within PP5, which form scaffolds to mediate protein–protein the TPR domain may perform several roles. Firstly, the interactions and often the assembly of multiprotein TPR domain is responsible for the 25-fold stimulation complexes. TPR-containing proteins include the of protein phosphatase activity by polyunsaturated fatty anaphase promoting complex (APC) subunits cdc16, acids such as arachidonic acid (Chen and Cohen, 1997; cdc23 and cdc27, the NADPH oxidase subunit p67 Skinner et al., 1997). The interaction of arachidonic acid phox, hsp90-binding immunophilins, transcription fac- with the TPR domain induces an allosteric conformational tors, the PKR protein kinase inhibitor, and peroxisomal change within PP5 that abolishes the suppression of and mitochondrial import proteins. Here, we report catalytic activity by the TPR domain (Chen and Cohen, the crystal structure of the TPR domain of a protein 1997). The activity of the arachidonic acid-stimulated PP5 phosphatase, PP5. Each of the three TPR motifs of is similar to that of the isolated PP5 phosphatase domain this domain consist of a pair of antiparallel α-helices that has been proteolytically cleaved from the TPR domain. of equivalent length. Adjacent TPR motifs are packed A second role of the TPR domain of PP5 is to mediate together in a parallel arrangement such that a tandem interactions between PP5 and hsp90 within an hsp90– TPR motif structure is composed of a regular series of glucocorticoid receptor complex, and also with the kinase antiparallel α-helices. The uniform angular and spatial domain of the ANP-guanylate cyclase receptor in vivo, arrangement of neighbouring α-helices defines a helical findings which are consistent with a role for PP5 in signal structure and creates an amphipathic groove. Multiple- transduction (Chinkers, 1994; Silverstein et al., 1997). TPR motif proteins would fold into a right-handed However, it is not known whether interactions between super-helical structure with a continuous helical groove the TPR domain of PP5 and hsp90 stimulate the PP5 suitable for the recognition of target proteins, hence phosphatase activity. In addition to its interactions with defining a novel mechanism for protein recognition. PP5, hsp90 also associates with the TPR-containing The spatial arrangement of α-helices in the PP5–TPR domain is similar to those within 14-3-3 proteins. immunophilins FKBP52 and Cyp40, in a mutually Keywords: Protein crystallography/protein phosphatase/ exclusive manner (Owens-Grillo et al., 1996; Ratajckak protein phosphorylation/signal transduction/ and Carrello, 1996). tetratricopeptide repeat The TPR motif was first identified as a tandemly repeated degenerate 34 amino acid sequence in the cell division cycle genes cdc16, cdc23 and cdc27 which encode subunits of the anaphase promoting complex Introduction (APC) (Hirano et al., 1990; Sikorski et al., 1990; King et al., 1995). The role of the APC is to target cell cycle Reversible protein phosphorylation on serine and threonine proteins for ubiquitin-dependent degradation at both the residues is essential for the regulation of numerous cellular onset of anaphase and at the exit of mitosis. Mutations functions and signal transduction pathways. Control of within the TPR motifs of these proteins cause mitotic this process is achieved by the modulation of the activities arrest at the metaphase to anaphase transition. It is now and substrate specificities of the protein kinases and realised that over 25 proteins present in organisms as phosphatases, which catalyse the opposing phosphoryl- diverse as bacteria and humans contain TPR motifs. ation and dephosphorylation reactions, respectively. The In addition to cell cycle regulation, processes such as protein Ser/Thr phosphatase PP5 is the prototype of the fourth subfamily of the PPP-family of protein transcription control, mitochondrial and peroxisomal pro- phosphatases, which also includes PP1, PP2A and PP2B tein transport, neurogenesis, protein kinase inhibition, Rac- (calcineurin) (Barford, 1996; Cohen, 1997). PP5 is con- mediated activation of NADPH oxidase, and protein served from yeast to humans being predominantly, folding involve TPR motifs (Lamb et al., 1995; Ponting, 1192 © Oxford University Press Structure of tetratricopeptide repeats Fig. 1. Schematic of TPR-containing proteins. TPR motifs (shown as boxes) are observed as multiple tandem repeats and also separated by sequence insertions. 1996). Moreover, a 10 tandem TPR motif protein (UTY) in TPR7 of cdc27 reduced its ability to interact with cdc23 was recently identified as one of the 12 novel genes of but did not affect its interactions with itself or with cdc16 the non-recombining region of the human Y-chromosome (Lamb et al., 1994). (Lahn and Page, 1997). In order to understand the molecular basis for TPR- Within TPR-containing proteins, the TPRs are usually mediated protein recognition, we have determined the arranged in tandem arrays of 3–16 motifs, although crystal structure of the TPR domain of PP5 to 2.5 Å individual, or blocks, of TPR motifs may be dispersed resolution. The results provide insight into the structure throughout the protein sequence (Figure 1). Mutagenesis of multiple TPR motif proteins, the basis for disruptive and deletion studies have revealed roles for TPR motifs mutations within the TPR-containing subunits of the APC in mediating protein–protein interactions. Proteins with and NADPH oxidase and reveal a novel mechanism for multiple copies of TPR motifs function as scaffolding protein–protein interactions. proteins and coordinate the assembly of proteins into multi-subunit complexes. Such proteins include Ssn6, Results and discussion cdc16, cdc23, cdc27 and rapsyn (Ponting and Phillips, 1996). Rapsyn for example, has been proposed to act Structure determination and overall structure as a molecular link between the nicotinic acetylcholine The crystal structure of the N-terminal TPR domain of receptor and the dystrophin–dystroglycan complex (Apel PP5 was determined to 2.5 Å resolution using multiple et al., 1995). Specific blocks of TPR motifs mediate isomorphous replacement and anomalous scattering interactions with particular target proteins and have been methods. Electron density maps obtained using experi- assigned specific biological functions. For example, the mental phases were readily interpretable (Figure 2) and N-terminal three TPR motifs of Ssn6 associate with the the polypeptide chain was traced within continuous density. co-repressor Tup1, whereas other combinations of TPR The three tandem TPR motifs of PP5 reveal a novel motifs mediate interactions with different transcription protein fold. Each TPR motif is composed of a pair of factors, which accounts for the diverse gene expression antiparallel α-helices, termed helices A and B, associated patterns regulated by Ssn6 (Smith et al., 1995; Tzamarias together with a packing angle of ~24° between helix axes and Struhl, 1995). TPR motifs 5–7 of p58, an inhibitor of (Figure 3A–C). The structure of each TPR motif is the RNA-dependent protein kinase (PKR), are responsible virtually identical; main-chain atoms of TPR1 superimpose for interactions with PKR, while the N-terminal TPR with those of TPR2 and TPR3 with a root mean square motifs direct homotypic interactions (Gale et al., 1996). deviation (r.m.s.d.) of 0.35 Å and 0.8 Å, respectively. The TPR motifs 1–3 of the peroxisomal import protein PAS8p three TPR motifs are organised into a parallel arrangement, interact with the peroxisomal targeting signal (Terlecky such that sequentially adjacent α-helices are antiparallel et al., 1995). Within the multi-subunit APC, a mutation in a manner reminiscent of a concertina (Figure 3A–C). 