The structure of an Iws1/Spt6 complex reveals an interaction domain conserved in TFIIS, Elongin A and Med26
2010; Springer Nature; Volume: 29; Issue: 23 Linguagem: Inglês
10.1038/emboj.2010.272
ISSN1460-2075
AutoresMarie-Laure Diebold, Michael Koch, Erin Loeliger, Vincent Cura, Fred Winston, J. Cavarelli, Christophe Romier,
Tópico(s)Fungal and yeast genetics research
ResumoArticle5 November 2010free access The structure of an Iws1/Spt6 complex reveals an interaction domain conserved in TFIIS, Elongin A and Med26 Marie-Laure Diebold Marie-Laure Diebold Département de Biologie et Génomique Structurales, IGBMC (Institut de Génétique et Biologie Moléculaire et Cellulaire), UDS, CNRS, INSERM, Illkirch Cedex, France Search for more papers by this author Michael Koch Michael Koch Institut für Biochemie, Universität zu Köln, Köln, Germany Search for more papers by this author Erin Loeliger Erin Loeliger Department of Genetics, Harvard Medical School, Boston, MA, USA Search for more papers by this author Vincent Cura Vincent Cura Département de Biologie et Génomique Structurales, IGBMC (Institut de Génétique et Biologie Moléculaire et Cellulaire), UDS, CNRS, INSERM, Illkirch Cedex, France Search for more papers by this author Fred Winston Fred Winston Department of Genetics, Harvard Medical School, Boston, MA, USA Search for more papers by this author Jean Cavarelli Jean Cavarelli Département de Biologie et Génomique Structurales, IGBMC (Institut de Génétique et Biologie Moléculaire et Cellulaire), UDS, CNRS, INSERM, Illkirch Cedex, France Search for more papers by this author Christophe Romier Corresponding Author Christophe Romier Département de Biologie et Génomique Structurales, IGBMC (Institut de Génétique et Biologie Moléculaire et Cellulaire), UDS, CNRS, INSERM, Illkirch Cedex, France Search for more papers by this author Marie-Laure Diebold Marie-Laure Diebold Département de Biologie et Génomique Structurales, IGBMC (Institut de Génétique et Biologie Moléculaire et Cellulaire), UDS, CNRS, INSERM, Illkirch Cedex, France Search for more papers by this author Michael Koch Michael Koch Institut für Biochemie, Universität zu Köln, Köln, Germany Search for more papers by this author Erin Loeliger Erin Loeliger Department of Genetics, Harvard Medical School, Boston, MA, USA Search for more papers by this author Vincent Cura Vincent Cura Département de Biologie et Génomique Structurales, IGBMC (Institut de Génétique et Biologie Moléculaire et Cellulaire), UDS, CNRS, INSERM, Illkirch Cedex, France Search for more papers by this author Fred Winston Fred Winston Department of Genetics, Harvard Medical School, Boston, MA, USA Search for more papers by this author Jean Cavarelli Jean Cavarelli Département de Biologie et Génomique Structurales, IGBMC (Institut de Génétique et Biologie Moléculaire et Cellulaire), UDS, CNRS, INSERM, Illkirch Cedex, France Search for more papers by this author Christophe Romier Corresponding Author Christophe Romier Département de Biologie et Génomique Structurales, IGBMC (Institut de Génétique et Biologie Moléculaire et Cellulaire), UDS, CNRS, INSERM, Illkirch Cedex, France Search for more papers by this author Author Information Marie-Laure Diebold1,‡, Michael Koch2,‡, Erin Loeliger3, Vincent Cura1, Fred Winston3, Jean Cavarelli1 and Christophe Romier 1 1Département de Biologie et Génomique Structurales, IGBMC (Institut de Génétique et Biologie Moléculaire et Cellulaire), UDS, CNRS, INSERM, Illkirch Cedex, France 2Institut für Biochemie, Universität zu Köln, Köln, Germany 3Department of Genetics, Harvard Medical School, Boston, MA, USA ‡These authors contributed equally to this work *Corresponding author. Département de Biologie et Génomique Structurales, IGBMC (Institut de Génétique et Biologie Moléculaire et Cellulaire), UDS, CNRS, INSERM, 1 rue Laurent Fries, B.P. 10142, Illkirch Cedex 67404, France. Tel.: +33 38 854 5798; Fax: +33 38 865 3276; E-mail: [email protected] The EMBO Journal (2010)29:3979-3991https://doi.org/10.1038/emboj.2010.272 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Binding of elongation factor Spt6 to Iws1 provides an effective means for coupling eukaryotic mRNA synthesis, chromatin remodelling and mRNA export. We show that an N-terminal region of