Description of the fold

The C-terminal domain of Tup1 folds into a seven-bladed β propeller (residues 332–710) preceded by a 50 amino acid N-terminal subdomain (residues 283–331) that packs against the side of the propeller (Figure 1A and B). The propeller fold is characterized by seven blades that are pseudosymmetrically arranged around a central axis. Each blade of the propeller is a β sheet composed of four antiparallel β strands. Following the convention for WD40 propellers (Wall et al., 1995; Lambright et al., 1996), the blades are numbered 1–7 and, within each blade, the strands are labeled A, B, C and D from the inside to the outside of the propeller. The loops connecting the strands are labeled according to the two strands that they connect. The ‘top’ face of the propeller is defined as the face that contains the DA loops connecting consecutive blades whereas the ‘bottom’ face is the opposite surface. Six copies of the WD40 repeat were originally identified in the Tup1 sequence (Williams and Trumbly, 1990); however, an additional repeat, repeat 1, which is separated from the N-terminus of repeat 2 by an ~50 amino acid linker has also been noted (Komachi et al., 1994). The crystal structure confirms that Tup1 has seven copies of the WD40 sequence motif. Although the sequences of the WD40 regions of Tup1 and Gβ are only 23% identical, the propeller of Tup1 overlays remarkably well with that of Gβ, with an r.m.s.d. of 1.3 Å over 282 C_α atoms (Figure 3B). Furthermore, the global properties of the seven-bladed, WD40-repeat propeller, which were described for Gβ (Wall et al., 1995; Sondek et al., 1996), are also present in the Tup1 propeller structure. These similarities include the fact that the WD40 sequence repeat originally identified does not correspond to an individual blade. Beginning at the N-terminus of the first WD40 sequence repeat, the first strand is the outer strand (D) of blade 7 and then continues into strands A, B and C of the next blade, blade 1 (Figure 1A and C). The outer strand (D) of blade 1 comes from the next WD40 repeat and the pattern continues, with a single sequence repeat encompassing parts of two consecutive blades. The propeller ends with strand C of blade 7 from the final WD40 repeat. Thus, the propeller is closed by contacts in blade 7 between strand 7D from the first repeat and strand 7C from the seventh repeat, as is observed in Gβ (Wall et al., 1995; Sondek et al., 1996).

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Fig. 3. (A) Superposition of all seven blades of Tup1. The C_α backbone for each of the seven blades was aligned with respect to strands 3B and 3C. The four conserved side chains involved in the intrablade hydrogen-bonding network, referred to as the structural tetrad, are shown for blade 3 as ball-and-stick models colored according to atom type and hydrogen bonds are indicated with dashed lines. A representative blade from Gβ, blade 4, is shown for reference. Blade 1, light green; blade 2, red; blade 3, green; blade 4, purple; blade 5, pink; blade 6, cyan; blade 7, orange; Gβ, brown. (B) Superposition of the WD40 propellers. The C_α backbones for Gβ (blue; 1TBG) and Tup1 (yellow), which align with an r.m.s.d. of 1.3 Å, are shown in the same view as Figure 1A and labeled. Least-squares alignments for (A) and (B) were done in O (Jones et al., 1991) and figures generated with RIBBONS (Carson, 1997).

The structure of a single blade is highly conserved both within the Tup1 structure and between Tup1 blades and individual blades in Gβ. For example, blade 3 of Tup1 superimposes with other Tup1 blades with C_α–C_α r.m.s.ds of 0.21–0.88 Å, and with a typical blade of Gβ, blade 4, with an r.m.s.d. of 0.69 Å (Figure 3A). Therefore, the blades of Tup1 are as structurally similar to Gβ as they are to each other. A hallmark of blades in WD40-repeat propellers is the hydrogen-bonding network, or ‘structural tetrad’, observed between some of the most conserved residues in the WD40 repeat (Wall et al., 1995; Sondek et al., 1996). This tetrad is formed between Trp in strand C, Ser/Thr in strand B, His in the DA loop, and the nearly invariant Asp in the tight turn between strands B and C (Figures ​(Figures1C1C and ​and3A).3A). The interface between neighboring blades within the propeller domain is primarily hydrophobic, with residues on the adjacent faces of the β sheets in van der Waals contact. The overall packing of the blades within the propeller leads to the formation of a narrow channel along the ~7-fold symmetry axis of the propeller. The channel is ~12 Å in diameter (C_α to C_α) and is filled with solvent molecules that are bound by the carbonyl oxygens and amide groups from the inner strands (A) of each blade. Side chains from small residues, such as serine, cysteine and alanine, also line the channel, and a few larger side chains even extend into the interior of the channel, most notably Lys351 and Asp492. Side chain atoms from Asn354, Arg586 and Asn682, which are located on the bottom surface of the propeller and line the opening to the channel, form a cluster of hydrogen-bond acceptors and donors.

