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• W2894880237 abstract "Scientific Report12 March 2019Open Access Transparent process Structural basis of HEAT-kleisin interactions in the human condensin I subcomplex Kodai Hara Corresponding Author Kodai Hara [email protected] orcid.org/0000-0002-1586-6312 Department of Physical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan Search for more papers by this author Kazuhisa Kinoshita Kazuhisa Kinoshita Chromosome Dynamics Laboratory, RIKEN, Wako, Saitama, Japan Search for more papers by this author Tomoko Migita Tomoko Migita Department of Physical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan Search for more papers by this author Kei Murakami Kei Murakami Department of Physical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan Search for more papers by this author Kenichiro Shimizu Kenichiro Shimizu Department of Physical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan Search for more papers by this author Kozo Takeuchi Kozo Takeuchi Chromosome Dynamics Laboratory, RIKEN, Wako, Saitama, Japan Search for more papers by this author Tatsuya Hirano Corresponding Author Tatsuya Hirano [email protected] orcid.org/0000-0002-4219-6473 Chromosome Dynamics Laboratory, RIKEN, Wako, Saitama, Japan Search for more papers by this author Hiroshi Hashimoto Hiroshi Hashimoto orcid.org/0000-0003-1503-6789 Department of Physical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan Search for more papers by this author Kodai Hara Corresponding Author Kodai Hara [email protected] orcid.org/0000-0002-1586-6312 Department of Physical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan Search for more papers by this author Kazuhisa Kinoshita Kazuhisa Kinoshita Chromosome Dynamics Laboratory, RIKEN, Wako, Saitama, Japan Search for more papers by this author Tomoko Migita Tomoko Migita Department of Physical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan Search for more papers by this author Kei Murakami Kei Murakami Department of Physical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan Search for more papers by this author Kenichiro Shimizu Kenichiro Shimizu Department of Physical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan Search for more papers by this author Kozo Takeuchi Kozo Takeuchi Chromosome Dynamics Laboratory, RIKEN, Wako, Saitama, Japan Search for more papers by this author Tatsuya Hirano Corresponding Author Tatsuya Hirano [email protected] orcid.org/0000-0002-4219-6473 Chromosome Dynamics Laboratory, RIKEN, Wako, Saitama, Japan Search for more papers by this author Hiroshi Hashimoto Hiroshi Hashimoto orcid.org/0000-0003-1503-6789 Department of Physical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan Search for more papers by this author Author Information Kodai Hara *,1, Kazuhisa Kinoshita2, Tomoko Migita1, Kei Murakami1, Kenichiro Shimizu1, Kozo Takeuchi2,3, Tatsuya Hirano *,2 and Hiroshi Hashimoto1 1Department of Physical Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan 2Chromosome Dynamics Laboratory, RIKEN, Wako, Saitama, Japan 3Present address: Hamamatsu Photonics K. K., Hamamatsu, Shizuoka, Japan *Corresponding author. Tel: +81 54 264 5646; Fax: +81 54 264 5644; E-mail: [email protected] *Corresponding author. Tel: +81 48 467 9531; Fax: +81 48 462 4673; E-mail: [email protected] EMBO Reports (2019)20:e47183https://doi.org/10.15252/embr.201847183 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 Condensin I is a multi-protein complex that plays an essential role in mitotic chromosome assembly and segregation in eukaryotes. It is composed of five subunits: two SMC (SMC2 and SMC4), a kleisin (CAP-H), and two HEAT-repeat (CAP-D2 and CAP-G) subunits. Although balancing acts of the two HEAT-repeat subunits have been demonstrated to enable this complex to support the dynamic assembly of chromosomal