Functional characterization and comparative analysis of gene repression-mediating domains interacting with yeast pleiotropic corepressors Sin3, Cyc8 and Tup1

Transcriptional corepressors Sin3, Cyc8 and Tup1 are important for downregulation of gene expression by recruiting various histone deacetylases once they gain access to defined genomic locations by interaction with pathway-specific repressor proteins. In this work we systematically investigated whether 17 yeast repressor proteins (Cti6, Dal80, Fkh1, Gal80, Mig1, Mot3, Nrg1, Opi1, Rdr1, Rox1, Sko1, Ume6, Ure2, Xbp1, Yhp1, Yox1 and Whi5) representing several unrelated regulatory pathways are able to bind to Sin3, Cyc8 and Tup1. Our results show that paired amphipathic helices 1 and 2 (PAH1 and PAH2) of Sin3 are functionally redundant for some regulatory pathways. WD40 domains of Tup1 proved to be sufficient for interaction with repressor proteins. Using length variants of selected repressors, we mapped corepressor interaction domains (CIDs) in vitro and assayed gene repression in vivo. Systematic comparison of CID minimal sequences allowed us to define several related positional patterns of hydrophobic amino acids some of which could be confirmed as functionally supported by site-directed mutagenesis. Although structural predictions indicated that certain CIDs may be α-helical, most repression domains appear to be randomly structured and must be considered as intrinsically disordered regions (IDR) adopting a defined conformation only by interaction with a corepressor. Supplementary Information The online version contains supplementary material available at 10.1007/s00294-023-01262-6.


