Structural basis for centromere maintenance by Drosophila CENP ‐A chaperone CAL 1
2020; Springer Nature; Volume: 39; Issue: 7 Linguagem: Inglês
10.15252/embj.2019103234
ISSN1460-2075
AutoresBethan Medina‐Pritchard, Vasiliki Lazou, Juan Zou, Olwyn Byron, Maria Alba Abad, Juri Rappsilber, Patrick Heun, A. Arockia Jeyaprakash,
Tópico(s)DNA Repair Mechanisms
ResumoArticle5 March 2020Open Access Transparent process Structural basis for centromere maintenance by Drosophila CENP-A chaperone CAL1 Bethan Medina-Pritchard Bethan Medina-Pritchard orcid.org/0000-0002-6736-9203 Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Vasiliki Lazou Vasiliki Lazou Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Juan Zou Juan Zou Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Olwyn Byron Olwyn Byron School of Life Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Maria A Abad Maria A Abad Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Juri Rappsilber Juri Rappsilber orcid.org/0000-0001-5999-1310 Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany Search for more papers by this author Patrick Heun Patrick Heun Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author A Arockia Jeyaprakash Corresponding Author A Arockia Jeyaprakash [email protected] orcid.org/0000-0002-1889-8635 Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Bethan Medina-Pritchard Bethan Medina-Pritchard orcid.org/0000-0002-6736-9203 Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Vasiliki Lazou Vasiliki Lazou Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Juan Zou Juan Zou Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Olwyn Byron Olwyn Byron School of Life Sciences, University of Glasgow, Glasgow, UK Search for more papers by this author Maria A Abad Maria A Abad Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Juri Rappsilber Juri Rappsilber orcid.org/0000-0001-5999-1310 Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany Search for more papers by this author Patrick Heun Patrick Heun Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author A Arockia Jeyaprakash Corresponding Author A Arockia Jeyaprakash [email protected] orcid.org/0000-0002-1889-8635 Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Author Information Bethan Medina-Pritchard1, Vasiliki Lazou1, Juan Zou1, Olwyn Byron2, Maria A Abad1, Juri Rappsilber1,3, Patrick Heun1 and A Arockia Jeyaprakash *,1 1Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, UK 2School of Life Sciences, University of Glasgow, Glasgow, UK 3Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany ‡Lead author *Corresponding author. Tel: +44 1316 507113; E-mail: [email protected] The EMBO Journal (2020)39:e103234https://doi.org/10.15252/embj.2019103234 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 Abstract Centromeres are microtubule attachment sites on chromosomes defined by the enrichment of histone variant CENP-A-containing nucleosomes. To preserve centromere identity, CENP-A must be escorted to centromeres by a CENP-A-specific chaperone for deposition. Despite this essential requirement, many eukaryotes differ in the composition of players involved in centromere maintenance, highlighting the plasticity of this process. In humans, CENP-A recognition and centromere targeting are achieved by HJURP and the Mis18 complex, respectively. Using X-ray crystallography, we here show how Drosophila CAL1, an evolutionarily distinct CENP-A histone chaperone, binds both CENP-A and the centromere receptor CENP-C without the requirement for the Mis18 complex. While an N-terminal CAL1 fragment wraps around CENP-A/H4 through multiple physical contacts, a C-terminal CAL1 fragment directly binds a CENP-C cupin domain dimer. Although divergent at the primary structure level, CAL1 thus binds CENP-A/H4 using evolutionarily conserved and adaptive structural principles. The CAL1 binding site on CENP-C is strategically positioned near the cupin dimerisation interface, restricting binding to just one CAL1 molecule per CENP-C dimer. Overall, by demonstrating how CAL1 binds CENP-A/H4 and CENP-C, we provide