Spt6 (Spt6N) is responsible for interaction with Iws1. The crystallographic structures of Encephalitozoon cuniculi Iws1 and the Iws1/Spt6N complex reveal two conserved binding subdomains in Iws1. The first subdomain (one HEAT repeat; HEAT subdomain) is a putative phosphoprotein-binding site most likely involved in an Spt6-independent function of Iws1. The second subdomain (two ARM repeats; ARM subdomain) specifically recognizes a bipartite N-terminal region of Spt6. Mutations that alter this region of Spt6 cause severe phenotypes in vivo. Importantly, the ARM subdomain of Iws1 is conserved in several transcription factors, including TFIIS, Elongin A and Med26. We show that the homologous region in yeast TFIIS enables this factor to interact with SAGA and the Mediator subunits Spt8 and Med13, suggesting the molecular basis for TFIIS recruitment at promoters. Taken together, our results provide new structural information about the Iws1/Spt6 complex and reveal a novel interaction domain used for the formation of transcription networks. Introduction Eukaryotic RNA polymerase II (RNAPII) requires the timely and concerted recruitment of many factors to enhance pre-initiation complex (PIC) formation, promoter clearance, transcription elongation and to overcome the transcriptional barriers imposed by chromatin (Saunders et al, 2006; Li et al, 2007). Furthermore, recruitment by elongating RNAPII of factors involved in mRNA processing, mRNA surveillance and mRNA export enables a direct and tight coupling between transcription, mRNA maturation and mRNA export (Perales and Bentley, 2009). The Iws1/Spt6 complex participates in this coupling by acting in transcription elongation, chromatin remodelling and mRNA export. Spt6 is a putative histone chaperone that interacts directly with histone H3, and appears to promote nucleosome reassembly at core promoters and within the body of genes in the wake of RNAPII (Bortvin and Winston, 1996; Winkler et al, 2000; Kaplan et al, 2003; Adkins and Tyler, 2006). Spt6 is also an elongation factor that enhances elongation rates of RNAPII both in vitro and in vivo (Endoh et al, 2004; Yoh et al, 2007; Ardehali et al, 2009). Yet, the role of Spt6 in elongation cannot be fully recapitulated by its histone chaperone activity as elongation enhancement also occurs on naked DNA templates (Endoh et al, 2004; Yoh et al, 2007). A recent study has shown that both Spt6 and Iws1 are associated genome-wide across transcribed regions (Mayer et al, 2010). Interaction between Spt6 and the Iws1/Spn1 protein has been reported both in yeast and in mammals (Krogan et al, 2002; Lindstrom et al, 2003; Yoh et al, 2007). The involvement of Spt6 in mRNA export requires its association with Iws1, as Iws1 interacts directly with the mRNA export factor REF1/Aly (Yoh et al, 2007). Furthermore, either depletion of Iws1 or a mutation within the SH2 domain of Spt6, which impairs the ability of the Iws1/Spt6 complex to interact with elongating RNAPII, results in splicing defects and nuclear retention of bulk poly(A)+ mRNAs in mammalian cells (Yoh et al, 2007). Mammalian Iws1 is also required for the recruitment of the lysine methyltransferase, HYPB/Setd2, which trimethylates H3K36 across transcribed regions (Yoh et al, 2008). Iws1 also functions independently of Spt6 by binding constitutively to the postassembly regulated CYC1 gene, thus preventing the recruitment of the Swi/Snf complex and repressing transcription of this gene (Fischbeck et al, 2002; Zhang et al, 2008). A single amino-acid change in yeast Iws1 (K192N) prevents its recruitment to the CYC1 gene, leading to the constitutive recruitment of Swi/Snf and increased transcription, even in non-inducing conditions. The direct interaction of Iws1 with the Ser5-phosphorylated RNAPII has been suggested for the constitutive recruitment of Iws1 (Zhang et al, 2008). Despite the wealth of data on Spt6 and Iws1, their functional roles remain largely unknown at the molecular level. So far, only the structures of the Spt6 tandem SH2 domains (Diebold et al, 2010; Sun et al, 2010) and of the putative bacterial ancestor of Spt6, Tex (Johnson et al, 2008), have shed some light on the structural organization of Spt6. As for Iws1, part of the conserved region of Iws1 is