Introduction
To prevent gene expression in eukaryotes under adequate conditions, repressor proteins may counteract activators (e. g. by shielding activation domains) or trigger the local formation of a chromatin structure, which is inhibitory against transcriptional activation. Formation of local inactive chromatin can be achieved by recruitment of pleiotropic corepressors associated with histone deacetylases (HDACs; Hildmann et al. 2007). In the yeast Saccharomyces cerevisiae, corepressors were genetically identified by isolation of mutations sin3, cyc8 and tup1 showing complex transcriptional deregulation of several unrelated pathways (reviewed by Grzenda et al. 2009;Malavé and Dent 2006).
Sin3 was initially characterized in yeast as a negative regulator of mating type switch (repressor of HO: Swiindependent; Sternberg et al. 1987;Wang et al. 1990) but is strongly conserved in all eukaryotes, being required as an antagonist of cellular proliferation in mammals (Adams et al. 2018). In yeast, several unrelated regulatory systems such as phospholipid biosynthesis, phosphate acquisition, sporulation and silencing of hidden mating type loci are affected by Sin3 (Vidal et al. 1991), leading to a number of alias gene designations. Sin3 is devoid of enzymatic activities but instead functions as a versatile interaction scaffold, using its paired amphipathic helices (PAH1-PAH4; Wang et al. 1990) for binding to pathway-specific repressors (e. g. Ume6 and Opi1; Kadosh and Struhl 1997;Wagner et al. 2001) and HDAC interaction domains (HID; Laherty et al. 1997;Grigat et al. 2012) for recruitment of histone deacetylases. Thus, Sin3 is of central importance for formation of a high-molecular-weight complex of at least 14 subunits (also designated Rpd3L; Carrozza et al. 2005), comprising auxiliary proteins Pho23, Sap30 and Sds3, among others. It has been shown that PAH1 and PAH2 form a bundle of four α-helical segments (also designated "wedged helical bundle"; Spronk et al. 2000), defining a hydrophobic cleft into which Sin3-interaction domains (SID) of repressor proteins can be inserted (Brubaker et al. 2000;Sahu et al. 2008). Importantly, a truncated variant of Sin3A has been identified by exome sequencing of human breast cancer samples, indicating that Sin3A can function as a tumour suppressor in certain tissues (Watanabe et al. 2018).
Cyc8 (= Ssn6) and Tup1 (= Flk1) also negatively influence various regulons in yeast, being required for repression of respiratory functions, glucose-regulated genes, mating functions and DNA damage repair (summarized by Malavé and Dent 2006). Cyc8 and Tup1 form a complex comprised of a Tup1 tetramer, which is associated with a single Cyc8 subunit (Varanasi et al. 1996). Some functional redundancy of Sin3 and Cyc8-Tup1 is supported by the finding of synthetic lethality of mutations sin3 and cyc8 (Jäschke et al. 2011). To become recruited to defined target promoters, Cyc8 und Tup1 must interact with specific DNA-binding proteins for which 10 TPR motifs (tetratricopeptide repeat) at the N-terminus of Cyc8 (Schultz et al. 1990;Tzamarias and Struhl 1995) or, presumably, 7 WD40 repeats (= β-transducin repeats; Williams and Trumbly 1990;Komachi et al. 1994) at the C-terminal domain of Tup1 are responsible. Importantly, repression mediated by a LexA-Tup1 fusion is effective even in the absence of Cyc8 while, vice versa, LexA-Cyc8 fails to down-regulate gene expression when Tup1 is missing (Tzamarias and Struhl 1994). It can be concluded that Tup1 is ultimately responsible for gene repression, supported by the finding that Tup1 preferentially binds to underacetylated histones H3 and H4 (Watson et al. 2000). Similar to Sin3, Cyc8 and Tup1 are also able to interact with HDACs (Rpd3, Hda1, Hos1 and Hos2; Wu et al. 2001;Davie et al. 2003). Cyc8-Tup1 may also trigger gene repression by counteracting transcriptional activation (Wong and Struhl 2011). In addition, Tup1 physically interacts with the cyclin-dependent kinase Srb10/Srb11 of the repression-mediating module of the mediator complex (Zaman et al. 2001;Schüller and Lehming 2003). Since SRB10 and SRB11 are required for full repression by gene fusions LexA-Cyc8 and LexA-Tup1 (Kuchin and Carlson 1998), Cyc8-Tup1 functionally interferes with the mediator and the RNA polymerase II holoenzyme complex.
Although functional studies of Sin3 and Cyc8-Tup1 mainly focus on gene repression, a simple classification as corepressors would ignore phenotypes which clearly provide evidence also for positive roles. Vidal et al. (1991) comparatively characterized expression of various unrelated genes (such as PHO5, HO, STE6 and SPO11) in wild-type and sin3 mutant strains and observed that Sin3 is involved in full repression and maximal activation of these genes, a finding which was also confirmed for genes of phospholipid biosynthesis (Wagner et al. 2001;Kliewe et al. 2017). Similarly, Cyc8-Tup1 can also positively influence the activity of heme-dependent activator Hap1 (Zhang and Guarente 1994), the retrograde-regulated citrate synthase gene CIT2 (Conlan et al. 1999) and the galactose-inducible GAL1 gene (Papamichos-Chronakis et al. 2002), once the adequate inducing conditions have become effective. It can be concluded that Sin3, Cyc8 and Tup1 fulfil dual functions as transcriptional corepressors (recruitment of negatively acting HDACs) and as coactivators (recruitment of SAGA; Papamichos- Chronakis et al. 2002;Parnell et al. 2021), depending on the regulatory situation studied.
Since we have previously shown that a pathway-specific repressor such as Opi1 can bind to Sin3 and Cyc8 (Jäschke et al. 2011), we wished to investigate whether other repressors show a similar diversity of interactions. We thus selected various repressor proteins affecting several unrelated regulatory pathways and performed a dual analysis by studying interaction with corepressors in vitro and gene repression in vivo. Our results show that minimal repression domains can be bound by more than a single corepressor while structural predictions provide no precise evidence for a conserved folding pattern common to all repressor proteins tested.

Yeast strains, media and growth conditions
Assays for gene repression in vivo were performed with S. cerevisiae strain RTS-lexA, containing an integrated CYC1-lacZ reporter gene with 4 lexA operator sites in its upstream region (MATα leu2 his3 trp1 ura3::lexA Op -CYC1-lacZ::URA3; Lettow et al. 2022). Transformants with effector plasmids encoding LexA-repressor fusions (based on pRT-lexA: 2 µm LEU2 MET25 PR -HA 3 -lexA DBD -NLS) were cultivated under double-selective conditions in synthetic complete media (SCD-Ura-Leu, 2% glucose). Gene repression in vivo was assayed by performing three independent transformations of strain RTS-lexA and using four colonies in each case (12 individual cultivations of transformants and enzyme assays).
The QuikChange site-directed mutagenesis kit (Agilent) together with pairs of mutagenic primers was used to introduce missense mutations into the YOX1 coding sequence, replacing selected natural codons against an alanine-specific codon (sequences of mutagenic primers are shown in Supporting Online Table S3). The authenticity of YOX1 mutational variants was verified by DNA sequencing (LGC Genomics, Berlin, Germany).