The greatest differences between the Tup1 and Gβ propellers occur in the loop regions. In most WD40 repeats, the length of the β strands and loops connecting the β strands are relatively constant, resulting in a typical length for a WD40 repeat of 40 amino acids. In general, variation in the length of a WD40 repeat occurs because of insertions in the loops. In Gβ, the loops within the WD40 repeats generally differ by no more than three residues. In contrast, the lengths of the loops in Tup1 are more variable, with some significantly longer than those found in Gβ. The lengths of the WD40 repeats of Tup1 vary with insertions of 6–60 amino acids in each of four loops (Figure 1C and D). In the Tup1 crystal structure, the density for each of these loops was poor and was often interpretable in only one of the three molecules of the asymmetric unit (see Materials and methods); thus, these loops are likely to be flexible. Within each blade, the AB and CD loops are the most flexible whereas the BC loops are fixed in length and less conformationally variable (Figure 3). An exception to the conformational invariance of the BC loop occurs in blade 1. This BC loop deviates from those in all of the other blades of both Tup1 and Gβ because it is shorter by one residue, and an Asn replaces the nearly invariant Asp, which is the most conserved residue in all WD40 repeats. This Asp is present in the BC loops of blades 2–7, where it participates in the structural tetrad, as described above, and forms a hydrogen bond with the backbone to stabilize the tight turn. Although the substitution of Asn for Asp is conservative, the shortened BC loop of blade 1 alters the position of Asn364 such that it can not make contacts analogous to the usual Asp (Figure 3B). Instead, in blade 1, Asn364 stabilizes the DA loop connecting blades 1 and 2 as it leads into strand 2A through hydrogen bonds with Leu444 N and Asp443 OD1. One additional consequence of the shortened BC loop is the unusual backbone conformation of the next residue, Lys365, which is classified as a Ramachandran outlier.

The N-terminal 50 amino acids do not form an independent globular domain, but rather form a subdomain that is joined to the propeller by β sheet interactions that extend one blade into a six-stranded sheet. Starting at the N-terminus of the model, the protein forms an extended strand (residues 283–286) followed by one turn of a 3₁₀ helix and then another extended strand (residues 296–298) followed by a second turn of a 3₁₀ helix. These two extended strands are antiparallel to each other and perpendicular to the axis of the propeller (Figure 1A and B). The second 3₁₀ helix is preceded by a loop that leads into the final two β strands of the N-terminal subdomain, n1 and n2. Strands n1 (residues 312–316) and n2 (residues 322–325) are antiparallel and interact using the typical hydrogen bonds between backbone atoms that are found in β sheets. In addition, strand n2 forms backbone hydrogen-bond contacts with strand 6D, extending blade 6 into a six-stranded antiparallel β sheet (Figure 2A). Interaction between the N-terminal and propeller subdomains is primarily mediated by hydrophobic residues from the N-terminal subdomain contacting side chains located near the interfaces of neighboring blades (Figure 2B). For example, residues from strand n2 of the blade 6 extension, Tyr321, Ile323 and Tyr325, are in van der Waals contact with side chains that line the faces of the sheets between neighboring blades 6 and 7. Phe301 makes non-polar contacts with Pro664, a side chain on the face of blade 6 that is adjacent to blade 5. The largest hydrophobic pocket is between blades 4 and 5, which is lined by residues Lys576, Asp597, Ser599, Lys601, Thr625 and Ile627, and accommodates His294, which is a side chain from the second extended strand in the N-terminal subdomain. These extensive non-polar contacts are further buttressed by polar contacts scattered across the interface. The interface between the N-terminal subdomain and the propeller buries 2395 Å² of surface area. In contrast to the close association of the N-terminal subdomain of Tup1 with the propeller, the residues in Gβ that precede the propeller form a helical subdomain that makes few contacts with the propeller domain (Wall et al., 1995; Lambright et al., 1996). Instead, the N-terminal helix of Gβ is tightly associated with an accessory protein, Gγ, which interacts extensively with the propeller.

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Fig. 2. Packing of the N-terminal fragment against the propeller. (A) Extended β sheet formed by blade 6 and strands n1 and n2 from the N-terminal fragment. Hydrogen bonds are indicated with dashed lines. (B) Stereoview of contacts between the N-terminal fragment (283–331) and the propeller. Side chain and backbone atoms are superimposed on the C_α trace. Some side chain labels were omitted for clarity. The approximate boundaries of each blade are shown in different colors (blade 5, light green; blade 6, blue; blade 7, red; N-terminal fragment, gray). This figure was generated with RIBBONS (Carson, 1997).