homologous in sequence to the N-terminal domains of the elongation factors TFIIS and Elongin A, and of the co-activator Med26 (Wery et al, 2004; Ling et al, 2006). In TFIIS, this domain enables TFIIS recruitment to gene promoters by the co-activators SAGA and Mediator, in which it favours the formation of active PICs (Pan et al, 1997; Wery et al, 2004; Prather et al, 2005; Guglielmi et al, 2007; Kim et al, 2007). Yet, the NMR structures of the yeast and mouse TFIIS N-terminal domains adopt totally different folds (PDB code 1EO0; Booth et al, 2000; PDB code 1WJT, RIKEN Structural Genomics/Proteomics Initiative), precluding our understanding of the role of this domain. Here, we present the biochemical, crystallographic and functional characterization of Iws1 and the Iws1/Spt6 complex from Encephalitozoon cuniculi and Saccharomyces cerevisiae. We show that Iws1 possesses two binding subdomains (HEAT and ARM subdomains) defined by the N- and C-termini of its conserved region. Another study has also recently identified these domains from the crystallographic analysis of S. cerevisiae Iws1 (Pujari et al, 2010). The HEAT subdomain has determinants for recognizing large negatively charged ions or small molecules such as phosphates, suggesting that it may serve to recognize phosphoproteins. This subdomain is specific to Iws1 and is most likely responsible for the Spt6-independent function of Iws1. The ARM subdomain of Iws1 specifically recognizes an N-terminal region of Spt6 (Spt6N). In yeast, we show that mutations that alter this region of Spt6 cause severe phenotypes, suggesting that the interaction between Spt6 and Iws1 is critical in vivo. Importantly, the ARM subdomain of Iws1 encompasses the region of sequence homology with TFIIS, Elongin A and Med26. We show that the homologous subdomain from yeast TFIIS is able to form complexes with the Spt8 and Med13 subunits of SAGA and the Mediator, suggesting a molecular basis for TFIIS recruitment at promoters. These complexes combine interactions that are observed within the Iws1/Spt6N complex, along with interactions that are specific to the TFIIS/Spt8 and TFIIS/Med13 complexes. Taken together, our results provide a structural characterization of the Iws1/Spt6 complex and highlight a specific interaction domain shared by other transcriptional effectors. Results A small N-terminal region of Spt6 is sufficient to interact with Iws1 Yeast (S. cerevisiae; sc) and human (Homo sapiens; hs) Spt6 proteins are very large polypeptides that are not easily amenable to biochemical and structural studies. To overcome this problem, we use the fungi-related intracellular parasite, E. cuniculi (ec), as a model organism as its proteins are generally shorter than their eukaryotic orthologues (Katinka et al, 2001; Romier et al, 2007; Diebold et al, 2010). Spt6 and Iws1 are present in E. cuniculi and show strong sequence conservation with their yeast and human orthologues despite their shorter length (Figure 1A; Supplementary Figure 1). Figure 1.A small N-terminal region of Spt6 is sufficient to retain the full length or the conserved domain of Iws1. (A) Schematic view of the putative domain architecture of Spt6 and Iws1. The domains of Spt6 and Iws1 characterized in this study are shown as light orange boxes. This domain in Iws1 is composed of two subdomains (HEAT subdomain and ARM subdomain) and corresponds to the conserved region of Iws1 sufficient for Iws1 function in yeast (Fischbeck et al, 2002). The Iws1 invariant lysine (scK192/ecK90) involved in the Spt6-independent function of Iws1 is marked by an 'X'. The Iws1 region homologous to TFIIS, Elongin A and Med26 N-terminal domains is hatched. The domains (HtH, YqgF, HhH and S1) that have been putatively assigned to the Spt6 core domain based on the structure of the bacterial Tex protein (Johnson et al, 2008) are shown. The two SH2 domains from the tandem SH2 domains at the C-terminus of Spt6 (Diebold et al, 2010; Sun et al, 2010) are indicated. The results of co-expression experiments shown in (B) are summarized below the proteins. E. cuniculi (ec) and S. cerevisiae (sc) numbering are shown. (B) Deciphering of E. cuniculi Iws1/Spt6 complex formation upon (co-) expression in E. coli of various constructs of both proteins