1193 A.K.Das, P.T.W.Cohen and D.Barford Fig. 2. Stereo view of the electron density map of the PP5–TPR domain calculated to 2.5 Å resolution using experimental phases with the refined coordinates superimposed. The figure was produced using O (Jones et al., 1991). Within a tandem array of TPR motifs, the packing of PP5, while sharing weak sequence similarity to a TPR helix A against adjacent B-helices is defined by the same motif, are folded into an extended α-helix (α7). This helix spatial and angular parameters both within, and between, packs against helix B of TPR3 in a similar arrangement adjacent TPR motifs. Hence, in general, each α-helix to that of the helices within the TPR domain (Figure 3A– shares two immediate α-helix neighbours and the protein C). The consequence of a regular repeat of α-helices that fold may be defined as an overlapping array of three- are related by a crossover angle of 24° is to generate a right- helix bundles. handed helical conformation that creates an amphipathic Multiple sequence alignments of the TPRs present channel (Figure 4). The surface of the channel is formed in 25 proteins reveals a highly degenerate sequence. predominantly by the side chains of amino acids in helix Although there is no position characterised by an invariant A of each TPR motif with relatively little contribution residue, a consensus sequence pattern of small and large from residues of helix B. In contrast, the opposite side of hydrophobic residues has been defined (Figure 3D). Small the channel is formed by residues from both TPR α-helices. hydrophobic residues are commonly observed at positions 8, 20 and 27 within the motif. Position 32 is frequently a Structural relationship to 14-3-3 proteins proline and large hydrophobic residues are also located at The antiparallel arrangement of α-helices of the TPR particular positions. Analysis of the structure of the TPR domain of PP5 is reminiscent of the antiparallel α-helices domain of PP5 provides a rationale for this consensus of the 14-3-3 protein, a homodimeric molecule with sequence pattern (Figure 3C and D). Residues 8 and 20 nine α-helices in each subunit (Liu et al., 1995; Xiao are located at the position of closest contact between the et al., 1995) (Figure 5). Unlike TPR proteins however, A and B α-helices of a TPR, whereas residue 27 on helix the α-helices of 14-3-3 proteins vary in length and in B is located at the interface of 3 helices (A, B and A) their relative packing geometries and only three of the within a 3-helix bundle. Proline 32 is located at the seven α-helices of the TPR domain superimpose closely C-terminus of helix B, and the large consensus hydro- (r.m.s.d. of 1.2 Å) with equivalent α-helices of 14-3-3 phobic residues form the interfaces between adjacent (Figure 5). TPR-containing proteins and 14-3-3 proteins α-helices. Mutations at position 8 of TPR3 of the NADPH share common structural and functional properties, oxidase subunit p67 phox and within TPRs 5 and 7 of the despite their lack of sequence similarities (Aitken, 1996). APC subunit cdc23 result in the disruption of protein The latter proteins interact with proteins phosphorylated function, presumably due to the incorrect packing of on Ser/Thr residues, most probably via an amphipathic neighbouring α-helices (Figure 3D) (Sikorski et al., 1993; groove present within each 14-3-3 subunit that is de Boer et al., 1994). The Gly to Glu substitution in p67 similar to the groove observed in the PP5–TPR domain. phox is associated with chronic granulomatous disease Within the PP5–TPR domain crystals, the protein forms and impairment of NADPH oxidase activity (de Boer a homodimer as a result of the antiparallel association et al., 1994). The TPR region of p67 phox is the site of of symmetry-related α-7 helices and the interaction of NADPH oxidase recognised by the small G-protein Rac, residues C-terminal to α-7 with residues forming the hence disruption of TPR3 may prevent Rac-stimulation channel surface of the same symmetry-related molecule of the enzyme activity (Diekmann et al., 1994; Ponting, (Figure 4). Similar to the crystal structure of the τ- 1996). In yeast, individual mutations at position 8 of isoform of 14-3-3 (Xiao et al., 1995), a sulfate ion is TPR5 and TPR7 of cdc23 (Sikorski et al., 1993) and bound within this groove by a cluster of basic residues, position 20 of TPR9 of Cut9 (Samejima and Yanagida, and this sulfate ion interacts with the channel-bound 1994) (a cdc16 homologue) result in mitotic arrest at the peptide. The interaction of a peptide within the groove metaphase to anaphase transition, most likely as a result of the TPR domain, as observed in the PP5–TPR of a blockade in the ubiquitin-dependent degradation of domain crystals, may provide a model for the recognition inhibitors of this transition. of phosphopeptides by 14-3-3 proteins. However, the The 35-residues C-terminal to the three TPR motifs of TPR-mediated dimer does not occur in full-length PP5 1194 Structure of tetratricopeptide repeats Fig. 3. (A and B) Two orthogonal views of ribbon representations of the TPR domain of PP5. Each TPR motif and the α-7 helix are labelled. The sequence of α-7 shows weak similarity to a TPR motif. Helix A of TPR1 is extended by nine residues N-terminal to the TPR domain. (C) Stereo view of the molecule. Within TPR2 and helix A of TPR3, consensus TPR motif residues (Figure 3D) are drawn to illustrate their role in inter-helix packing. The residue numbering refers to the TPR motif numbering. Primes denote residue numbering in TPR3. Main-chain atoms of any pair of helices superimpose onto equivalent atoms of all other pairs of helices within an r.m.s.d. of 1.1 Å. Figures drawn using MOLSCRIPT (Kraulis, 1991). (D) Sequence alignment of the three TPR motifs of PP5. Consensus TPR motif residues are shown with red and green backgrounds for small and large hydrophobic residues, respectively. The position of the consensus proline residue is indicated with a P. The sites of mutations in the NADPH oxidase subunit p67 phox (position 8; de Boer et al., 1994), cdc23 (position 8; Sikorski et al., 1993) and cdc16 (position 20; Samejima and Yanagida, 1994) are indicated with blue arrows. Numbers above the alignment refer to TPR motif residues and at the side, to the PP5 residues. The figure was prepared using ALSCRIPT (Barton, 1993). 