In vitro-interaction assays
GST-and HA-modified proteins were synthesized in E. coli strain BL21-CodonPlus DE3-RP (Agilent), overproducing selected tRNAs to avoid poor translation.
Crude protein extracts from induced E. coli transformants were prepared by sonication. Similar amounts of released GST fusion proteins (according to GST enzyme assays) were bound to glutathione (GSH) sepharose and incubated with yeast or bacterial total protein extracts containing HA fusions of Sin3, Cyc8 or Tup1. Washing conditions and elution of GST fusions together with prey proteins using free GSH have been described (Wagner et al. 2001). Eluted proteins were analyzed by SDS/PAGE and subsequently transferred to a PVDF membrane. HA-tagged proteins could be visualized by treatment with anti-HA-peroxidase conjugate (monoclonal antibody 12CA5 conjugate; Sigma-Aldrich) and POD chemiluminescent substrate, using a digital imager (ChemoStar, Intas).

Pathway-specific repressor proteins are able to physically interact with pleiotropic corepressors Sin3, Cyc8 and Tup1
Transcriptional repressor Opi1 negatively regulates structural genes of yeast phospholipid biosynthesis by interaction with pleiotropic corepressors Sin3 and Cyc8 (Jäschke et al. 2011) both of which are able to counteract gene expression by recruitment of various histone deacetylases. This redundancy of corepressor interaction may be specific for Opi1 but may be also effective for additional repressor proteins. To find out whether functional redundancy is indeed a general mechanism of gene repression, we systematically investigated in vitro interaction of repressors affecting several unrelated regulatory pathways in yeast (Supporting Online Material, Table S1) with corepressors Sin3, Cyc8 and Tup1 by GST pull-down experiments. GST fusions of 18 full-length repressors (exception: Ume6, for which the minimal domain interacting with Sin3 was used) were synthesized in E. coli, bound to glutathione sepharose and subsequently incubated with protein extracts containing epitope-tagged corepressors HA-Sin3, HA-Cyc8 and HA-Tup1, respectively. Interaction studies were initially performed with corepressor-containing protein extracts from yeast (not shown) and then repeated with bacterial extracts. These in vitro studies summarized in Fig. 1 confirmed several previously described individual interactions but indeed revealed a complex pattern of interaction between corepressors and repressors some of which may bind three corepressors (5; Ume6, Cti6, Rox1, Yox1 and Dal80), two corepressors (9; Opi1, Yhp1, Mot3, Whi5, Fkh1, Rdr1, Gal80, Ure2 and Sko1) or a single corepressor (3; Xbp1, Mig1 and Nrg1). 13 out of 18 repressor proteins were able to interact with Cyc8, 12 with Sin3 and 11 with Tup1. Eight repressors were able to bind to both subunits of the Cyc8-Tup1 corepressor (Ume6, Cti6, Rox1, Yox1, Dal80, Gal80, Ure2 and Sko1), indicating that redundancy of interaction is effective even within the same complex. These studies were performed with proteins entirely produced by E. coli, indicating that direct interactions have been detected in these experiments. Among repressor proteins assayed for interaction, only Mth1 could not bind to a corepressor. Thus, Mth1 was not considered for subsequent investigations. In contrast to a previous publication reporting that Sko1 merely interacts with Tup1 (Pascual-Ahuir et al. 2001) we found that Sko1 could bind Cyc8 and Tup1.
In addition to in vitro binding studies we also investigated whether repressor proteins which can bind at least one corepressor are able to fulfil repressor function in the living cell, thus decreasing gene expression in vivo. An episomal expression plasmid (pRT-lexA) containing a MET25 Prom -HA-lexA DBD -NLS cassette with a versatile multi-cloning site was constructed and used to generate lexA DBD -repressor fusions. To assay gene repression in vivo, derivatives of pRT-lexA encoding lexA DBD -repressor fusions were transformed into a yeast strain containing an integrated reporter gene with a lexA operator sequence upstream of UAS1 and UAS2 of a CYC1-lacZ promoter fusion (Kadosh and Struhl 1997). Once recruited to the lexA Op upstream of the native CYC1 promoter, a functional repressor should significantly decrease expression of the reporter gene lacZ, leading to reduced activity of β-galactosidase. As is also shown in Fig. 1, lexA DBD -repressor fusions were indeed able to downregulate expression of the reporter gene, although to varying degrees (2.6-fold repression mediated by Dal80 vs. 11.5-fold repression by Yox1). As is evident from the comparison of Yox1 and Dal80, which may both bind to Sin3, Cyc8 and Tup1, the strength of gene repression in vivo does not simply correlate with the number of corepressors a given repressor may contact.