axes in vertebrate cells, its underlying mechanisms remain poorly understood. Here, we report the crystal structure of a human condensin I subcomplex comprising hCAP-G and hCAP-H. hCAP-H binds to the concave surfaces of a harp-shaped HEAT-repeat domain of hCAP-G. Physical interaction between hCAP-G and hCAP-H is indeed essential for mitotic chromosome assembly recapitulated in Xenopus egg cell-free extracts. Furthermore, this study reveals that the human CAP-G-H subcomplex has the ability to interact with not only double-stranded DNA, but also single-stranded DNA, suggesting functional divergence of the vertebrate condensin I complex in proper mitotic chromosome assembly. Synopsis Condensin I has a central role in mitotic chromosome assembly and segregation. The crystal structure of a human condensin I subcomplex reveals that the interaction between hCAP-G and hCAP-H is essential for mitotic chromosome assembly and DNA binding. The crystal structure of the hCAP-G-H condensin I subcomplex shows an “open” conformation. The interaction between hCAP-G and hCAP-H is required for proper assembly of mitotic chromosomes. hCAP-G-H interacts not only with double-stranded DNA, but also single-stranded DNA. Introduction Immediately before cell division, chromatin that resides in the nucleus is converted into a set of rod-shaped structures to support their faithful segregation into daughter cells. The condensin complexes play a central role in this process, known as mitotic chromosome assembly or condensation, and also participate in diverse chromosome functions such as gene regulation, recombination, and repair 1, 2. Moreover, hypomorphic mutations in the genes encoding condensin subunits have been implicated in the human disease microcephaly 3. Many eukaryotes have two different types of condensin complexes, namely, condensins I and II. Condensin I, for example, consists of a pair of structural maintenance of chromosomes (SMC) ATPase subunits (SMC2 and SMC4) and three non-SMC regulatory subunits (CAP-D2, CAP-G, and CAP-H). SMC2 and SMC4 dimerize through their hinge domains to form a V-shaped heterodimer, and CAP-H, which belongs to the kleisin family of proteins, bridges SMC head domains through its C- and N-terminal regions. CAP-D2 and CAP-G, both of which are composed of arrays of short amphiphilic helices known as HEAT repeats, bind to the central region of CAP-H 4, 5. Although many if not all prokaryotic species have a primitive type of condensin composed of an SMC homodimer and two other regulatory subunits including a kleisin subunit, the HEAT-repeat subunits are unique to eukaryotic condensins and not found in prokaryotic condensins. Biochemical studies using purified condensin I holocomplexes identified several ATP-dependent activities in vitro, such as positive supercoiling of DNA 6-8, DNA compaction 9, translocation along dsDNA 10, and DNA loop extrusion 11. Mechanistically, how these activities are supported by condensin I remains poorly understood. Indeed, condensin I can interact with DNA in many different ways. For instance, like cohesin and prokaryotic SMC complexes, it encircles double-stranded DNA (dsDNA) within its tripartite ring composed of the SMC dimer and kleisin 12-14. It has also been reported that a mouse SMC2-SMC4 hinge domain binds single-stranded DNA (ssDNA), but not dsDNA 15, whereas a budding yeast non-SMC subcomplex composed of YCG1/CAP-G, YCS4/CAP-D2, and BRN1/CAP-H binds dsDNA, but not ssDNA 16. A recent study reported the crystal structure of a budding yeast non-SMC subcomplex consisting of YCG1 and BRN1 bound to dsDNA 17. Another study using Xenopus egg cell-free extracts found that the pair of HEAT-repeat subunits plays an essential role in the dynamic assembly of mitotic chromosome axes 18. In the current study, we determined the crystal structure of a human subcomplex composed of