1195 A.K.Das, P.T.W.Cohen and D.Barford Fig. 4. Solvent accessible surface of the PP5–TPR domain showing the electrostatic potential and amphipathic TPR-groove. Atoms of the α-7 helix of the symmetry-related molecule are shown interacting with the α-7 helix and the C-terminus with the amphipathic groove. The sulfate ion is labelled. The figure was drawn using GRASP (Nicholls and Honig, 1991). Fig. 5. Comparison of the structures of the TPR domain of PP5 (cyan) and a 14-3-3 protein (Liu et al., 1995) (yellow). Helices 4–6 of 14-3-3 were superimposed onto equivalent main-chain atoms of helices B, A and B of TPR motifs 1 and 2 with a r.m.s.d. of 1.2 Å. The figure was drawn using MOLSCRIPT (Kraulis, 1991). which is a monomer, as judged by size exclusion multiple-motif TPR proteins, we constructed a model of chromatography. 12 tandem TPR motifs. We assumed that the packing parameters between adjacent TPR motifs of the tandem Protein recognition by multiple TPR motif proteins repeat would be similar to those observed in the TPR Our model of the three TPR motifs of PP5 provides a domain of PP5. The resultant model indicates that tan- framework for predicting the structures of other TPR- demly arranged TPR motifs are organised into a regular motif-containing proteins and for interpreting functional right-handed super-helix with a helical repeat of studies of these proteins. To understand the properties of approximately seven TPR motifs, a pitch of 60 Å and a 1196 Structure of tetratricopeptide repeats Fig. 6. Model of a multiple TPR motif containing protein. (A) View perpendicular to the helix axis of 12 TPR motifs. The main-chain is colour ramped from blue at the N-terminus to red at the C-terminus. (B) View parallel to the helix axis of an 8-motif TPR protein. A and B helices are coloured yellow and cyan, respectively. (C) Surface representation of a 7-motif TPR helix indicating that a poly-leucine α-helix may be accommodated within the extended helical groove of the protein. The surface of alternate TPR motifs are coloured white and green. The figures were drawn using MOLSCRIPT (Kraulis, 1991) and GRASP (Nicholls and Honig, 1991). 1197 A.K.Das, P.T.W.Cohen and D.Barford cadherins, the tumor suppressor gene product Adeno- Table I. Data collection, phase determination and refinement statistics matous Polyposis Coli and Tcf-family transcription factors. Significantly, the crystal structure of the armadillo Native Derivatives repeats of β-catenin shares structural similarities with TPR a b EMTS TMLA proteins as it consists of a right-handed super-helix of α-helices possessing a long positively charged groove Space group I422 I422 I422 predicted to be the site of multi-protein–protein inter- Cell parameters a (Å) 90.06 90.06 90.94 c (Å) 104.45 104.45 105.52 actions (Huber et al., 1997). Z (N) 16 16 16 In conclusion, the structure of the TPR domain of c d X-ray source PX96 PX96 PX95 PP5 provides a basis for understanding protein–protein Resolution (Å) 2.45 2.45 2.45 interactions mediated by TPR-motif-containing proteins, Unique reflections (N) 7832 7959 5033 revealing a novel mechanism for molecular recognition. Completeness (%) 96.8 97.5 66 Redundancy 2.9 4.36 2.5 The model of TPR-mediated protein recognition that we R (I) (%) 7.3 7.2 8.0 sym present here will serve as a basis for the design of Phasing power – 1.26/1.70 0.64/0.56 experiments to elucidate the roles of individual TPR motifs (centric/acentric) and of specific amino acid residues in this mechanism. Number of sites – 3 1 Future studies will be aimed at revealing the basis for the Figure of merit of MIRAS from 24–2.5Å: 0.42 recognition of hsp90 and polyunsaturated fatty acids by (0.83 after solvent flattening) Refinement the TPR domain of PP5 and the mechanism of regulation Resolution range (Å) 15–2.45 of protein phosphatase activity. Reflections used 6751 (1045) (R set) free R /R 0.201/0.298 cryst free Materials and methods Protein (solvent) 1281 (78) atoms (N) Protein purification and crystallization Sulfate ions (N) 2 The cDNA encoding the N-terminal domain (Arg 16 to Leu 181) of R.m.s.d. bond lengths 0.012/1.754 human PP5, preceded and terminated by the vector sequences MARIRA (Å)/angles (°) and LGMM, respectively, was expressed from the pT7.7 vector in Escherichia coli at 20°C (Chen and Cohen, 1997). The TPR domain is EMTS, ethyl mercurithiosalicylate 1.0 mM incubation for 3 h. within residues 28–129 and the phosphatase catalytic domain of PP5 starts TMLA, trimethyl lead acetate, 20 mM incubation for 3 days. at approximately residue 186 (Chen et al., 1994). Protein purification to PX96: Station PX9.6, SRS, Daresbury, UK. homogeneity was performed using five purification steps: (i) S-Sepharose PX95: Station PX9.5, SRS, Daresbury, UK. cation exchange chromatography; (ii) Mono-Q anion exchange chromato- R Σ Σ |I –I |/ Σ Σ I where I and I are the ith and the sym h i (h) i(h) h i i(h) i(h) (h) graphy; (iii) phenyl-TSK hydrophobic interaction chromatography; (iv) mean measurements of the intensity of reflection h. gel filtration using a Pharmacia Superose S75 column and (v) Mono-S Root mean square (F /E) where F is the heavy atom structure H H cation exchange chromatography. The purified protein was dialysed into factor amplitude and E is the residual lack of closure error. 