As discussed above, the protein used in the structure determination contains a deletion that replaces residues 389–431 with the three amino acid linker, KDP, which joins residues 282–388 to residues 432–713. In a comparison of seven Tup1 sequences from different species of fungi, only the S.cerevisiae and K.lactis Tup1 proteins contain an extended linker between blades 1 and 2. The linkers from these two Tup1 proteins bear little sequence similarity to one another, other than an abundance of polar residues. This linker is unlikely to play a functional role because the C.albicans Tup1 protein, which lacks this linker region, represses a genomic a-specific reporter gene in S.cerevisiae (Braun and Johnson, 1997). Moreover, in yeast cells lacking endogenous Tup1, a full-length Tup1 protein with the deletion of residues 389–431 complements for several and perhaps all of the Tup1 functions (K.Komachi and A.D.Johnson, personal communication), further suggesting that this region is not required for the known functions of the Tup1 protein. The linker in S.cerevisiae Tup1 is therefore likely to be a flexible loop that does not significantly contribute to the repressor properties of Tup1, particularly repression of the mating-type genes. In the crystal structure reported here, poor density is observed for the residues adjacent to the deleted linker, whereas the ordered region of the loop extends away from the top surface of the propeller and adopts a conformation that is distinct from all of the other DA loops between adjacent blades (Figure 3A). Although the conformation of the linker in the intact protein has not been determined, the linker is likely to be a flexible loop that does not form significant contacts with the rest of the C-terminal domain.

Although the C-terminal domain of Tup1 crystallizes with three molecules in the asymmetric unit, there is no evidence that the contacts observed between NCS-related molecules reflect formation of a possible biologically relevant multimer of the C-terminal domain. Little surface area is buried at the interface between NCS-related molecules, ranging from 192 to 407 Å², indicating that the interaction energy is very weak (Janin and Rodier, 1995). In solution, both the linker-deleted fragment used in this study and a fragment of Tup1 containing residues 253–713 are monomeric, as determined by sedimentation equilibrium analytical ultracentrifugation (data not shown). Although the full-length Tup1 protein is a tetramer whose oligomerization is mediated by the N-terminal 91 residues (Varanasi et al., 1996), overexpression of the monomeric C-terminal domain in yeast partially suppresses the mating-type defects in cells whose TUP1 and SSN6 genes have been disrupted (Komachi et al., 1994). One interpretation of this result is that Tup1 can function, albeit weakly, as a monomer.

Mapping of Tup1 mutations

Studies of protein fragments and mutant proteins have shown definitively that the C-terminal domain of Tup1 interacts with at least one promoter-specific DNA-binding protein, Matα2 (Komachi et al., 1994; Komachi and Johnson, 1997). Eleven point mutations in Tup1 that specifically affect its interaction with Matα2 have been isolated (Komachi and Johnson, 1997). All of these mutations are located on the top face of the propeller, as shown in Figure 1A–C. Mutations found in the DA loops include C348R, Y445C, N673S and S674P; mutations found in the BC turns include E463N and K650N; and mutations found in strand A, near the top face, include S448P, Y489H, Y580H, L634S, I676T and I676V. With the exception of blade 4, mutations affecting interactions with Matα2 have been identified in every blade. Because of the repetitive and symmetric nature of the propeller fold, all of the DA loops and BC turns are found on the top surface of the propeller, whereas all of the AB and CD loops are found on the bottom surface of the propeller. All of these critical side chains cluster on the top surface of the propeller, near the center, and are accessible to solvent. Model building confirms that the side chain substitutions that disrupt α2-mediated repression are not structural mutations, because the mutant side chains can be modeled as one of the common side chain rotamers without steric clashes. Surface localization of these eleven mutations strongly supports the model that the Matα2 protein interacts with Tup1 via the top surface of the propeller (Komachi and Johnson, 1997).

A striking similarity in protein–protein interaction interfaces is seen when the putative Tup1–α2 interface is compared with both the Gβγ–Gα and Gβγ–phosducin complexes. In each of the complexes with Gβγ, the interaction between Gβγ and the partner protein is mediated by two surfaces on Gβγ: a side surface, which is unique for each complex, and a top surface, which is similar for both complexes (Wall et al., 1995; Lambright et al., 1996). The residues in Gβ that mediate these contacts with each of the partner proteins are highlighted in Figure 1D. Because the Tup1 mutations discussed above do not affect the general repression function of Tup1 (Komachi and Johnson, 1997), Tup1 most likely interacts with downstream proteins in the repression pathway, such as components of the transcription machinery, using a different subset of residues, perhaps a different face or side of the propeller. The location of the S448P mutation in strand A, pointing into the solvent channel, and the positioning of four more critical residues in strand A, around the top entrance into the channel, raises the possibility that Tup1 may also use at least a portion of the channel to interact with Matα2. The channel could accommodate an extended strand without major conformational adjustments, similar to the β hairpin that projects into the central channel in galactose oxidase, a seven-bladed propeller that does not contain WD40 repeats (Ito et al., 1994).