key insights into the minimalistic principles underlying centromere maintenance. Synopsis Epigenetic maintenance of centromeres requires specific deposition of histone H3 variant CENP-A. X-ray crystallography explains how the evolutionary distinct histone chaperone CAL1 combines both CENP-A recognition and centromere targeting activities of the respective mammalian counterparts, HJURP and Mis18 complex. Crystal structures of Drosophila CAL1 bound to CENP-A/H4 dimers show that CAL1 employs evolutionarily conserved and adaptive interactions to recognise CENP-A/H4. CAL1 binding shields key CENP-A/H4 surfaces required for nucleosome assembly, a key requirement for preventing untimely CENP-A loading. CAL1 interactions with its centromeric receptor CENP-C involve a short helical CAL1 segment spanning residues 890–913, and the CENP-C cupin domain. CAL1 binds CENP-C at a site stabilised by cupin domain dimerization, thereby restricting binding to one CAL1 molecule per CENP-C dimer. Introduction Centromeres are specialised chromosomal regions that act as a platform for the assembly of kinetochores, the microtubule anchoring sites essential for chromosome segregation during mitosis and meiosis (Musacchio & Desai, 2017). Unlike budding yeast where DNA sequence is sufficient to define centromere identity, centromeres in most other eukaryotes are defined by the enrichment of unique nucleosomes containing the histone H3 variant CENP-A (Sekulic & Black, 2012; Zasadzinska & Foltz, 2017). As a consequence, maintenance of CENP-A-containing nucleosomes is essential for preserving centromere identity through generations of cell cycles. This is achieved through an epigenetic mechanism that relies on CENP-A as an epigenetic mark (Westhorpe & Straight, 2014; McKinley & Cheeseman, 2016; Musacchio & Desai, 2017; Zasadzinska & Foltz, 2017). Unlike canonical chromatin maintenance, centromeric chromatin maintenance is decoupled from DNA replication. As a result, CENP-A levels on the sister chromatids are reduced by half during replication (Jansen et al, 2007; Hemmerich et al, 2008; Dunleavy et al, 2009; Mellone et al, 2011; Lidsky et al, 2013). To ensure stable centromere maintenance, CENP-A nucleosomes must return to their original levels through active CENP-A deposition. The timing of CENP-A deposition varies among species; however, the underlying mechanisms appear to share significant similarity (Zasadzinska & Foltz, 2017). A central player in this process is the CENP-A-specific chaperone HJURP in human and its homologue Scm3 in fungi (Kato et al, 2007; Foltz et al, 2009; Pidoux et al, 2009; Sanchez-Pulido et al, 2009; Dunleavy et al, 2011). Both HJURP and Scm3 can bind the CENP-A–histone H4 (CENP-A/H4) heterodimer in its pre-nucleosomal form, and these complexes are then targeted to centromeres by the Mis18 complex (Fujita et al, 2007; Moree et al, 2011; Dambacher et al, 2012; Hayashi et al, 2014; McKinley & Cheeseman, 2014; Nardi et al, 2016; Stellfox et al, 2016; French et al, 2017; Hori et al, 2017). While the human Mis18 complex is composed of Mis18α, Mis18β and Mis18BP1, the fission yeast Mis18 complex consists of Mis18, Mis16, Eic1 and Eic2, where Eic1 and Eic2 are proposed to be functional equivalents of human Mis18BP1 (Fujita et al, 2007; Hayashi et al, 2014; Subramanian et al, 2014). The timing of Mis18 complex assembly, its centromere targeting, and subsequent CENP-A deposition are suggested to be tightly controlled by the kinase activities of CDK and Plk1 (Silva et al, 2012; McKinley & Cheeseman, 2014; Stankovic et al, 2017; French & Straight, 2019). While we know the identity of key players involved in centromere maintenance, molecular and mechanistic understanding of their intermolecular cooperation are just emerging (Nardi et al, 2016; Stellfox et al, 2016; Pan et al, 2017; Spiller et al, 2017). Strikingly, Drosophila species have regional centromeres defined by the presence of CENP-A (also called CID in this organism), but lack clear homologues of HJURP and the subunits of the Mis18 complex. Instead, fly-specific CAL1 appears to combine