10 mM Tris–HCl pH 7.5, 50 mM NaCl, 2 mM dithiothreitol (DTT) and R Σ{|F |–|F |} / Σ|F | where |F | and |F | are the observed and cryst o c o o c concentrated to 9 mg/ml (assuming an extinction coefficient at 280 nm calculated structure factor amplitudes respectively. R is calculated free of 1.0 per 1 mg/ml of protein). Crystallizations were performed at 4°C for a randomly chosen reflection omitted from the refinement, and using the vapour diffusion method with a precipitant of 1.8 M ammonium R is calculated for the remaining reflections included in refinement. cryst sulfate, 4% (v/v) 2-methyl-2,4-pentanediol (MPD), 100 mM HEPES pH 7.5 and 2 mM DTT. Tetragonal crystals appeared within 7 days and width of 42 Å (Figure 6A). The inside face of the TPR grew to the dimensions 0.30.20.05 mm. All X-crystallographic data were collected at 100 K. Crystals were transferred into a cryoprotection helix is formed by α-helix A of each TPR motif with the buffer consisting of 2.0 M ammonium sulfate, 4% MPD, 100 mM B α-helix located on the outside of the TPR helix (Figure HEPES pH 7.5, 50 mM NaCl and 17.5% glycerol, before flash freezing 6B). The groove observed in the three-TPR motif structure in a nitrogen gas stream. of PP5 (Figures 3 and 4) is extended to form a continuous groove on the inside of the TPR helix, and is ideally Structure determination suited to accept an α-helix of a target protein (Figure 6C). All data were collected using MAR imaging plates at stations PX9.5 and PX9.6, SRS, Daresbury and processed using DENZO and SCALEPACK Five to six TPR motifs could contribute to the interactions (Otwinowski and Minor, 1997). The protein phases were obtained from with a bound α-helix. Amino acid insertions between mercury (EMTS)- and lead (TMLA)-derivatives (Table I). Heavy-atom TPRs may be readily accommodated on the outer face of site refinement and phase calculations were performed using SHARP the super-helix, which could also allow the assembly of (de La Fortelle and Bricogne, 1997). The phases calculated using SHARP were improved using iterative solvent flattening (solvent content of 0.57) protein complexes or, as has been proposed for cdc27, by SOLOMON (Abraham and Leslie, 1996) and the histogram matching perform other biological functions such as DNA recogni- algorithm by DM (Cowtan, 1994). The electron density map calculated tion (Hirano et al., 1990). The TPR helix formed by TPR- to 2.5 Å resolution was readily interpreted and model building performed containing proteins would allow such proteins to interact by means of O (Jones et al., 1991), and atomic coordinates were refined simultaneously with multiple target proteins, utilising using X-PLOR (Brunger, 1992). The refined model has 90.5% of the residues in the most favoured region and 9.5% in the additionally specific combinations of TPR motifs within the super- allowed region of the Ramachandran plot. The N-terminal three residues helix, consistent with mutagenesis and deletion studies and C-terminal four residues that are not visible in the electron density and with the assembly function of a scaffolding protein map are assumed to be disordered. All other residues are clearly visible (Smith et al., 1995; Terlecky et al., 1995; Tzamarias and within the electron density maps. The mercury heavy-atoms sites were located at Cys 139 and two sites corresponding to two positions adopted Struhl, 1995; Gale et al., 1996). The role played by TPR by the side-chain of Cys 77. The lead site is coordinated by the side proteins to coordinate multi-subunit assembly is similar chains of Cys 77 and Glu 56 from a symmetry-related molecule. to that played by the cytoskeletal protein β-catenin, which The structure of the multiple-TPR-containing super-helix was gener- is composed of 12 copies of a 42 residue armadillo ated by superimposing equivalent main-chain atoms of TPR1 onto TPR3 repeat. β-catenin binds to numerous proteins including of PP5 to create a five-TPR-motif model. TPR1 of PP5 was then 1198 Structure of tetratricopeptide repeats superimposed onto TPR5 of the model to produce a seven-TPR-motif Jones,T.A., Zou,J.Y., Cowan,S.W. and Kjeldgaard,M. (1991) Improved model. This process was repeated until 12 TPR motifs were generated. methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr., A47, 110–119. King,R.W., Peters,J.M., Tugendreich,S., Rolfe,M., Hieter,P. and Acknowledgements Kirschner,M.W. (1995) A 20S complex containing cdc27 and cdc16 catalyses the mitosis-specific conjugation of ubiquitin to cyclin B. Cell, We thank M.Groves for helpful discussions, M.X.Chen for construction 81, 279–288. of the PP5–TPR domain expression vector, S.J.Gamblin and R.Liddington Kraulis,P. (1991) MOLSCRIPT: a program to produce both detailed and for the 14-3-3 protein coordinates and staff at stations PX9.5 and PX9.6, schematic plots of protein structures. J. Appl. Crystallogr., 24, 946–950. SRS, Daresbury for access to synchrotron facilities. The work was Lahn,B.T. and Page,D.C. (1997) Functional coherence of the human T supported by MRC and Wellcome grants to D.B. and MRC funds to chromosome. Science, 278, 675–680. P.T.W.C. The TPR-domain coordinates and structure factors have been Lamb,J.R., Michaud,W.A., Sikorski,R.S. and Hieter,P.A. 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(1994) Autosomal recessive chronic 16224–16230. granulomatous disease with absence of the 67-kD cytosolic NADPH Skinner,J., Sinclair,C., Romeo,C., Armstrong,D., Charbonneau,H. and oxidase component: Identification of mutation and detection of carriers. Rossie,S. (1997) Purification of a fatty acid-stimulated protein–serine/ Blood, 83, 531–536. threonine phosphatase from bovine brain and its identification as a de La Fortelle,E. and Bricogne,G. (1997) Maximum-likelihood heavy- homolog of protein phosphatase 5. J. Biol. Chem., 272, 22464–22471. atom parameter refinement for the multiple isomorphous replacement Smith,R.L., Redd,M.J. and Johnson,A.D. (1995) The tetratricopeptide and multiwavelength anomalous diffraction methods. Methods repeats of Ssn6 interact with the homeo domain of alpha-2. Genes Dev., Enzymol., 276, 472–494. 9, 2903–2910. Diekmann,D., Abo,A., Johnston,C., Segal,A.W. and Hall,A. (1994) Terlecky,S.R., Nuttley,W.M., McCollum,D., Sock,E. and Subramani,S. Interaction of Rac with p67 phox and regulation of phagocytic NADPH (1995) The Pichia pastoris peroxisomal protein PAS8p is the receptor oxidase activity. Science, 265, 531–533. for the C-terminal tripeptide peroxisomal targeting signal. EMBO J., 14, Gale,M.,Jr, Tan,S.-L., Wambach,M. and Katze,M.G. (1996) Interaction 3627–3634. of the interferon-induced PKR protein kinase with inhibitory proteins Tzamarias,D. and Struhl,K. (1995) Distinct TPR motifs of CYC8 are IPK P58 and vaccinia virus K3L is mediated by unique domains: involved in recruiting the CYC8–TUP1 corepressor complex of implications for kinase regulation. Mol. Cell Biol., 16, 4172–4181. differentially regulated promoters. Genes Dev., 9, 821–831. Goebl,M. and Yanagida,M. (1991) The TPR snap helix: a novel protein Xiao,B., Smerdon,S.J., Jones,D.H., Dodson,G.G., Soneji,Y., Altken,A. and repeat motif from mitosis to transcription. Trends Biochem. Sci., 16, Gamblin,S.J. (1995) Structure of a 14-3-3 protein and implications for 173–177. coordination of multiple signalling pathways. Nature, 376, 188–191. Hirano,T., Kinoshita,N., Morikawa,K. and Yanagida,M. (1990) Snap helix with knobs and hole: essential repeats in S.pombe nuclear protein nuc2. Received November 21, 1997; revised January 7, 1998; Cell, 60, 319–328. accepted January 8, 1998 Huber,A.H., Nelson,W.J. and Weis,W.I. (1997) Three-dimensional structure of the armadillo repeat region of β-Catenin. Cell, 90, 871–882. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR‐mediated protein–protein interactions