and purification by affinity chromatography. All samples are analysed on SDS–PAGE. The fainter band for His-ecSpt6N53−71 in lane 12 compared with lane 13 is due to the lower amount of soluble complex obtained. Construct boundaries are indicated. Spt6 degradation products are marked with an '*'. (C) Characterization of the S. cerevisiae Iws1/Spt6N interaction based on the data obtained with the E. cuniculi proteins. The faint band for His-scSpt6N229−269 in lane 1 is most likely due to the poor solubility of this construct when expressed alone. Iws1 degradation products are marked with an '*'. Molecular weights are shown and are the same throughout the figures. Download figure Download PowerPoint Our experiments show that full-length E. cuniculi Spt6 and Iws1 form a complex when co-expressed in Escherichia coli. Singly expressed ecSpt6, N-terminally tagged with a poly-histidine sequence, is retained on a cobalt affinity resin (Figure 1B, lane 1). In contrast, singly expressed, but untagged ecIws1 does not bind to the same resin, showing that no non-specific binding occurs (Figure 1B, lane 2). However, upon co-expression of his-tagged ecSpt6 with untagged ecIws1, both proteins are retained on the resin, revealing the formation of a complex between these two proteins (Figure 1B, lane 3). Additional co-expression experiments defined regions in each protein sufficient for their interaction. First, the conserved region of Iws1 (ecIws1 residues 55–198), which is sufficient for the essential functions of Iws1 in yeast (Fischbeck et al, 2002), is also sufficient for binding to ecSpt6 (Figure 1B, lane 5). In the case of ecSpt6, as observed for its mammalian homologue (Yoh et al, 2007), the N-terminal region (ecSpt6 residues 1–71; ecSpt6N1−71), but not the remaining C-terminal region (ecSpt6 residues 71–894), is involved in Iws1 binding (Figure 1B, compare lanes 6 and 8). Multiple alignment of Spt6 sequences shows that its N-terminal region contains a small conserved region (ecSpt6 residues 53–71; ecSpt6N53−71; Supplementary Figure 1A). Co-expression experiments showed that this small conserved region of Spt6 is sufficient for binding ecIws1 (Figure 1B, lanes 10–13). These results were confirmed by looking for a similar interaction between the yeast proteins: an scSpt6N229−269 construct, similar to ecSpt6N53−71, was able to form a complex with either full-length scIws1 or its conserved region (residues 144–314) (Figure 1C). The conserved region of Iws1 is formed by HEAT and ARM repeats To characterize precisely the interaction between Spt6 and Iws1, we solved the X-ray crystallographic structures of these proteins and their complex from E. cuniculi. Owing to rapid proteolytic N-terminal degradations occurring during purification, only the stable ecIws155−198 construct was considered and could be readily crystallized on its own in different space groups. For Spt6, due to yield and C-terminal proteolytic degradation problems with the full-length protein, only N-terminal constructs (encoding ecSpt6N residues 1–71, 34–71 and 53–71) were used in complex with ecIws155−198. Three different crystal forms were obtained: one form with the ecSpt6N34−71 construct and two forms with the ecSpt6N53−71 construct. All crystal forms were unrelated, providing an unbiased view of the interaction between Spt6N and Iws1. The structure of ecIws155−198 was solved by multiple anomalous dispersion using selenomethionine derivatives. The model was refined against a 2.25 Å resolution native data set to an R-factor of 20.4% and an R-free of 26.5% (Supplementary Table I). The three structures of the complexes were then solved by molecular replacement using the structure of free Iws1. The Spt6N regions were built into the additional density and the structures of the ecSpt6N53−71/ecIws155−198 (forms 1 and 2) and ecSpt6N34−71/ecIws155−198 complexes were refined to 1.95, 2.10 and 1.75 Å resolution to R-factors of 21.0, 20.0 and 19.4% and R-frees of 23.6, 24.7 and 23.6%, respectively (Supplementary Table II). For Spt6N and Iws1, almost all residues could be seen in density, apart from a few N- and/or C-terminal residues of both proteins. A single exception was observed for the longer ecSpt6N34−71 construct in which the first 11 N-terminal residues