Sin3-binding repressor proteins interact with PAH1 or PAH2
Although the four PAH domains of Sin3 were proposed as sites of protein-protein-interactions (Wang et al. 1990), previous studies with a limited number of proteins indicated that PAH1 and PAH2 are mainly responsible for repressor recruitment (Wagner et al. 2001;Washburn and Esposito 2001;Sahu et al. 2008). We thus systematically investigated which of its domains is contacted by 10 repressor proteins able to bind full-length Sin3 in vitro (not shown: Ume6 for which PAH2 was described as its target site, Washburn and Esposito 2001;Cti6, which binds to PAH1 and PAH2, Aref and Schüller 2020).
Epitope-tagged length variants of Sin3 were synthesized in S. cerevisiae and protein extracts subsequently used for interaction studies with immobilized GST fusions of repressor proteins. As is depicted in Fig. 2, several repressors are able to bind PAH1 and PAH2 in vitro (Opi1, Yox1, Rox1, Fkh1, Rdr1, Xbp1 and Whi5) while others show specificity for PAH1 (Dal80, Yhp1) or PAH2 (Mot3). As an exception, Whi5 could also interact with additional Sin3 length variants both of which contain the HDAC interaction domain 1, known to recruit the yeast major HDAC Rpd3 (Laherty et al. 1997;Grigat et al. 2012).
Although we have previously found that Opi1 contacts PAH1 of Sin3 (Wagner et al. 2001), these results show that PAH2 can be bound as well. To obtain evidence for the in vivo significance of Opi1-Sin3 interactions, we investigated regulated expression of a reporter gene dependent on   Kadosh and Struhl 1997). GST fusion proteins were immobilized on GSH sepharose and incubated with bacterial protein extracts, containing HA-Sin3 (pSW11; aa 1-1536, full-length), HA-Cyc8 (pFK77, aa 1-398, encoding TPR motifs) or HA-Tup1 (pFK76; full-length). Gene repression in vivo was measured in transformants of strain RTS-lexA (reporter gene lexA Op -CYC1-lacZ) containing plasmids which encode lexA fusions. Specific β-galactosidase activities are given in nmol oNPG hydrolyzed per min per mg of protein. Protein extracts prepared from at least 12 independent transformants were assayed. GST and lexA fusion plasmids are compiled in Supporting Online Material (Table S2). In vivo gene repression (RF, repression factor) was calculated as the ratio of specific β-galactosidase activities measured in transformants with empty vector pRT-lexA and individual lexArepressor fusions, respectively. n. t. not tested, SD standard deviation, + in vitro interaction, -no interaction the Opi1-controlled UAS element of phospholipid biosynthetic genes (ICRE-CYC1-lacZ, inositol/choline response element; Wagner et al. 2001) in the presence of several Sin3 deletion variants (Wang and Stillman 1993). As is shown in Table 1, our assays confirmed the severe deregulation of ICRE-dependent gene expression in a sin3 deletion strain (increased expression under repressing conditions, decreased expression under derepressing conditions). Removal of each single PAH from Sin3 still allowed a significant repression of the reporter gene while gene expression in the absence of both PAH1 and PAH2 was substantially increased. It should be mentioned that loss of PAH1 also prevents full derepression of the ICRE-dependent reporter gene. In summary, we conclude that PAH1 and PAH2 are indeed redundant for mediating Opi1-dependent gene repression. A similar functional redundancy may be also effective for other repressors shown to bind in vitro to both domains, PAH1 and PAH2.

Tup1-binding repressor proteins interact with WD40 repeats
It has been shown that repressor interactions with Cyc8 are mediated by TPR domains clustered at its N-terminus (Schultz et al. 1990;Struhl 1994, 1995;Jäschke et al. 2011). Although WD40 repeats of Tup1 are generally presumed to mediate interaction with repressor proteins, experimental evidence for this assumption has been reported only in details for alpha2 (α2; Komachi et al. 1994).