CAP-G bound by a short fragment of CAP-H. The structure established molecular interactions between human CAP-G and CAP-H, and implicated these interactions in the ability of condensin I to support mitotic chromosome assembly. Furthermore, the human CAP-G-H subcomplex bound both dsDNA and ssDNA, suggesting the functional divergence of the eukaryotic condensin I complex. Results and Discussion Structure of the human CAP-G-H subcomplex The consensus sequence of HEAT repeats at the primary structure level is not tight. The original report by Neuwald and Hirano 19 assigned nine HEAT repeats in vertebrate CAP-G, whereas a subsequent re-assignment by Yoshimura and Hirano 5 identified 19 HEAT repeats that span the near-entire length of human CAP-G (hCAP-G). Furthermore, the secondary structural prediction server PrDOS 20 predicted that hCAP-G has two long disordered regions (amino acid residues 477–553 and 896–1,015) and five short disordered regions (residues 1–12, 81–93, 382–393, 660–687, and 812–821) (Fig 1A, upper). On the other hand, human CAP-H (hCAP-H) has five regions that are conserved among its orthologs among eukaryotic species (motifs I-V) (Fig 1A, lower). A previous biochemical study revealed that the N-terminal and C-terminal halves of hCAP-H bind to hCAP-D2 and hCAP-G, respectively 4. As the most C-terminally located motif V was predicted to bind to SMC2 21, we thought that motif IV (residues 461–503) may be responsible for binding to hCAP-G. With this information, we aimed to express and purify hCAP-G complexed with a fragment of hCAP-H. We found that the N-terminal domain of hCAP-G (residues 1–478) connected to the C-terminal domain of hCAP-G (residues 554–900), and a fragment of hCAP-H containing motif IV (residues 460–515) was able to be co-expressed and co-purified (Fig 1B). This hCAP-G-H subcomplex was successfully crystalized and its structure was determined at 3.0 Å resolution (Table 1). Two molecules of the hCAP-G-H subcomplex are present in the crystallographic asymmetric unit (Fig EV2A). Their structures are essentially identical, but 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) is bound to only one of the two molecules. In the current report, we describe the HEPES-bound hCAP-G-H subcomplex (a, b-molecules) as a representative structure (Fig 1C). Consistent with the recent assignment based on its amino acid sequence 5, hCAP-G displays a “harp-shaped” structure composed of 19 HEAT repeats (H1-H19), in which H12 and H15 have long disordered loops (residues 479–553 and 661–691, respectively) (Figs 1C and EV1A and EV2B). hCAP-H, which comprises three α-helices (α2, α3′, and α4), binds to the concave surfaces of hCAP-G (Figs 1C and EV1B). This overall structure in which a kleisin fragment binds to the concave surfaces of a harp-shaped HEAT-repeat domain is highly reminiscent of other cohesin subunits and its regulators 22-24, as well as budding and fission yeast condensin subunits (YCG1-BRN1 and CND3/CAP-G-CND2/CAP-H) 17. It should be noted that the hCAP-G used in this study shares only 16 and 21% amino acid identity with YCG1 and CND3, respectively, and that the hCAP-H fragment bound to hCAP-G shares only 25 and 29% identity with BRN1 and CND2, respectively. Although there is great divergence in their amino acid sequences, two basic residues (K60 and R848) located at the N- and C-terminal lobes of hCAP-G, which correspond to DNA-binding residues K70 (YC1) and R849 (YC2) of YCG1, respectively, are structurally well conserved (Fig EV1A). Similarly, four basic residues (R435, R437, K456, and K457) of hCAP-H, which correspond to K409 (BC1), R411 (BC1), K456 (BC2), and K457 (BC2) of BRN1, respectively, are also conserved, but we were unable to visualize these residues because they were not included in the crystallized recombinant protein (Fig EV1B). Kschonsak et al 17 recently demonstrated that