could not be seen. Structural analysis of ecIws155−198 in the different crystal forms reveals that the Iws1 conserved region is formed by a single HEAT repeat followed by two ARM repeats (ARM1 and ARM2) (Figure 2). HEAT and ARM motifs are related protein/protein interaction modules that are often found repeated several times in many proteins (Andrade et al, 2001). The HEAT motif is composed of an α-helical hairpin formed by two α-helices (αA and αB), whereas the ARM motif is composed of a short α-helix (α1), which is almost perpendicular to a helical hairpin formed by two α-helices (α2 and α3). Only minor structural differences are observed for Iws1 between all crystal forms, implying that binding of Spt6N does not induce large conformational changes. Figure 2.Structure of E. cuniculi Iws1. (A) Ribbon representation of ecIws155−198 crystal structure. α-helices of the HEAT, ARM1 and ARM2 motifs are coloured red, blue and yellow, respectively. Loops are coloured green. The two subdomains of Iws1 are indicated. The invariant K90 is displayed as sticks and coloured according to atom type. The anion bound to K90 Nε is shown as a cyan sphere. The first and last residues observed in the density are labelled. This colour scheme is used throughout the figures unless otherwise stated. The ribbon figures have been made with PYMOL (version 0.99; DeLano Scientific). (B) Multiple sequence alignment of the conserved region of Iws1 (top five rows) with the TFIIS, Elongin A and Med26 N-terminal domains (bottom three rows; not shown for the HEAT repeat, which is Iws1 specific). mm, Mus musculus; sc, S. cerevisiae; hs, H. sapiens. Sequence similarities are indicated by shading. Observed α-helices in the ecIws155−198 structure are shown above the sequences as cylinders coloured as in (A). Numbering above the sequences correspond to E. cuniculi, whereas the numbering at the end of each row relates to the different organisms. K90 and the residues involved in its packing at the interface of the HEAT and ARM1 repeats are labelled with yellow stars. Residues forming the highly conserved putative phosphate-binding domain of Iws1, together with K90, are labelled by magenta triangles. Residues involved in Spt6 IR1 and IR2 binding are labelled with yellow diamonds and cyan circles, respectively. Alignment features are identical in all figures unless otherwise stated. Alignments were created with ALINE (Bond and Schuttelkopf, 2009). Download figure Download PowerPoint A conserved binding subdomain (HEAT subdomain) formed by the Iws1 HEAT repeat and part of the ARM1 repeat contains the invariant ecK90/scK192 In yeast Iws1, mutation of the invariant lysine K192 to asparagine leads to the rescue of TBP mutants that possess postrecruitment defects, and prevents constitutive recruitment of Iws1 to the CYC1 gene promoter in an Spt6-independent manner (Fischbeck et al, 2002; Zhang et al, 2008). The equivalent lysine of ecIws1, K90, is found at the start of helix αB in the HEAT repeat (Figures 2A and 3A). The K90 side chain is found at the interface of the tips of both HEAT and ARM1 repeats. The aliphatic part of the K90 side chain is at the centre of a hydrophobic core formed by the side chains of HEAT αA I73, ARM1 α21 W122 and ARM1 α31 I136. Furthermore, K90 Nε is fixed through a hydrogen bond with the carboxylate of invariant HEAT αA D77 that also hydrogen bonds with the K90 main chain amide (Figure 3A). Figure 3.The HEAT-subdomain Iws1. (A) Close-up view of K90 interactions at the interface of Iws1 HEAT and ARM1 repeats. The side chains involved in hydrophobic interactions and hydrogen bonding are shown. (B) Close-up view of the HEAT subdomain formed by Iws1 HEAT and ARM1 repeats. Residues forming the surface are displayed. The bromide anion (Br) is shown as a cyan sphere. (C) Stereo view of the structure at the bromide-binding site. The bromide (Br) and water molecules are shown as cyan and red spheres, respectively. The 2Fo–Fc electron density map of the refined structure is shown and contoured at 1.5 σ. Download figure Download PowerPoint Importantly, the hydrogen bond formed between the carboxylate of D77 and the K90 Nε positions the latter atom precisely at the bottom of a pocket formed by both tips of the HEAT and ARM1 