the roles of both HJURP and the Mis18 complex: pre-nucleosomal CENP-A recognition and its targeting to the centromere for deposition, respectively (Phansalkar et al, 2012). Targeting CAL1 to non-centromeric DNA in Drosophila cells can recruit CENP-A and establish centromeres capable of assembling kinetochore proteins and microtubule attachments (Chen et al, 2014). These observations and the ability of CAL1 to bind CENP-A/H4 and CENP-C with its N- and C-terminal regions, respectively, collectively established CAL1 as a “self-sufficient” CENP-A-specific assembly factor in Drosophila (Schittenhelm et al, 2010; Chen et al, 2014). However, structure-level mechanistic understanding of how CAL1 binds CENP-A/H4 and CENP-C to facilitate the establishment and maintenance of centromeres is yet to be determined. The simplistic nature of the centromere maintenance pathway in Drosophila makes it a unique model system to understand the fundamentally conserved structural principles underlying centromere maintenance. In this study, we present the structural basis for the recognition of CENP-A/H4 and CENP-C by CAL1. Our analysis reveals that although CAL1 does not share noticeable sequence similarity with its human or fission yeast counterpart, it recognises CENP-A/H4 using both conserved and adaptive structural principles. We also provide the structural framework of interactions responsible for CENP-C recognition by CAL1. Our structural analysis, together with validation of structure-guided mutants in vitro and in cells, provides the molecular basis for the mechanism by which CAL1 single-handedly recognises and targets CENP-A to centromeres to maintain centromere identity in flies. Results The N-terminal region of CAL1 forms a heterotrimer with the histone fold domain of CENP-A and H4 Secondary structure prediction analysis indicated that CAL1 is likely to be a predominantly unstructured protein, although it includes an N-terminal domain spanning amino acid (aa) residues 1–200 predicted to fold into α helices (Fig EV1A and B). With the aim of structurally characterising the intermolecular interactions responsible for CAL1 binding to CENP-A/H4, we reconstituted a protein complex containing the N-terminal 160 aa of CAL1, a putative histone fold domain of CENP-A and H4 (His-CAL11–160–CENP-A101–225–H4) (Fig 1A) using recombinant proteins as previously reported (Chen et al, 2014). Limited proteolysis experiments performed on CAL11–160–CENP-A101–225–H4 complex using different proteases suggested that a CENP-A fragment containing aa 144–255 (CENP-A144–255) is sufficient to interact with CAL1 and H4. Subsequently, using CAL11–160, CENP-A144–255 and H4, we reconstituted a truncated protein complex (His-CAL11–160–CENP-A144–225–H4). The molecular weights (MW) measured for His-CAL11–160–CENP-A101–225–H4 and His-CAL11–160–CENP-A144–225–H4 using size-exclusion chromatography combined multi-angle light scattering (SEC-MALS) are 47.0 ± 0.9 and 43.4 ± 0.8 kDa, respectively (Fig EV1C). These values match with calculated MW for a 1:1:1 heterotrimeric assembly for both complexes (46.7 and 41.7 kDa, respectively) and are in agreement with our previous report (Roure et al, 2019). This observation is also in agreement with the subunit stoichiometry of the human pre-nucleosomal CENP-A/H4 in complex with HJURP (Hu et al, 2011). Click here to expand this figure. Figure EV1. CAL1 is predicted to be predominantly unstructured Disordered regions of CAL1 as predicted by Disopred (http://bioinf.cs.ucl.ac.uk/psipred). Residue number on x-axis and probability of disorder on y-axis. Secondary structure composition and multiple sequence alignment of CAL1 N-terminus as performed using Psipred (http://bioinf.cs.ucl.ac.uk/psipred) and MUSCLE (Madeira et al, 2019). Numbers correspond to Drosophila melanogaster. Drosophila melanogaster (D. mel), Drosophila grimshawi (D. gri), Drosophila mojavensis (D. moj), Drosophila virilis (D. vir), Drosophila persimilis (D. per), Drosophila pseudoobscura pseudoobscura (D. pse), Drosophila ananassae (D. ana), Drosophila erecta (D. ere), Drosophila yakuba (D. yak) and Drosophila simulans (D. sim). SEC-MALS analysis of His-CAL11–160–CENP-A101–225–H4 and His-CAL11–160–CENP-A144–225–H4. Absorption at 280 nm (mAU, left y-axis) and molecular mass (kDa, right y-axis) are plotted against elution volume (ml, x-axis). Measured MW and the calculated subunit stoichiometry based on the predicted MW of different subunit compositions. Samples were analysed using Superdex 200 increase 10/300 in 50 mM HEPES pH 8.0, 2 M NaCl and 1 mM TCEP. Download figure Download PowerPoint Figure 1. N-terminal 160 amino acids of CAL1 wrap around CENP-A/H4 heterodimer to form a heterotrimeric assembly Schematic representation of structural features of CAL1, CENP-A and H4. Filled boxes represent folded domains. Overall structure of His-CAL11–160–CENP-A101–225–H4 (crystal form I). CAL1 is shown in blue, CENP-A in maroon and H4 in green. Overall structure of His-CAL11–160–CENP-A144–225–H4 (crystal form II). CAL1 is shown in blue, CENP-A in maroon and H4 in green. Download figure Download PowerPoint Structure determination of the CAL11–160–CENP-A/H4 complex Extensive crystallisation trials with CAL11–160–CENP-A101–225–H4 and CAL11–160–CENP-A144–225–H4 yielded two different crystal forms: form I that diffracted X-rays to about 3.5 Å and form II that diffracted anisotropically to about 4.4 Å (Table 1). Molecular replacement was performed for the dataset collected from form I using the coordinates of Drosophila melanogaster (dm) H3/H4 heterodimer (deduced from the structure of dm nucleosome core particle, PDB: 2PYO) (Clapier et al, 2008). Molecular replacement solution yielded initial phases sufficient for subsequent rounds of model building and refinement (Fig EV2A). The final model included residues 17–47 of CAL1, 147–220 of CENP-A and 27–98 of H4 and was refined to an R factor 27.2% and Rfree factor 28.6% (Fig 1B and Table 1). Although we used a CAL1 fragment spanning residues 1–160 in the crystallisation experiment, the calculated electron density map accounted only for CAL1 residues 17–47. Considering these crystals took more than a year to form, we concluded that CAL1 was proteolytically cleaved, which may have facilitated the crystallisation of a truncated complex. Table 1. Data collection and refinement statistics CAL11–160–CENP-A/H4 Form I CAL11–160–CENP-A/H4 Form II CENP-C1264–1411 CAL1841–979–CENP-C1264–1411 Data collection Space group R 3 2 :H P 63 2 2 P 41 21 2 P 21 21 21 Cell dimensions a, b, c (Å) 178.25, 178.25, 133.28 199.70, 199.70, 76.75 57.20, 57.20, 92.96 86.27, 86.44, 88.46 α, β, γ (°) 90, 90, 120 90, 90, 120 90, 90, 90 90, 90, 90 Wavelength 0.97625 0.91587 0.97623 0.97625 Resolution (Å) 66.79–3.47 (3.59–3.47) 172.9–4.38 (5.19–4.38) 28.6–1.82 (1.88–1.82) 28.9–2.27 (2.35–2.27) R merge 0.147 (1.96) 0.129 (0.95) 0.0548 (0.598) 0.076 (0.663) R pim 0.058 (0.759) 0.046 (0.526) 0.013 (0.177) 0.022 (0.202) I/σI 10.46 (1.15) 9.61 (2.4) 34.13 (3.55) 21.16 (3.40) Completeness (%) 99.93 (100.00) 89 (81)a 99.73 (97.87) 98.41 (94.61) Redundancy 7.6(7.7) 8.9 (8.8) 18.0 (11.9) 13.1 (11.3) Refinement No. of reflections 80,934 (8,111) 20,689 (307) 14,426 (1,381) 404,275 (33,273) Rwork (%)/Rfree (%) 27.2/28.6 30.6/32.3 19.4/23.5 23.7/26.6 No. of atoms Protein 2,803 1,715 1,065 4,229 Average B Protein 136.6 193 40.8 71.1 R.m.s deviations Bond length (Å) 0.004 0.005 0.006 0.006 Bond angles (°) 0.87 1.2 085 1.20 Ramachandran values Favoured (%) 90.3 89.7 97.79 97.1 Disallowed (%) 1.2 1.7 0.00 0.19 Statistics for the highest-resolution shell are shown in parentheses. a Ellipsoidal completeness (%) from STARANISO (see also Materials and Methods). Click here to expand this figure. Figure EV2. Stereo images of the electron density maps and corresponding final models of CAL1–CENP-A/H4 from crystal form I and II 2Fo–Fc electron density map contoured at 1 sigma for the vicinity of CAL1 residues W22 and F29 for crystal form I. CENP-A in maroon and H4 in green. 