Yox1
The homeodomain-containing repressor Yox1 is phosphorylated by the cyclin-dependent kinase Cdc28/Cdk1 and counteracts gene activation by the MADS box transcription factor Mcm1, which is responsible for expression of genes with an ECB promoter motif (early cell cycle box; Pramila et al. 2002). Since Yox1 mediated strong gene repression in vivo and interacted with corepressors Sin3, Cyc8 and Tup1 (Fig. 1), we performed a detailed analysis of its molecular functions. Length variants of Yox1 were fused with GST and incubated with bacterial protein extracts containing Sin3, Cyc8 and Tup1, respectively. As is shown in Fig. 4, a short region comprising aa 220-280 of Yox1 following its homeodomain was able to interact with Sin3, Cyc8 and Tup1 in vitro and could also repress expression of a lexA Op -dependent reporter gene in vivo when fused with LexA. Corepressor interaction was also observed with an even shorter Yox1 domain (aa 235-280). For a more precise analysis, we introduced missense mutations into Yox1 length variant aa 220-280 at selected positions, focusing on several hydrophobic amino acids at positions reminiscent of a heptad-like sequence pattern. Gene repression was strongly weakened when residues V257, L262, V266 and I270 L271 were replaced with alanine (Fig. 4). As is evident from a double mutation replacing R272 D273, not only hydrophobic residues are important for functional repression, indicating that at least Yox1 aa 257-273 define the functional core of its repression domain.

Dal80
The zinc finger repressor Dal80 (degradation of allantoin; Cunningham and Cooper 1993) negatively regulates genes required for acquisition of nitrogen from poor sources by binding to URS GATA sequence motifs but its mechanism of repression has not been described previously. As demonstrated above (Fig. 1), Dal80 can interact with corepressors Sin3, Cyc8 and Tup1. GST fusions of Dal80 length variants showed that its N-terminal part comprising the zinc finger domain was unable to interact with corepressors (Fig. 5a). In contrast, truncations representing the C-terminus and internal sequences could bind to Sin3 and Cyc8. Indeed, aa 151-200, which are common to these truncations were able to interact with three corepressors and could also mediate strong gene repression in vivo (repression factor 7.3), which is substantially more effective than observed with entire Dal80. Possibly, fulllength Dal80 contains sequences counteracting maximal

Xbp1
The DNA-binding repressor Xbp1 prevents expression of cyclin genes of the G1 phase and thus causes delay of cell cycle progression (Mai and Breeden 2000). Xbp1 contains an Mbp1-related DNA-binding domain and was shown to interact with Sin3 but not with Cyc8 and Tup1 (Fig. 1). In contrast to other repressor proteins studied here, Xbp1 contains at least two non-overlapping domains mediating corepressor interaction and gene repression in vivo (aa 1-430 and aa 430-647; Fig. 5b). Using additional Xbp1 truncations, we could finally map a repression domain of 74 aa at the ultimate C-terminus of the protein. No further mapping was done for the length variant aa 1-430.

Ure2
This negative regulator of nitrogen catabolite repression is devoid of an obvious DNA-binding domain but contains two domains related to glutathione-S-transferases (GST) of unknown significance. In the presence of a favorable nitrogen source, Ure2 prevents gene activation by GATA factors Gln3 and Gat1 (Cooper 2002). Ure2 can convert into the conformational variant [URE3], forming polymeric amyloid prion-like filaments being able to self-propagate but unable to mediate gene repression (Baxa et al. 2002). Interestingly, the prion-forming domain of Ure2 (aa 1-80; Baxa et al. 2002) is part of its Cyc8 and Tup1 interaction domain, which also mediates efficient gene repression in vivo (5.2fold; Fig. 5c). In contrast, GST-related domains of Ure2 (aa 106-228 and aa 201-354) were unable to bind corepressors.

Rox1
Oxygen limitation (hypoxia) requires activation of certain respiratory enzymes such as cytochrome and cytochrome oxidase which, however, are repressed by Rox1 under aerobic conditions. Rox1 contains an N-terminal HMG box sequence motif (high mobility group) mediating its binding to promoter sites upstream of hypoxic genes such as ANB1 and CYC7 (Deckert et al. 1999). Rox1 is able to interact with corepressors Sin3, Cyc8 and Tup1 in vitro, utilizing a repression domain comprising amino acids 124-200 (Supporting Online Fig. 1a).