repeats, making it solvent accessible. Strikingly, most of the constituent residues of this pocket are highly conserved (Figures 2B and 3B). Inspection of this pocket reveals the direct binding of an ion to K90 Nε, which is further coordinated by the N133 main chain amide as well as one or two water molecules. In most structures, the bound ion is most likely a chloride ion provided by the protein buffer. In one structure, however, a much larger electron density was observed reflecting the replacement of the chloride ion by a negatively charged bromide ion provided by the crystallization conditions (Figure 3B and C). Interestingly, this ion-binding pocket would be large enough to accommodate a phosphate at the position of the bromide. Furthermore, the atoms coordinating the bromide ion (K90 Nε, N133 main chain amide and two water molecules) would be perfectly placed to coordinate an incoming phosphate. As one of the coordinating water molecules is located at the entrance of the pocket, it could also be replaced by the oxygen of a phosphorylated residue. Taken together, these results suggest that this conserved binding domain of Iws1 could be specific for phosphoproteins. To further investigate the role of K90, four mutants were created: K90R, K90A, K90D and K90N. All mutants could be expressed and purified. Despite a two- to four-fold decrease in yield for most of the mutants, none showed an increased tendency to aggregate as assessed by dynamic light scattering analyses. However, temperature-dependent unfolding of these proteins using the ThermoFluor technology showed that the K90D and K90N mutants have greater instability (Supplementary Figure 2A). We next tested the effect of these mutants on the formation of the complex with Spt6N. Upon co-expression of the mutants with ecSpt6N53−71, no complex was observed with the K90D mutant and the level of complex formation appeared to be reduced with the K90N mutant (Supplementary Figure 2B, lanes 1–5). Surprisingly, none of the mutants impaired the binding of the longer ecSpt6N1−71 construct (Supplementary Figure 2B, lanes 7–11). Taken together, our results suggest that the effects of the K90 mutants are local and suggest a more extensive interface between Iws1 and Spt6N. Bipartite binding of Spt6N to Iws1 ARM repeats (ARM subdomain) Analysis of the structures of the Iws1/Spt6N complex confirms the hypothesis of extended Iws1–Spt6N interactions, showing that the region of ecSpt6N interacting with Iws1 encompasses residues 45–67, forming contacts with both ARM repeats, but not with the HEAT repeat (Figure 4A and B). This region is composed of an N-terminal α-helix (αN) that interacts with Iws1 ARM2, pointing towards the ARM2 α22 helix. αN stops abruptly at the invariant glycine 58, which enables the peptide to go round the Iws1 α22 helix. The C-terminal part of the Spt6N peptide adopts a rather extended conformation followed by a short-helical turn (αC) and interacts with both ARM repeats (Figure 4B). Figure 4.Crystal structure of E. cuniculi Iws1/Spt6N complex. (A) Multiple sequence alignment of the Iws1-binding region of Spt6N. Both Spt6N IR1 and IR2 sub-regions are indicated below the sequences. Residues whose side chains are involved in Iws1 binding are labelled with yellow stars. Spt6N α-helices observed in the Iws1/Spt6N structures are shown as orange cylinders. (B) Ribbon representation of the Spt6N34−71/Iws155−198 structure. Spt6N is coloured orange. Most Spt6N side chains interacting with Iws1 are shown. (C) Close-up view of the interaction between Spt6N αN and Iws1 α32 helices. (D) Close-up view of Spt6N IF motif binding to Iws1 hydrophobic cavity. (E) Close-up view of Spt6N IR2 binding to Iws1. The red sphere represents a water molecule. (F) GRASP (Nicholls et al, 1991) representation of the electrostatic potential at the surface of E. cuniculi Iws1. The electrostatic potentials −8 and +8 kBT (kB, Boltzmann constant; T, temperature) are coloured red and blue, respectively. The Spt6N region binding to Iws1 is shown as orange ribbon. K90 Nε is located within a cavity and is labelled (K90). Download figure Download PowerPoint As such, the interaction region (IR) of Spt6N with Iws1 can be divided into two sub-regions that we termed IR1 (Spt6N αN up