2Fo–Fc electron density map contoured at 1 sigma for CAL1 bound to CENP-A/H4 for crystal form II. CAL1 is shown in blue, CENP-A in maroon and H4 in green. Download figure Download PowerPoint The refined model obtained using crystal form I was used as a template in molecular replacement to determine the structure of crystal form II (Figs 1C and EV2B). The difference electron density map calculated using the molecular replacement solution revealed unambiguous density for most main chain atoms of CAL11–160. Considering the modest resolution of the structure, intermolecular interactions stabilising the CAL1–CENP-A/H4 complex were further analysed using chemical cross-linking mass spectrometry (CLMS). Purified recombinant CAL11–160–CENP-A101–225–H4 complex was cross-linked using EDC (solid lines), a zero-length cross-linker that covalently links carboxylate groups of Asp or Glu residues with primary amines of Lys and N-terminus, or hydroxyl group of Ser, Thr and Tyr, or BS3 (dashed lines), a cross-linker that covalently links amine to amine or hydroxyl group of Ser, Thr and Tyr. The cross-linked peptides were analysed by mass spectrometry to identify intra- and intermolecular contacts (Fig EV3). Notably, the data revealed intramolecular cross-links between the N- and C-terminal regions of CAL11–160, particularly between Ser19 and Lys20 and Glu139 and Glu155, suggesting a direct interaction between these regions (Fig EV3). This information was particularly helpful in tracing the backbone atoms of residues beyond CAL1 residue 47 within the electron density map. Click here to expand this figure. Figure EV3. Intra- and intermolecular contacts identified between CAL1 and CENP-A/H4 using cross-linking mass spectrometry (CLMS) SDS–PAGE analysis of His-CAL11–160-CENP-A101–225-H4 cross-linked with both EDC (left) and BS3 (right) cross-linker. Linkage map showing the sequence position and cross-linked residue pairs between His-CAL11–160, CENP-A101–225 and H4. Cross-linked samples were resolved with SDS–PAGE and then analysed by MS. CAL1 is shown in blue, CENP-A in maroon and H4 in green. Solid lines represent EDC cross-links, while dashed lines show BS3 cross-links. High-resolution representative fragmentation spectra displayed using XiSpec (Ref: PMID: 29741719) for cross-linked peptides seen between CAL11–160 and CAL11–160, CAL11–160 and CENP-A101–225 and CAL11–160 and H4. Download figure Download PowerPoint Overall structure of CENP-A/H4 assembly The structures obtained from two different crystal forms together provide key insights into the overall architecture of the assembly (Fig 1B and C). Structural superposition analysis showed that CENP-A/H4 heterodimer (form I) aligns well with H3/H4 heterodimer (PDB: 2PYO) with a root mean square deviation (RMSD) of 1 Å (Fig EV4A). This suggests that both H3 and CENP-A use an identical mode of H4 binding. However, CENP-A α1, H4 α3 and C-terminal tail show conformational variations in the CAL1-bound CENP-A/H4 complex, likely due to CAL1 binding (Fig EV4B). Particularly, in the H3/H4 structure, the C-terminal tail of H4 folds back and makes contacts with the H3 α3, resembling CAL1 interaction at the equivalent region of CENP-A in the CAL1/CENP-A/H4 structure. The H4 C-terminal tail possibly swings away from this site upon CAL1 binding. Overall structure of dm CENP-A/H4 (form I) is very similar to human CENP-A/H4 (PDB: 3NQJ) (Sekulic et al, 2010) with a RMSD of 1 Å (Fig EV4C). However, noticeable conformational variation is seen in loop L1, possibly to accommodate the amino acid variations between HJURP and CAL1 (Fig EV4C). Click here to expand this figure. Figure EV4. Mode of H4 recognition by CENP-A by CAL1 Structural superposition of CENP-A/H4 with H3/H4 (Clapier et al, 2008). CENP-A is shown in maroon, H4 in green and H3/H4 in silver. Arrows indicate conformational changes. Structural superposition of CAL1–CENP-A/H4 (form I) with H3/H4 (Clapier et al, 2008). CAL1 is shown in blue, CENP-A in maroon, H4 in green and H3/H4 in silver. Arrows indicate conformational changes; dotted arrow highlights conformational changes in the loop regions. Structural superposition of hs CENP-A/H4 (Sekulic et al, 2010) with dm CENP-A/H4. dm CENP-A in maroon, dm H4 in green and hs CENP-A/H4 