the corresponding amino acid residues of YCG1-BRN1 function in dsDNA binding, and proposed a “safety-belt mode” by which a peptide loop produced by two regions of BRN1, namely a latch and buckle, encircles the bound DNA and prevents its dissociation. The previous study strongly suggests that the hCAP-G-H subcomplex also uses these residues to bind to dsDNA (see below). Figure 1. Domain architecture and overall structure of the hCAP-G-H subcomplex hCAP-G is composed of 1,015 amino acids and contains 19 HEAT repeats. hCAP-H is composed of 730 amino acids and contains 5 conserved motifs (I: hSMC2 binding region, II: hCAP-D2 binding region, III: DNA-binding region, IV: hCAP-G binding region, V: hSMC4 binding region). Scheme of the hCAP-G-H subcomplex. Residues 1–478 of hCAP-G were fused to residues 554–900 of hCAP-G. hCAP-G (1–478, 554–900) was co-expressed in E. coli and co-purified for crystallography. Cartoon diagram of the crystal structure of hCAP-G (orange) in complex with a fragment of hCAP-H (green). Unstructured, disordered regions are indicated by the dots. The 19 HEAT repeats (H1-H19) and 2 disordered loops (H12 loop and H15 loop) of hCAP-G, and 4 helices (α2, α3′, and α4) of hCAP-H are labeled. The N- and C-termini of CAP-G and CAP-H are also indicated. A molecule of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) is shown by the orange-colored stick model. A 90-degree rotated version is shown on the right. Comparison of the hCAP-G-H subcomplex with its related structures. Superimposition of the structures of hCAP-G-H (orange), S. cerevisiae YCG1-BRN1 (PDB ID: 5OQQ; blue), and S. pombe CND3-CND2 (PDB ID: 5OQR; green) is presented as a Cα-tracing model. Comparison of the DNA-bound form with DNA-free forms. Superimposition of the structures of DNA-bound YCG1-BRN1 (PDB ID: 5OQN; red), hCAP-G-H (orange), YCG1-BRN1 (blue), and CND3-CND2 (green) is presented as in (D). Download figure Download PowerPoint Table 1. X-ray crystallography: data collection and refinement statistics Native Au (peak) Data collection Space group P21 P21 Cell dimensions a, b, c (Å) 122.4, 61.9, 130.9 122.5, 61.2, 131.3 α, β, γ (°) 90.0, 93.4, 90.0 90.0, 93.6, 90.0 Resolution (Å) 19.81–3.00 (3.12–3.00) 19.73–3.38 (3.58–3.38) No. total/unique reflections 132,620/39,503 364,483/27,655 Rmerge 0.070 (0.600) 0.161 (0.879) Rpim 0.045 (0.375) 0.046 (0.254) CC1/2 0.998 (0.675) 0.998 (0.806) I/σI 12.7 (2.0) 12.9 (3.3) Completeness (%) 98.8 (97.1) 99.4 (99.1) Redundancy 3.4 13.2 Refinement Rwork/Rfree 21.2/27.1 No. atoms Protein 12,341 Ligand 15 Water 6 B-factors Protein 74.80 Ligand 98.40 Water 51.10 R.m.s. deviations Bond lengths (Å) 0.002 Bond angles (°) 0.684 Values in parentheses are for the highest resolution shell. Click here to expand this figure. Figure EV1. Secondary structures and structure-based sequence alignment of human CAP-G and CAP-H Secondary structures and structure-based sequence alignment of human CAP-G (hCAP-G), Xenopus laevis CAP-G (XCAP-G), and Saccharomyces cerevisiae YCG1. The secondary structural elements of hCAP-G and YCG1 are drawn above and below the sequence alignments, respectively. Identical and homologous residues are shown on black and gray backgrounds, respectively. The colored circles indicate residues of hCAP-G that interact with hCAP-H (green) or bind to HEPES (red). Residues of YCG1 that interact with BRN1 and dsDNA are labeled with light blue and red, respectively. The YC1 and YC2 regions indicate residues essential for DNA binding defined by Kschonsak et al 17. Structure-based sequence alignment of hCAP-H, XCAP-H, and BRN1. The secondary structural elements of hCAP-H and BRN1 are drawn above and below the sequence alignments, respectively. Identical and homologous residues are shown on black and gray backgrounds, respectively. The colored circles indicate residues of hCAP-H that interact with hCAP-G (orange) and residues of BRN1 that interact with YCG1 (purple) or dsDNA (red). BC1, BC2, latch, and buckle regions defined by Kschonsak et al 17, and motifs III and IV of CAP-H are also shown in Fig 1A. Download figure Download PowerPoint We next performed superimpositions between the hCAP-G-H subcomplex and its budding/fission yeast counterparts, YCG1-BRN1/CND3-CND2, using PyMoL (http://www.pymol.org/). Structural alignments between hCAP-G-H and YCG1-BRN1/CND3-CND2 each had a root mean square deviation (RMSD) value of 4.293 and 5.272 Å for 3,990 and 4,049 superimposable atoms, respectively (Fig 1D, orange and blue or green), whereas the RMSD value between YCG1-BRN1 and CND3-CND2 was 3.145 Å for 8,075 superimposable atoms (Fig 1D, blue and green). These superimpositions revealed that the main chain structure of hCAP-G-H is different from that of its yeast counterpart, explaining why we were unable to determine the structure of hCAP-G-H by molecular replacement using its yeast counterpart structures. In addition, we performed superimpositions between DNA-bound YCG1-BRN1 and DNA-free forms. The RMSD value between the DNA-bound YCG1-BRN1 and hCAP-G-H was 5.316 Å for 4,126 superimposable atoms (Fig 1E, red and orange), whereas the RMSD value between DNA-bound YCG1-BRN1 and YCG1-BRN1/CND3-CND2 was 2.062 and 2.732 Å for 12,997 and 8,199 superimposable atoms, respectively (Fig 1E, red and blue or green). These superimpositions suggested that the overall structure of DNA-bound YCG1-BRN1 is identical to the structure of DNA-free YCG1-BRN1. There are several notable differences between the human and yeast structures on comparison of our structure with the previous one. First, some secondary structures of the hCAP-G-H subcomplex are different from those of the yeast counterpart. The H12 loop is a common disordered loop also found in the yeast counterpart, but the H15 disordered loop present in hCAP-G is missing in its yeast counterpart (Figs 1C and EV1A and EV2B). The hCAP-H sequence (residues 499–503), which corresponds to the buckle region of BRN1 (residues 498–504) and CND2 (residues 519–523), is also disordered in our hCAP-G-H structure (Fig EV1B). Notably, the corresponding α3 helix of BRN1 does not exist in hCAP-H. Instead, hCAP-H has the α3′ helices, producing a disordered loop that connects between the α2 and α3′ helices (Fig EV1B). Overall, the hCAP-G-H subcomplex is structurally more flexible than the YCG1-BRN1 subcomplex. Indeed, the b-factors of hCAP-G-H were higher than those of the yeast counterpart, especially the middle HEAT-repeat domain connecting the N- and C-terminal regions of hCAP-G (Fig EV2C). Second, hCAP-H is more loosely bound to hCAP-G than the yeast counterpart, resulting in the more opened conformation of the HEAT-repeat subunit hCAP-G. The distance between R257 and V754 of hCAP-G is 17.06 Å (Fig 2A), whereas the corresponding distance between R287 and F749 of YCG1 is 6.25 Å (Fig EV3A). The distance between K154 and K889 of hCAP-G is 35.29 Å (Fig 2A), whereas the corresponding distance between R170 and K895 of YCG1 is 23.02 Å (Fig EV3A). The previous electron microscopy study also demonstrated that the HEAT-repeat subunit of cohesin loader, Scc2, adopted several flexible conformations 25. These observations of the structural flexibility led us to speculate that our hCAP-G-H structure represents a snapshot of an “open conformation”, whereas the structure of its yeast counterpart represents a snapshot of a “closed conformation”. Click here to expand this figure. Figure EV2. Structure of the hCAP-G-H subcomplex Two molecules of the hCAP-G-H subcomplex in the asymmetric unit, shown by orange (hCAP-G; a) and green (hCAP-H; b), and blue (hCAP-G; c) and pink (hCAP-H; d) ribbon representations. The pink stick model indicates HEPES. Note that HEPES bound only one of the two hCAP-G molecules (a-molecule) present