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Copyright © European Molecular Biology Organization 1998
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0261-4189
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10.1093/emboj/17.5.1192
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Abstract

The EMBO Journal Vol.17 No.5 pp.1192–1199, 1998 The structure of the tetratricopeptide repeats of protein phosphatase 5: implications for TPR-mediated protein–protein interactions although not exclusively, nuclear in location (Chen et al., Amit K.Das, PatriciaT.W.Cohen and 1994). In mammals, PP5 has a widespread tissue distribu- David Barford tion. Elevated levels of PP5 in rapidly growing cells Laboratory of Molecular Biophysics, University of Oxford, compared with serum-deprived cells suggest a role in cell Rex Richards Building, South Parks Road, Oxford, OX1 3QU and growth (Cohen et al., 1996). MRC Protein Phosphorylation Unit, Department of Biochemistry, The primary structure of PP5 reveals a C-terminal University of Dundee, Dundee, DD1 4HN, UK catalytic domain and an N-terminal tetratricopeptide repeat Corresponding author (TPR) domain containing three tandem TPR motifs e-mail: [email protected] (Becker et al., 1994; Chen et al., 1994; Chinkers, 1994) (Figure 1). TPRs occur in over 25 proteins of diverse The tetratricopeptide repeat (TPR) is a degenerate 34 biological functions and are known to mediate protein– amino acid sequence identified in a wide variety of protein interactions (Goebl and Yanagida, 1991; Sikorski proteins, present in tandem arrays of 3–16 motifs, et al., 1991; Lamb et al., 1995) (Figure 1). Within PP5, which form scaffolds to mediate protein–protein the TPR domain may perform several roles. Firstly, the interactions and often the assembly of multiprotein TPR domain is responsible for the 25-fold stimulation complexes. TPR-containing proteins include the of protein phosphatase activity by polyunsaturated fatty anaphase promoting complex (APC) subunits cdc16, acids such as arachidonic acid (Chen and Cohen, 1997; cdc23 and cdc27, the NADPH oxidase subunit p67 Skinner et al., 1997). The interaction of arachidonic acid phox, hsp90-binding immunophilins, transcription fac- with the TPR domain induces an allosteric conformational tors, the PKR protein kinase inhibitor, and peroxisomal change within PP5 that abolishes the suppression of and mitochondrial import proteins. Here, we report catalytic activity by the TPR domain (Chen and Cohen, the crystal structure of the TPR domain of a protein 1997). The activity of the arachidonic acid-stimulated PP5 phosphatase, PP5. Each of the three TPR motifs of is similar to that of the isolated PP5 phosphatase domain this domain consist of a pair of antiparallel α-helices that has been proteolytically cleaved from the TPR domain. of equivalent length. Adjacent TPR motifs are packed A second role of the TPR domain of PP5 is to mediate together in a parallel arrangement such that a tandem interactions between PP5 and hsp90 within an hsp90– TPR motif structure is composed of a regular series of glucocorticoid receptor complex, and also with the kinase antiparallel α-helices. The uniform angular and spatial domain of the ANP-guanylate cyclase receptor in vivo, arrangement of neighbouring α-helices defines a helical findings which are consistent with a role for PP5 in signal structure and creates an amphipathic groove. Multiple- transduction (Chinkers, 1994; Silverstein et al., 1997). TPR motif proteins would fold into a right-handed However, it is not known whether interactions between super-helical structure with a continuous helical groove the TPR domain of PP5 and hsp90 stimulate the PP5 suitable for the recognition of target proteins, hence phosphatase activity. In addition to its interactions with defining a novel mechanism for protein recognition. PP5, hsp90 also associates with the TPR-containing The spatial arrangement of α-helices in the PP5–TPR domain is similar to those within 14-3-3 proteins. immunophilins FKBP52 and Cyp40, in a mutually Keywords: Protein crystallography/protein phosphatase/ exclusive manner (Owens-Grillo et al., 1996; Ratajckak protein phosphorylation/signal transduction/ and Carrello, 1996). tetratricopeptide repeat The TPR motif was first identified as a tandemly repeated degenerate 34 amino acid sequence in the cell division cycle genes cdc16, cdc23 and cdc27 which encode subunits of the anaphase promoting complex Introduction (APC) (Hirano et al., 1990; Sikorski et al., 1990; King et al., 1995). The role of the APC is to target cell cycle Reversible protein phosphorylation on serine and threonine proteins for ubiquitin-dependent degradation at both the residues is essential for the regulation of numerous cellular onset of anaphase and at the exit of mitosis. Mutations functions and signal transduction pathways. Control of within the TPR motifs of these proteins cause mitotic this process is achieved by the modulation of the activities arrest at the metaphase to anaphase transition. It is now and substrate specificities of the protein kinases and realised that over 25 proteins present in organisms as phosphatases, which catalyse the opposing phosphoryl- diverse as bacteria and humans contain TPR motifs. ation and dephosphorylation reactions, respectively. The In addition to cell cycle regulation, processes such as protein Ser/Thr phosphatase PP5 is the prototype of the fourth subfamily of the PPP-family of protein transcription control, mitochondrial and peroxisomal pro- phosphatases, which also includes PP1, PP2A and PP2B tein transport, neurogenesis, protein kinase inhibition, Rac- (calcineurin) (Barford, 1996; Cohen, 1997). PP5 is con- mediated activation of NADPH oxidase, and protein served from yeast to humans being predominantly, folding involve TPR motifs (Lamb et al., 1995; Ponting, 1192 © Oxford University Press Structure of tetratricopeptide repeats Fig. 1. Schematic of TPR-containing proteins. TPR motifs (shown as boxes) are observed as multiple tandem repeats and also separated by sequence insertions. 