in silver. Dotted arrow highlights conformational changes in the loop regions. (left panel) Quantifications of Ni-NTA pull-down of His-CAL11–160 WT and indicated mutants with CENP-A101–225–H4. Bar graph shows average ratio of the band intensities between CENP-A101–225/H4 and His-CAL11–160 (n = 7 experiments). (right panel) Quantifications of Ni-NTA pull-down of His-CAL11–160 WT with CENP-A101–225–H4 and indicated mutants. Bar graph shows average ratio of the band intensities between CENP-A101–225/H4 and His-CAL11–160 (n = 4 experiments). Data information: In (D), (left panel) data presented as mean ± SD of 7 experiments, P-values were calculated using a Mann–Whitney test. (right panel) Data presented as mean ± SD of 4 experiments, P-values were calculated using unpaired two-tailed t-test. (*P < 0.05, ***P < 0.001, ****P < 0.0001). Download figure Download PowerPoint CAL1 binds CENP-A/H4 heterodimers through multiple physical contacts CAL11–160 is almost entirely made of α helices that make multiple contacts with CENP-A/H4 heterodimer by wrapping around it (Figs 1C and 2A). Most CENP-A contacts are made by CAL1 helices α1 and α2 and loop L1, which interact with the CENP-A helices α2, α1 and loop L1, respectively, involving a total interface area of about 940 Å2. Particularly, while the N-terminal half of the CAL1 α1 helix packs against CENP-A α2 involving electrostatic (CAL1 R18 with CENP-A Q90) and hydrophobic (involving CAL1 L11 and M14) interactions, the C-terminal half, mainly aa W22 and F29, is sandwiched between CENP-A α2 and H4 α3 (Fig 2A). CAL1 L1 crosses over CENP-A L1 to facilitate CAL1 α2 interaction with CENP-A α3. In addition, CAL1 α4 contacts both CENP-A α2 and α3 involving an interface area of about 80 Å2. These CAL1–CENP-A interactions appear to be further stabilised by CAL1 α5 and α6 which together with CAL1 α1 make an intramolecular helical bundle resembling a latch that restrains the position of α1 helix (Fig 1C). Figure 2. Hydrophobic interactions between CAL1 α1 and CENP-A α2 are critical for CENP-A/H4 binding in vitro Crystal structure of His-CAL11–160–CENP-A144–225–H4 highlighting key residues involved in interaction. CAL1 is shown in blue, CENP-A in maroon and H4 in green. Multiple sequence alignment performed with MUSCLE (Madeira et al, 2019) showing conservation of CAL1 homologues in different fly species. Numbers correspond to Drosophila melanogaster. Drosophila melanogaster (D. mel), Drosophila grimshawi (D. gri), Drosophila mojavensis (D. moj), Drosophila virilis (D. vir), Drosophila willistoni (D. wil), Drosophila persimilis (D. per), Drosophila pseudoobscura pseudoobscura (D. pse), Drosophila ananassae (D. ana), Drosophila erecta (D. ere), Drosophila yakuba (D. yak) and Drosophila simulans (D. sim). SEC-MALS of His-CAL11–50–CENP-A101–225–H4. Absorption at 280 nm (mAU, left y-axis) and molecular mass (kDa, right y-axis) are plotted against elution volume (ml, x-axis). Measured molecular weight (MW) and the calculated subunit stoichiometry based on the predicted MW of different subunit compositions. Samples were analysed using a Superdex 200 increase in 50 mM HEPES pH8.0, 2 M NaCl and 1 mM TCEP. Ni-NTA pull-down of His-CAL11–160 WT and indicated mutants with CENP-A101–225–H4. SDS–PAGE shows input and protein bound to beads. Quantifications shown in Fig EV4D. Download figure Download PowerPoint Hydrophobic interactions involving CAL1 W22 and F29 are critical for CENP-A/H4 binding Considering the extent of contacts made by the N-terminal 50 aa of CAL1, we checked whether CAL11–50 is sufficient to interact with CENP-A/H4. Using recombinant His-CAL11–50, H4 and CENP-A101–225, we confirmed complex formation (Fig 2B). Further characterisation using SEC-MALS showed that CAL11–50–CENP-A101–225–H4 is a 1:1:1 complex with a measured MW of 39.6 ± 0.7 kDa (calculated MW 34.1 kDa) (Fig 2B). Within CAL1, the conserved residues in α1: W22 and F29, and in α2: F43 are completely buried in the complex, so we hypothesised that these interactions are crucial for CENP-A/H4 binding (Fig 2A). To test this, we produced recombinant