in the asymmetric unit. Schematic illustration of the structure and domain organization of hCAP-G. Two antiparallel helices (A and B helices) comprising each HEAT repeat are colored in orange and light orange, respectively. The binding sites of hCAP-H and DNA are indicated by the green and red double-headed arrows, respectively. The H12 loop (residues 479–553) connecting the H12A and H12B helices, and the H15 loop (residues 660–690) connecting the H15A and H15B helices are shown by black loops. Comparison of the b-factors of the hCAP-G-H subcomplex with its related structures. The structures of hCAP-G-H (left), S. cerevisiae YCG1-BRN1 (PDB ID: 5OQQ; middle), and S. pombe CND3-CND2 (PDB ID: 5OQR; right) are shown as a ribbon model colored by b-factor. The b-factors are shown in warm (high b-factors) to cool colors (low b-factors). Download figure Download PowerPoint Figure 2. Structural details of the interaction between hCAP-G and hCAP-H A. The molecular surface of hCAP-G is shown in orange. hCAP-H is shown as a green ribbon model. The four major contact sites (I, II, III, and IV) are boxed. B–E. Zoomed-in views of sites I–IV. Residues of hCAP-G and hCAP-H are labeled in white or black and green, respectively. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Structural details of the interaction between YCG1 and BRN1 A. The molecular surface of YCG1 is shown in purple. BRN1 is shown as a light blue ribbon model. The five major contact sites (I, II, III, III′, and IV) are boxed. The representative structure was generated to use the a- and c-molecules from the reported structure of the YCG1-BRN1 subcomplex (PDB ID: 5OQQ). B–F. Zoomed-in views of sites I–IV. Residues of YCG1 and BRN1 are labeled in white or black and light blue, respectively. Download figure Download PowerPoint Structural details of the interaction between hCAP-G and hCAP-H hCAP-H interacts extensively with the concave surface of hCAP-G at four major sites (Fig 2A), whereas YCG1 interacts with BRN1 at five major sites (Fig EV3A) 17. At site I, a pocket of hCAP-G accommodates I461 and F463 of hCAP-H in a hydrophobic manner (Fig 2B). F469, Y472, and F473 of hCAP-H make mainly hydrophobic interactions with hCAP-G. In the YCG1-BRN1 subcomplex, I461, F463, E471, V474, and F475 of BRN1 form conserved hydrophobic interactions with YCG1 (Fig EV3B). At site II, hCAP-H interacts with hCAP-G by van der Waals forces. K475, T476, A479, T480, and I481 of CAP-H are accommodated in a shallow pocket of CAP-G (Fig 2C). At site II of the YCG1-BRN1 subcomplex, some residues (K478, T481, K482, I483, D484, and M485) of BRN1 also interact with YCG1 by van der Waals interactions. In particular, I483 and M485 of BRN1 are accommodated in the corresponding pockets of YCG1 (Fig EV3C). The site II interactions in both hCAP-G-H and YCG1-BRN1 primarily involve hydrophobic interactions, but the depths of their interaction pockets are substantially different: hCAP-G recognizes the small side chain of hCAP-H, whereas YCG1 may recognize the bulky side chains of BRN1. At site III, T495 of hCAP-H is accommodated in a shallow pocket of hCAP-G (Fig 2D). A pocket of hCAP-G accommodates L497 of hCAP-H through van der Waals contacts. Notably, W492 of hCAP-H interacts with R493 on the same helix. This interaction may stabilize the binding of W492 of hCAP-H to E188 of hCAP-G mediated by van der Waals forces. At site III of the YCG1-BRN1 subcomplex, YCG1 interacts with BRN1 by van der Waals interactions. R490 of BRN1 interacts with Y168 of YCG1 and Y496 of BRN1 forms a hydrophobic interaction with P218 of YCG1 (Fig EV3D). K491, H495, and L497 of BRN1 are accommodated in an elongated cleft of YCG1. Site III of the YCG1-BRN1 subcomplex includes deeper clefts than that of hCAP-G, enabling YCG1 to bind bulky residues of BRN1. Differences in site III may explain why amino acid sequences