1996). Moreover, a 10 tandem TPR motif protein (UTY) in TPR7 of cdc27 reduced its ability to interact with cdc23 was recently identified as one of the 12 novel genes of but did not affect its interactions with itself or with cdc16 the non-recombining region of the human Y-chromosome (Lamb et al., 1994). (Lahn and Page, 1997). In order to understand the molecular basis for TPR- Within TPR-containing proteins, the TPRs are usually mediated protein recognition, we have determined the arranged in tandem arrays of 3–16 motifs, although crystal structure of the TPR domain of PP5 to 2.5 Å individual, or blocks, of TPR motifs may be dispersed resolution. The results provide insight into the structure throughout the protein sequence (Figure 1). Mutagenesis of multiple TPR motif proteins, the basis for disruptive and deletion studies have revealed roles for TPR motifs mutations within the TPR-containing subunits of the APC in mediating protein–protein interactions. Proteins with and NADPH oxidase and reveal a novel mechanism for multiple copies of TPR motifs function as scaffolding protein–protein interactions. proteins and coordinate the assembly of proteins into multi-subunit complexes. Such proteins include Ssn6, Results and discussion cdc16, cdc23, cdc27 and rapsyn (Ponting and Phillips, 1996). Rapsyn for example, has been proposed to act Structure determination and overall structure as a molecular link between the nicotinic acetylcholine The crystal structure of the N-terminal TPR domain of receptor and the dystrophin–dystroglycan complex (Apel PP5 was determined to 2.5 Å resolution using multiple et al., 1995). Specific blocks of TPR motifs mediate isomorphous replacement and anomalous scattering interactions with particular target proteins and have been methods. Electron density maps obtained using experi- assigned specific biological functions. For example, the mental phases were readily interpretable (Figure 2) and N-terminal three TPR motifs of Ssn6 associate with the the polypeptide chain was traced within continuous density. co-repressor Tup1, whereas other combinations of TPR The three tandem TPR motifs of PP5 reveal a novel motifs mediate interactions with different transcription protein fold. Each TPR motif is composed of a pair of factors, which accounts for the diverse gene expression antiparallel α-helices, termed helices A and B, associated patterns regulated by Ssn6 (Smith et al., 1995; Tzamarias together with a packing angle of ~24° between helix axes and Struhl, 1995). TPR motifs 5–7 of p58, an inhibitor of (Figure 3A–C). The structure of each TPR motif is the RNA-dependent protein kinase (PKR), are responsible virtually identical; main-chain atoms of TPR1 superimpose for interactions with PKR, while the N-terminal TPR with those of TPR2 and TPR3 with a root mean square motifs direct homotypic interactions (Gale et al., 1996). deviation (r.m.s.d.) of 0.35 Å and 0.8 Å, respectively. The TPR motifs 1–3 of the peroxisomal import protein PAS8p three TPR motifs are organised into a parallel arrangement, interact with the peroxisomal targeting signal (Terlecky such that sequentially adjacent α-helices are antiparallel et al., 1995). Within the multi-subunit APC, a mutation in a manner reminiscent of a concertina (Figure 3A–C). 1193 A.K.Das, P.T.W.Cohen and D.Barford Fig. 2. Stereo view of the electron density map of the PP5–TPR domain calculated to 2.5 Å resolution using experimental phases with the refined coordinates superimposed. The figure was produced using O (Jones et al., 1991). Within a tandem array of TPR motifs, the packing of PP5, while sharing weak sequence similarity to a TPR helix A against adjacent B-helices is defined by the same motif, are folded into an extended α-helix (α7). This helix spatial and angular parameters both within, and between, packs against helix B of TPR3 in a similar arrangement adjacent TPR motifs. Hence, in general, each α-helix to that of the helices within the TPR domain (Figure 3A– shares two immediate α-helix neighbours and the protein C). The consequence of a regular repeat of α-helices that fold may be defined as an overlapping array of three- are related by a crossover angle of 24° is to generate a right- helix bundles. handed helical conformation that creates an amphipathic Multiple sequence alignments of the TPRs present channel (Figure 4). The surface of the channel is formed in 25 proteins reveals a highly degenerate sequence. predominantly by the side chains of amino acids in helix Although there is no position characterised by an invariant A of each TPR motif with relatively little contribution residue, a consensus sequence pattern of small and large from residues of helix B. In contrast, the opposite side of hydrophobic residues has been defined (Figure 3D). Small the channel is formed by residues from both TPR α-helices. hydrophobic residues are commonly observed at positions 8, 20 and 27 within the motif. Position 32 is frequently a Structural relationship to 14-3-3 proteins proline and large hydrophobic residues are also located at The antiparallel arrangement of α-helices of the TPR particular positions. Analysis of the structure of the TPR domain of PP5 is reminiscent of the antiparallel α-helices domain of PP5 provides a rationale for this consensus of the 14-3-3 protein, a homodimeric molecule with sequence pattern (Figure 3C and D). Residues 8 and 20 nine α-helices in each subunit (Liu et al., 1995; Xiao are located at the position of closest contact between the et al., 1995) (Figure 5). Unlike TPR proteins however, A and B α-helices of a TPR, whereas residue 27 on helix the α-helices of 14-3-3 proteins vary in length and in B is located at the interface of 3 helices (A, B and A) their relative packing geometries and only three of the within a 3-helix bundle. Proline 32 is located at the seven α-helices of the TPR domain superimpose closely C-terminus of helix B, and the large consensus hydro- (r.m.s.d. of 1.2 Å) with equivalent α-helices of 14-3-3 phobic residues form the interfaces between adjacent (Figure 5). TPR-containing proteins and 14-3-3 proteins α-helices. Mutations at position 8 of TPR3 of the NADPH share common structural and functional properties, oxidase subunit p67 phox and within TPRs 5 and 7 of the despite their lack of sequence similarities (Aitken, 1996). APC subunit cdc23 result in the disruption of protein The latter proteins interact with proteins phosphorylated function, presumably due to the incorrect packing of on Ser/Thr residues, most probably via an amphipathic neighbouring α-helices (Figure 3D) (Sikorski et al., 1993; groove present within each 14-3-3 subunit that is de Boer et al., 1994). The Gly to Glu substitution in p67 similar to the groove observed in the PP5–TPR domain. phox is associated with chronic granulomatous disease Within the PP5–TPR domain crystals, the protein forms and impairment of NADPH oxidase activity (de Boer a homodimer as a result of the antiparallel association et al., 1994). The TPR region of p67 phox is the site