to the invariant F57/G58 motif) and IR2 (C-terminal to G58) (Figure 4A). The interaction between the Spt6N IR1 region and Iws1 is dictated mostly by hydrophobic interactions. The Spt6N αN helix packs against the C-terminus of Iws1 ARM2 α32 helix (Figure 4C). This interaction is essentially hydrophobic and involves Iws1 V191 as well as several residues at the N-terminus of Spt6N αN. These latter residues are solvent exposed and their contribution to the hydrophobic core is mediated by their main chain Cβ and/or Cγ atoms. This most likely explains why the sequence of this helix is not evolutionarily conserved as only its α-helical propensity appears important. In agreement, secondary structure predictions of S. cerevisiae and H. sapiens Spt6N indicate an α-helix precisely at the position of ecSpt6N αN, ending at the invariant FG motif. At the C-terminal end of Spt6N αN, I56 and F57 interact with a hydrophobic cavity of Iws1 formed by residues L154, G160, V163, V184, W187 and V191 of the ARM2 repeat (Figure 4D). The perfect conservation of the hydrophobic character of the I56/F57 motif, as well as the ability of the ecSpt6N53−71 construct to retain its interaction with Iws1 despite the large truncation of the αN helix, demonstrates the importance of the binding of the I56/F57 motif to the hydrophobic cavity of Iws1. In contrast, the interaction between the Spt6N IR2 region and Iws1 is composed of both hydrophobic and hydrogen bond contacts. Following the I56/F57 motif, Spt6N G58 has the function of helix breaker, necessary to prevent steric clashes between Iws1 and an elongated αN helix of Spt6N. The four residues following G58 do not make extensive contacts with Iws1, but serve as a linker for the peptide to go around the α22 helix of ARM2. The next interaction between both proteins is mediated through the hydrogen bond formed between the hydroxyl of Spt6 Y63 and the carboxylate of Iws1 E124 (Figure 4E). This interaction is reinforced by hydrophobic contacts made between the Y63 side chain and Iws1 I162 and F165. Other interactions involve Spt6N tyrosine Y65 whose hydroxyl forms a water-mediated interaction with Iws1 L126 carboxyl, and Spt6 V66 and L67, which form hydrophobic contacts with residues of Iws1 ARM1 and ARM2 repeats. Interestingly, both the ion-binding HEAT subdomain and the Spt6N-binding ARM subdomain of Iws1 are connected by a positively charged channel (Figure 4F). Specifically, the K90-binding pocket and the Spt6 IR2-binding surface of Iws1 are relatively close, suggesting that binding of a protein to the former pocket could prevent binding of Spt6 to the latter surface, or vice versa. Point mutations within Spt6 Iws1-binding region cause Spt− and Ts− phenotypes in vivo We next investigated the interaction between Spt6 and Iws1 by mutational analysis, using the ecSpt6N1−71 construct, which encompasses the full region of interaction of ecSpt6N with Iws1. Based on our structural data, single and double mutants of ecSpt6N and ecIws1 were made. At the Spt6N IR1/Iws1 interface, Iws1 residues G160 and V191 were changed to bulkier residues (single mutants G160Y, V191Y and V191W), whereas Spt6N residues I56 and F57 were changed to smaller alanine residues (double mutant I56A/F57A). At the IR1/IR2 transition, both Spt6N G58 and G60 were changed to alanines (double mutant G58A/G60A). At the Spt6N IR2/Iws1 interface, Iws1 E124 was changed to either serine or alanine (E124S and E124A), whereas Spt6N Y63 and Y65 were changed to alanines (single mutant Y63A and double mutant Y63A/Y65A). Upon co-expression, none of the Iws1 mutants affected complex formation, showing that single mutations in Iws1 are not sufficient to destroy the complex with Spt6N (Figure 5A, lanes 1–6). In contrast, all Spt6N mutants, with the exception of the Y63A mutant, led to the loss of the complex (Figure 5A, lanes 8–12). Some of these Spt6N mutants appeared insoluble (Figure 5A, lanes 10 and 12), suggesting that the loss of the complex may be due to insolubility. However, the I56A/F57A mutant was still soluble, showing that, in this case, the loss of interaction with Iws1 was due to the mutations. In agreement, the same mutation introduced in the yeast Spt6N229−269 constr
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