Point mutations in the C-terminal domain of Tup1 that have differential effects on the expression of four Ssn6–Tup1-repressed reporter genes have also been reported (Carrico and Zitomer, 1998) (Figure 1C). These mutations derepress the a-mating type and hypoxic reporter genes while having little effect on the expression of genes that regulate flocculence and glucose response. Four of the five mutations replace the conserved Ser/Thr that is located in strand B in the core of each blade with a proline (T460P, S647P, S593P and T695P). This Ser/Thr is at the center of the structural tetrad, described above, making hydrogen bonds with the conserved Trp in strand C of the same blade and with the conserved His in the DA loop leading into strand A of the same blade (Figure 3A). The fifth mutation substitutes proline for Ser595, which is within hydrogen-bonding distance of the backbone carbonyl oxygens of residues 577 and 578. The wild-type side chain at each of the five positions is involved in polar intramolecular contacts, either directly or through a bound water that is conserved in each of the three molecules in the asymmetric unit. Model building shows that the substitution of proline for each of the five Ser/Thr residues disrupts the hydrogen bonds detailed above and causes steric clashes with nearby backbone and side chain atoms. The conclusion from this analysis is that these five mutations are likely to perturb the overall structure of the propeller and therefore do not identify specific residues in Tup1 that interact with promoter-specific DNA-binding proteins or with other general repression targets. The differential effects on repression observed for these mutations probably arise because the various promoters have different requirements for the WD40 domain of Tup1.

Analysis of conserved residues in seven Tup1 sequences

The sequences of all seven known fungal Tup1 homologs were compared in order to identify conserved residues that may be required for the in vivo function of Tup1. The sequence identity for each of the six homologs of S.cerevisiae Tup1 (residues 283–710) ranges from 50% for Dictyostelium discoideum (slime mold) Tup1 to 76% for K.lactis Tup1. The sequence identity over the 43 kDa C-terminal domain of Tup1 is extensive, with clusters of both high and low identity. A molecular surface representation of S.cerevisiae Tup1 that is colored by sequence identity highlights four regions of high sequence conservation (Figure 4). The largest region of sequence conservation is on the top surface centered around the channel and encompasses all of the DA and BC loops, in addition to all of the residues that were previously described as required for the interaction with Matα2 (Figure 4A). This area of high sequence conservation extends into the channel of propeller (Figure 4A). In contrast to the top surface, the bottom surface has only a small patch of sequence conservation that includes the AB loop of blade 2 (Figure 4B). The bottom surface also differs from the top surface because of a large region of negative electrostatic potential distributed over the entire bottom surface. The side surface of the propeller exhibits the lowest sequence conservation, with the exception of the outer strand (D) of blade 4 (Figure 4). Stretches of conserved buried residues are found in several of the blades in strand B; however, they presumably help to stabilize the overall protein fold rather than mediate protein–protein interactions (Figure 4). Most of the conserved buried residues, such as the Ser/Thr and Trp that are part of the conserved structural tetrad mentioned above, are characteristic of the WD40 repeat family (Neer et al., 1994).

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Fig. 4. Sequence identity of seven fungal Tup1 proteins mapped onto the molecular surface and C_α backbone of the C-terminal domain of the Tup1 structure. (A) View looking onto the top surface of the molecule, similar to the view in Figures ​Figures1A1A and ​and3B.3B. (B) View rotated 180° about the vertical axis in (A), looking onto the bottom surface of the molecule. The color gradient is a continuum from white to purple to pink, representing residues with <50% identity, residues with 75% identity and residues with 100% identity, respectively. Strand 4D displays significant sequence identity when compared with all of the other outer strands of the propeller. Tup1 sequences (P16649, P56094, P56093, O14432, Q09715, P78706 and O76734) were aligned using ClustalW 1.7 and displayed in GRASP (Nicholls et al., 1991).

The presence of four discrete regions of high sequence conservation suggests that all seven Tup1 homologs interact with one or more common proteins using these conserved surfaces. One possibility suggested by our structure and by the mutation data presented above is that the C-terminal domain of Tup1 uses residues centered around the channel on the top surface of the propeller to interact with the Matα2 protein. It is possible that this surface is conserved in all seven species where Tup1 homologs are found because they all have a Matα2-like protein. Matα2 homologs have been found in K.lactis (M.Redd and A.D.Johnson, unpublished data) and in C.albicans (Hull and Johnson, 1999); however, they have yet to be identified in other species of fungi. An intriguing possibility is that Tup1 uses one or more of the conserved surfaces to interact with proteins in the general repression pathway, such as components of the transcription machinery or chromatin.