of of symmetry-related α-7 helices and the interaction of NADPH oxidase recognised by the small G-protein Rac, residues C-terminal to α-7 with residues forming the hence disruption of TPR3 may prevent Rac-stimulation channel surface of the same symmetry-related molecule of the enzyme activity (Diekmann et al., 1994; Ponting, (Figure 4). Similar to the crystal structure of the τ- 1996). In yeast, individual mutations at position 8 of isoform of 14-3-3 (Xiao et al., 1995), a sulfate ion is TPR5 and TPR7 of cdc23 (Sikorski et al., 1993) and bound within this groove by a cluster of basic residues, position 20 of TPR9 of Cut9 (Samejima and Yanagida, and this sulfate ion interacts with the channel-bound 1994) (a cdc16 homologue) result in mitotic arrest at the peptide. The interaction of a peptide within the groove metaphase to anaphase transition, most likely as a result of the TPR domain, as observed in the PP5–TPR of a blockade in the ubiquitin-dependent degradation of domain crystals, may provide a model for the recognition inhibitors of this transition. of phosphopeptides by 14-3-3 proteins. However, the The 35-residues C-terminal to the three TPR motifs of TPR-mediated dimer does not occur in full-length PP5 1194 Structure of tetratricopeptide repeats Fig. 3. (A and B) Two orthogonal views of ribbon representations of the TPR domain of PP5. Each TPR motif and the α-7 helix are labelled. The sequence of α-7 shows weak similarity to a TPR motif. Helix A of TPR1 is extended by nine residues N-terminal to the TPR domain. (C) Stereo view of the molecule. Within TPR2 and helix A of TPR3, consensus TPR motif residues (Figure 3D) are drawn to illustrate their role in inter-helix packing. The residue numbering refers to the TPR motif numbering. Primes denote residue numbering in TPR3. Main-chain atoms of any pair of helices superimpose onto equivalent atoms of all other pairs of helices within an r.m.s.d. of 1.1 Å. Figures drawn using MOLSCRIPT (Kraulis, 1991). (D) Sequence alignment of the three TPR motifs of PP5. Consensus TPR motif residues are shown with red and green backgrounds for small and large hydrophobic residues, respectively. The position of the consensus proline residue is indicated with a P. The sites of mutations in the NADPH oxidase subunit p67 phox (position 8; de Boer et al., 1994), cdc23 (position 8; Sikorski et al., 1993) and cdc16 (position 20; Samejima and Yanagida, 1994) are indicated with blue arrows. Numbers above the alignment refer to TPR motif residues and at the side, to the PP5 residues. The figure was prepared using ALSCRIPT (Barton, 1993). 1195 A.K.Das, P.T.W.Cohen and D.Barford Fig. 4. Solvent accessible surface of the PP5–TPR domain showing the electrostatic potential and amphipathic TPR-groove. Atoms of the α-7 helix of the symmetry-related molecule are shown interacting with the α-7 helix and the C-terminus with the amphipathic groove. The sulfate ion is labelled. The figure was drawn using GRASP (Nicholls and Honig, 1991). Fig. 5. Comparison of the structures of the TPR domain of PP5 (cyan) and a 14-3-3 protein (Liu et al., 1995) (yellow). Helices 4–6 of 14-3-3 were superimposed onto equivalent main-chain atoms of helices B, A and B of TPR motifs 1 and 2 with a r.m.s.d. of 1.2 Å. The figure was drawn using MOLSCRIPT (Kraulis, 1991). which is a monomer, as judged by size exclusion multiple-motif TPR proteins, we constructed a model of chromatography. 12 tandem TPR motifs. We assumed that the packing parameters between adjacent TPR motifs of the tandem Protein recognition by multiple TPR motif proteins repeat would be similar to those observed in the TPR Our model of the three TPR motifs of PP5 provides a domain of PP5. The resultant model indicates that tan- framework for predicting the structures of other TPR- demly arranged TPR motifs are organised into a regular motif-containing proteins and for interpreting functional right-handed super-helix with a helical repeat of studies of these proteins. To understand the properties of approximately seven TPR motifs, a pitch of 60 Å and a 1196 Structure of tetratricopeptide repeats Fig. 6. Model of a multiple TPR motif containing protein. (A) View perpendicular to the helix axis of 12 TPR motifs. The main-chain is colour ramped from blue at the N-terminus to red at the C-terminus. (B) View parallel to the helix axis of an 8-motif TPR protein. A and B helices are coloured yellow and cyan, respectively. (C) Surface representation of a 7-motif TPR helix indicating that a poly-leucine α-helix may be accommodated within the extended helical groove of the protein. The surface of alternate TPR motifs are coloured white and green. The figures were drawn using MOLSCRIPT (Kraulis, 1991) and GRASP (Nicholls and Honig, 1991). 1197 A.K.Das, P.T.W.Cohen and D.Barford cadherins, the tumor suppressor gene product Adeno- Table I. Data collection, phase determination and refinement statistics matous Polyposis Coli and Tcf-family transcription factors. Significantly, the crystal structure of the armadillo Native Derivatives repeats of β-catenin shares structural similarities with TPR a b EMTS TMLA proteins as it consists of a right-handed super-helix of α-helices possessing a long positively charged groove Space group I422 I422 I422 predicted to be the site of multi-protein–protein inter- Cell parameters a (Å) 90.06 90.06 90.94 c (Å) 104.45 104.45 105.52 actions (Huber et al., 1997). Z (N) 16 16 16 In conclusion, the structure of the TPR domain of c d X-ray source PX96 PX96 PX95 PP5 provides a basis for understanding protein–protein Resolution (Å) 2.45 2.45 2.45 interactions mediated by TPR-motif-containing proteins, Unique reflections (N) 7832 7959 5033 revealing a novel mechanism for molecular recognition. Completeness (%) 96.8 97.5 66 Redundancy 2.9 4.36 2.5 The model of TPR-mediated protein recognition that we R (I) (%) 7.3 7.2 8.0 sym present here will serve as a basis for the design of Phasing power – 1.26/1.70 0.64/0.56 experiments to elucidate the roles of individual TPR motifs (centric/acentric) and of specific amino acid residues in this mechanism. Number of sites – 3 1 Future studies will be aimed at revealing the basis for the Figure of merit of MIRAS from 24–2.5Å: 0.42 recognition of hsp90 and polyunsaturated fatty acids by (0.83 after solvent flattening) Refinement the TPR domain of PP5 and the mechanism of regulation Resolution range (Å) 15–2.45 of protein phosphatase activity. Reflections used 6751 (1045) (R set) free R /R 0.201/0.298 cryst free Materials and methods Protein (solvent) 1281 (78) atoms (N) Protein purification and crystallization Sulfate ions (N) 2 The cDNA encoding the N-terminal domain (Arg 16 to Leu 181) of R.m.s.d. bond lengths 0.012/1.754 