His-CAL11–160 carrying either F43R, W22A, F29A, W22R, F29R, W22A/F29A or W22R/F29R mutations and tested their ability to interact with CENP-A/H4 complex in a nickel-NTA pull-down assay. His-CAL11–160 was mixed with molar excess of CENP-A/H4 complex. His-CAL11–160, and any proteins bound to it, was captured with nickel-NTA resin and subsequently analysed by SDS–PAGE. While the F43R mutation had small effect on CAL11–160 binding, the W22R and W22/F29 double mutations significantly reduced the ability of CAL11–160 to capture CENP-A/H4 compared with the WT protein (Figs 2C and EV4D left panel). To validate the requirement of these interactions in cells, we expressed CENP-A-GFP-LacI in U2OS cells containing a synthetic array with a LacO sequence integrated in a chromosome arm (Janicki et al, 2004) and analysed its ability to recruit CAL1-V5 (Roure et al, 2019). When CENP-A-GFP-LacI was tethered to the LacO site, CAL1WT was efficiently recruited (Fig 3A). Consistent with our in vitro binding assay, CENP-A-GFP-LacI recruited CAL1F43R threefold less efficiently when compared to CAL1WT. CAL1W22/F29A and CAL1W22/F29R showed an even stronger reduction in their ability to associate with CENP-A (Fig 3A). Figure 3. Hydrophobic interactions between CAL1 α1 and CENP-A α2 are critical for CENP-A/H4 binding in cells Representative fluorescence images and quantification of tethering assays. U2OS cells containing a LacO array were co-transfected with CENP-A-GFP-LacI with CAL1WT-V5 and also with CAL1-V5 carrying point mutations. Scale bar: 10 μm (n = 2 experiments). Representative fluorescence images and quantification of in vivo tethering assays. Drosophila Schneider S2 cells containing a LacO array were co-transfected with CENP-A-GFP-LacI with CAL1WT-V5 and also with CAL1-V5 carrying point mutations. Arrows point to the LacO site. Scale bar: is 5 μm (n = 3 experiments). Data information: In (A), data presented as mean ± SEM of 2 experiments, n ≥ 20 cells per experiment. P-values were calculated using a Mann–Whitney test. In (B), data presented as mean ± SEM of 3 experiments, n ≥ 45 cells per experiment, P-values were calculated using a Mann–Whitney test (****P < 0.0001). Download figure Download PowerPoint We also tested the recruitment of CAL1-V5 WT and mutants by co-transfecting them with CENP-A-GFP-LacI into a physiologically related Drosophila Schneider S2 cells containing a LacO array (Fig 3B). In agreement with the interaction studies in U2OS cells, association of CAL1F43R and CAL1W22/F29R with CENP-A was significantly reduced at the tethering site (Fig 3B). Overall, in vitro binding assays together with interaction studies in cells indicate that the interactions mediated by W22 and F29 are crucial for the recognition of CENP-A/H4 by CAL1. CAL1 uses conserved and adaptive interactions to recognise Drosophila CENP-A/H4 Structural superposition of CAL1–CENP-A/H4 onto its respective human and Kluyveromyces lactis structures, HJURP–CENP-A/H4 (PDB: 3R45) (Hu et al, 2011) and Scm3–CENP-A/H4 (PDB: 2YFV) (Cho & Harrison, 2011), showed that CAL1 employs a broadly similar mode of CENP-A recognition with a few striking differences (Fig 4A). All CENP-A chaperones compared here use their α1 helix to interact with α2 of CENP-A in an anti-parallel fashion, occluding the tetramerisation of CENP-A/H4 heterodimers. However, in CAL1 the upstream segment of α1 swings away from CENP-A as compared with its counterpart in HJURP and Scm3. Structural superposition-based sequence alignments showed a key amino acid variation in dm CENP-A at position 186 as compared with human and yeast CENP-A: Ala is replaced with Met, an amino acid with a long side chain, which appears to push CAL1 α1 away from it (Fig 4B). This apparent weakening of CAL1 α1–CENP-A α2 interaction is likely to be compensated by CAL1 α5 and α6 which together restrains the position of α1 helix by forming a helical bundle. Our efforts to measure the binding strengths of CAL1 with (CAL11–160) and without α helical elements (CAL11–50) to bind CENP-A/H4 di
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