Mot3
Similar to Rox1, the zinc finger protein Mot3 is required for repression of certain hypoxic genes among which are genes of ergosterol biosynthesis (Hongay et al. 2002). As shown above (Fig. 1), Mot3 binds to Cyc8 and Tup1 in vitro, mediated by two non-overlapping domains before and behind its zinc finger motif. However, efficient repression in vivo can be observed only with aa 231-347, which are thus considered as a genuine repression domain (Supporting Online Fig. 1b).

Rdr1
PDR5 encoding an ABC transporter of the plasma membrane responsible for efflux of various drugs is negatively regulated by the Zinc cluster repressor Rdr1 (Hellauer et al. 2002). Our mapping results show that an internal domain  (Table S2). Gene repression in vivo was measured in transformants of strain RTS-lexA (reporter gene lexA Op -CYC1-lacZ) which contain plasmids encoding lexA fusions. Empty vector pRT-lexA was used as a negative control for maximal reporter gene expression. Specific β-galactosidase activities are given in nmol oNPG hydrolyzed per min per mg of protein. Protein extracts prepared from at least 12 independent transformants were assayed. ZF zinc finger, MR Mbp1related DNA-binding domain, n. t. not tested, RF repression factor, SD standard deviation, + in vitro interaction, -no interaction comprising aa 364-455 of Rdr1 mediate interaction with Sin3 (Supporting Online Fig. 1c).

Sko1
Initially, Sko1 was identified as a negative regulator of the invertase gene SUC2 with a CREB-related leucine zipper (Nehlin et al. 1992). However, its major function is regulation of genes required to counteract hyperosmotic stress (such as the sodium exporter ENA1). Importantly, Sko1 can be converted into an activator by Hog1-dependent phosphorylation, which is now able to recruit complexes SAGA and SWI/SNF to its target promoters (Proft and Struhl 2002). In this work we demonstrate that Sko1 interaction with Cyc8 requires a central domain (aa 201-427), which also mediates gene repression in vivo (Supporting Online Fig. 1d).