human PP5, preceded and terminated by the vector sequences MARIRA (Å)/angles (°) and LGMM, respectively, was expressed from the pT7.7 vector in Escherichia coli at 20°C (Chen and Cohen, 1997). The TPR domain is EMTS, ethyl mercurithiosalicylate 1.0 mM incubation for 3 h. within residues 28–129 and the phosphatase catalytic domain of PP5 starts TMLA, trimethyl lead acetate, 20 mM incubation for 3 days. at approximately residue 186 (Chen et al., 1994). Protein purification to PX96: Station PX9.6, SRS, Daresbury, UK. homogeneity was performed using five purification steps: (i) S-Sepharose PX95: Station PX9.5, SRS, Daresbury, UK. cation exchange chromatography; (ii) Mono-Q anion exchange chromato- R Σ Σ |I –I |/ Σ Σ I where I and I are the ith and the sym h i (h) i(h) h i i(h) i(h) (h) graphy; (iii) phenyl-TSK hydrophobic interaction chromatography; (iv) mean measurements of the intensity of reflection h. gel filtration using a Pharmacia Superose S75 column and (v) Mono-S Root mean square (F /E) where F is the heavy atom structure H H cation exchange chromatography. The purified protein was dialysed into factor amplitude and E is the residual lack of closure error. 10 mM Tris–HCl pH 7.5, 50 mM NaCl, 2 mM dithiothreitol (DTT) and R Σ{|F |–|F |} / Σ|F | where |F | and |F | are the observed and cryst o c o o c concentrated to 9 mg/ml (assuming an extinction coefficient at 280 nm calculated structure factor amplitudes respectively. R is calculated free of 1.0 per 1 mg/ml of protein). Crystallizations were performed at 4°C for a randomly chosen reflection omitted from the refinement, and using the vapour diffusion method with a precipitant of 1.8 M ammonium R is calculated for the remaining reflections included in refinement. cryst sulfate, 4% (v/v) 2-methyl-2,4-pentanediol (MPD), 100 mM HEPES pH 7.5 and 2 mM DTT. Tetragonal crystals appeared within 7 days and width of 42 Å (Figure 6A). The inside face of the TPR grew to the dimensions 0.30.20.05 mm. All X-crystallographic data were collected at 100 K. Crystals were transferred into a cryoprotection helix is formed by α-helix A of each TPR motif with the buffer consisting of 2.0 M ammonium sulfate, 4% MPD, 100 mM B α-helix located on the outside of the TPR helix (Figure HEPES pH 7.5, 50 mM NaCl and 17.5% glycerol, before flash freezing 6B). The groove observed in the three-TPR motif structure in a nitrogen gas stream. of PP5 (Figures 3 and 4) is extended to form a continuous groove on the inside of the TPR helix, and is ideally Structure determination suited to accept an α-helix of a target protein (Figure 6C). All data were collected using MAR imaging plates at stations PX9.5 and PX9.6, SRS, Daresbury and processed using DENZO and SCALEPACK Five to six TPR motifs could contribute to the interactions (Otwinowski and Minor, 1997). The protein phases were obtained from with a bound α-helix. Amino acid insertions between mercury (EMTS)- and lead (TMLA)-derivatives (Table I). Heavy-atom TPRs may be readily accommodated on the outer face of site refinement and phase calculations were performed using SHARP the super-helix, which could also allow the assembly of (de La Fortelle and Bricogne, 1997). The phases calculated using SHARP were improved using iterative solvent flattening (solvent content of 0.57) protein complexes or, as has been proposed for cdc27, by SOLOMON (Abraham and Leslie, 1996) and the histogram matching perform other biological functions such as DNA recogni- algorithm by DM (Cowtan, 1994). The electron density map calculated tion (Hirano et al., 1990). The TPR helix formed by TPR- to 2.5 Å resolution was readily interpreted and model building performed containing proteins would allow such proteins to interact by means of O (Jones et al., 1991), and atomic coordinates were refined simultaneously with multiple target proteins, utilising using X-PLOR (Brunger, 1992). The refined model has 90.5% of the residues in the most favoured region and 9.5% in the additionally specific combinations of TPR motifs within the super- allowed region of the Ramachandran plot. The N-terminal three residues helix, consistent with mutagenesis and deletion studies and C-terminal four residues that are not visible in the electron density and with the assembly function of a scaffolding protein map are assumed to be disordered. All other residues are clearly visible (Smith et al., 1995; Terlecky et al., 1995; Tzamarias and within the electron density maps. The mercury heavy-atoms sites were located at Cys 139 and two sites corresponding to two positions adopted Struhl, 1995; Gale et al., 1996). The role played by TPR by the side-chain of Cys 77. The lead site is coordinated by the side proteins to coordinate multi-subunit assembly is similar chains of Cys 77 and Glu 56 from a symmetry-related molecule. to that played by the cytoskeletal protein β-catenin, which The structure of the multiple-TPR-containing super-helix was gener- is composed of 12 copies of a 42 residue armadillo ated by superimposing equivalent main-chain atoms of TPR1 onto TPR3 repeat. β-catenin binds to numerous proteins including of PP5 to create a five-TPR-motif model. TPR1 of PP5 was then 1198 Structure of tetratricopeptide repeats superimposed onto TPR5 of the model to produce a seven-TPR-motif Jones,T.A., Zou,J.Y., Cowan,S.W. and Kjeldgaard,M. (1991) Improved model. This process was repeated until 12 TPR motifs were generated. methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr., A47, 110–119. King,R.W., Peters,J.M., Tugendreich,S., Rolfe,M., Hieter,P. and Acknowledgements Kirschner,M.W. (1995) A 20S complex containing cdc27 and cdc16 catalyses the mitosis-specific conjugation of ubiquitin to cyclin B. Cell, We thank M.Groves for helpful discussions, M.X.Chen for construction 81, 279–288. of the PP5–TPR domain expression vector, S.J.Gamblin and R.Liddington Kraulis,P. (1991) MOLSCRIPT: a program to produce both detailed and for the 14-3-3 protein coordinates and staff at stations PX9.5 and PX9.6, schematic plots of protein structures. J. Appl. Crystallogr., 24, 946–950. SRS, Daresbury for access to synchrotron facilities. The work was Lahn,B.T. and Page,D.C. (1997) Functional coherence of the human T supported by MRC and Wellcome grants to D.B. and MRC funds to chromosome. Science, 278, 675–680. P.T.W.C. The TPR-domain coordinates and structure factors have been Lamb,J.R., Michaud,W.A., Sikorski,R.S. and Hieter,P.A. 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Journal

The EMBO Journal – Springer Journals

Published: Mar 2, 1998

Keywords: Protein crystallography; protein phosphatase; protein phosphorylation; signal transduction; tetratricopeptide repeat

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