Discussion
In this work we investigated 18 selected pathway-specific transcriptional repressor proteins for their diversity of interactions with pleiotropic corepressors Sin3, Cyc8 and Tup1. As previously shown for the intensively characterized negative regulator Opi1 of phospholipid biosynthesis (Jäschke et al. 2011), most repressors are able to contact more than a single corepressor. Principally, such versatile repressors could (I) use several distinct interaction domains or (II) use the same domain for all of these interactions. Previously, a separation of functions has been described for alpha2 being responsible for repression of a-and haploid-specific genes in S. cerevisiae. While the N-terminal repression-mediating domain of alpha2 interacts with various WD40 repeats of Tup1 (Komachi et al. 1994), TPR motifs of Cyc8 can bind to the C-terminal homeo domain of alpha2 (Smith et al. 1995 as well as WD40 motifs of Tup1 exhibiting a β-sheet of a seven-bladed propeller structure (Sprague et al. 2000). Consequently, a considerable structural flexibility and malleability of CIDs within repressor proteins is required. Indeed, circular dichroism (CD) experiments showed that CID peptides are unfolded in the absence of corepressors while a defined secondary structure is adopted in their presence (forming amphipathic α -helices together with Sin3; Spronk et al. 2000;Swanson et al. 2004;He et al. 2021). It can be concluded that CIDs are intrinsically disordered regions (IDRs) lacking a precise three-dimensional structure which, however, perform a disorder-toorder transition once their binding partners are present (Cumberworth et al. 2013). Results of mapping studies described here and elsewhere allowed us to compile 16 sequences of corepressor interaction domains being able to contact Sin3, Cyc8 and/ or Tup1 for mediating pathway-specific gene repression (Table 2). We thus wished to comparatively analyze and possibly to derive sequence motifs, which are common to CIDs. Focusing on Sin3 interaction domains (SID), a limited number of repressor-corepressor interactions were previously investigated structurally. These NMR studies led to several related consensus motifs, emphasizing the importance of hydrophobic residues with a defined spatial pattern (Φ = bulky hydrophobic residue; X = any nonproline residue; A = Ala; L = Leu): SID of Mad1 binding to PAH2 of mSin3A: ΦXXΦXAAXXΦ (Brubaker et al. 2000); SID of HBP1 binding to PAH2 of mSin3A: AAX ΦXXΦ (Swanson et al. 2004); SID of REST binding to PAH1 of mSin3B: LXXL ΦXΦA (Nomura et al. 2005); SID of Sap25 binding to PAH1 of mSin3A: type I, ΦXΦXXΦ; type II, ΦXXΦXΦ (Sahu et al. 2008); SID of Pf1 binding to PAH2 of mSin3A: ΦXXΦXAA (Kumar et al. 2011); SID of Myt1L binding to PAH1 of mSin3B: ΦXXΦXΦ (Marcum and Radhakrishnan 2020); SID of TGIF1 binding to PAH2 of mSin3A: ΦXXLΦXΦA; (He et al. 2021). Table 3 Compilation of sequence motifs from corepressor interaction domains exhibiting periodic heptad patterns of hydrophobic amino acids (data derived from Table 2) As an example, 1-4-1 means that hydrophobic residues are found at positions 1 and 4 of the first heptad and position 1 of the next heptad. Some sequence motifs partially overlap or agree with more than a single pattern and are thus shown repeatedly. Proline residues are depicted in bold Using the data of Table 2, we thus searched for sequence patterns exhibiting hydrophobic residues at various defined positions of a putative heptad motif (1-4-7; 1-4-1; 1-5-7; 1-5-1 and 1-5-2; for details see compilation in Table 3). At least a single sequence pattern could be identified within each of the 16 CIDs compiled while some of them match with all patterns (alpha2, Fkh1, Opi1, Rdr1 and Rfx1). Mutational analysis of hydrophobic amino acids within the single 1-5-2 pattern of Yox1 indeed confirmed their importance for gene repression in vivo (Fig. 4). However, not only hydrophobic but also charged residues such as Arg or Asp contribute to functional repression by Yox1. Electrostatic interactions have been also described for Mad1-mSin3B (salt bridge between Mad1 E23 and PAH2 K165; van Ingen et al. 2004) and TGIF1-mSin3A (salt bridge between TGIF1 E394 and PAH2 K326; He et al. 2021). Polar interactions by hydrogen bonding were identified for complexes TGIF1-mSin3A (TGIF1 Q380 and PAH2 Q336; He et al. 2021) and Pf1-mSin3A (Pf1 N221 and PAH2 K315/Y325).
Although the anti-parallel orientation of the two α-helices forming a single TPR motif partially resembles the wedged helix bundle typical for PAH1 and PAH2, much less is known about specific sequence requirements of TPR ligand binding (reviewed by Zeytuni and Zarivach 2012). Both domains form an independent folding unit with a conformation which is not substantially affected by binding of their CID ligands (Brubaker et al. 2000;D'Andrea and Regan 2003). The complex of a model peptide (MEEVD, representing the C-terminus of Hsp90) with an artificially designed TPR module was studied by X-ray crystallography, providing evidence for a combined influence of hydrophobic (M, V) and electrostatic interactions (E, D). Acidic residues are bound by a "carboxylate clamp", established by residues K, N and R within the TPR module (Cortajarena et al. 2010).
WD40 motifs with a seven-bladed propeller structure and blades formed by four anti-parallel β-sheets may exhibit the largest degree of structural versatility for ligand binding. Data summarized by Stirnimann et al. (2010) and Xu and Min (2011) show that distinct regions of the propeller can be involved in binding of partner proteins which may contain α-helical segments but can be also unstructured.
To compare conformations of repressors investigated here and elsewhere we finally used structure predictions of S. cerevisiae proteins by the recently described bioinformatic tool Alphafold (Jumper et al. 2021), focusing on their respective CIDs. As is shown in Supporting Online Fig. 2, no uniform structural motifs are detectable. While α-helices are dominant only within CIDs of Cti6, Opi1 and Rdr1, random structures partially mixed with helical or sheet motifs characterize CIDs of the remaining repressor proteins. As mentioned above, it appears plausible that most corepressor interaction domains are intrinsically disordered and become locally ordered only in the presence of a corepressor template providing a defined structural framework ("grafting").