between hCAP-H and BRN1 are not well conserved. At site IV, N504, V505, L508, and V509 of hCAP-H are accommodated in a pocket of hCAP-G (Fig 2E). Five residues (L511, H512, L513, K514, and P515) of hCAP-H are also accommodated in an elongated cleft of hCAP-G. I509, F513, and I514 of BRN1 corresponding to L508, H512, and L513 of hCAP-H also interact with YCG1 in a hydrophobic manner (Fig EV3F). At site IV, there are notable hydrogen bonds formed between H512 and L513 of hCAP-H and D647 of hCAP-G (Fig 3A). D647 is an acidic residue broadly conserved among the CAP-G/YCG1 orthologs (Fig EV1A). Of note, an aspartate side chain that makes hydrogen bonds with two backbone amides of residues in a pocket is commonly found in the prefusion state of hemagglutinin (HA) of the influenza virus 26, and a binding hotspot between the HEAT-repeat subunit SA2 of cohesin and kleisin subunit Scc1 22. Figure 3. Identification of residues required for interaction between hCAP-G and hCAP-H Zoomed-in view of site IV. Residues of hCAP-G and hCAP-H are shown in orange and green, respectively. The dashed red lines indicate hydrogen bonds. 3Q, 5Q, 3A, 5A, 2A, and Δ506–515 mutants of hCAP-H. Motif IV (residues 461–503) contains amino acid residues highly conserved among eukaryotic species (X, Xenopus laevis; Dr, Danio rerio; Cm, Cyanidioschyzon merolae; Sp, Schyzosaccharomyces pombe; Sc, Saccharomyces cerevisiae; Ec, Encephalitozoon cuniculi). To produce the IV-3Q, 5Q, 3A, 5A, and 2A mutants, the conserved aromatic amino acid residues (F463, F469, F473, F501, and Y503; labeled in dark blue) were substituted with glutamine (Q) or alanine (A) residues. The secondary structural element of hCAP-H is drawn below the sequence alignments. Interaction analysis between hCAP-G and hCAP-H. Bacterial cell lysates co-expressing hCAP-G (residues 1–478, 554–900) and hCAP-H (residues 394–515), either wild type (WT; lanes 2, 10 and 13), 3Q (F463Q, F469Q and F473Q; lane 3), 5Q (F463Q, F469Q, F473Q, F501Q and Y503Q; lane 4), 3A (F463A, F469A and F473A; lane 5), 5A (F463A, F469A, F473A, F501A and Y503A; lane 6), or 2A (F501A and Y503A; lane 11), or a C-terminal deletion mutant (506–514 residues were deleted from 394–515; lane 14) were applied to Ni-NTA agarose resin, and the bound fraction was analyzed by SDS–PAGE. Alternatively, a cell lysate co-expressing mutant hCAP-G (D647K) and wild-type hCAP-H was examined (lane 7). The uninduced cell lysate was also used as a negative control (lane 9). Download figure Download PowerPoint The YCG1-BRN1 subcomplex has an additional HEAT-kleisin interaction site, site III′ (Fig EV3E). At site III′, L498, P499, D501, F502, and F504 of BRN1 interact with YCG1 (Fig EV3E). Although no interactions corresponding to site III′ are found in the hCAP-G-H subcomplex, F501 and Y503 of hCAP-H corresponding to F502 and F504 of BRN1 are highly conserved among eukaryotic species. It is therefore possible that the hCAP-G-H subcomplex undergoes conformational changes (from an open form to a closed form), forming the site III′ interactions found in the YCG1-BRN1 subcomplex. Identification of residues required for interaction between hCAP-G and hCAP-H To identify residues required for interaction between hCAP-G and hCAP-H, we designed six mutants that targeted conserved, surface-exposed residues at the hCAP-G-H interface, and the amount of the hCAP-H fragment that co-purified with immobilized His6-tagged hCAP-G was evaluated (Fig 3B and C). As expected, three Gln substitutions (3Q) of F463, F469, and F473 of hCAP-H positioned at site I greatly impaired the interaction between hCAP-G and hCAP-H (Fig 3C, lanes 2 and 3). Three Ala substitutions (3A) of the same residues also diminished the interaction, suggesting that van der Waals interactions formed by these aromatic residues of CAP-H are essential for its interaction with hCAP-G (Fig 3C, lane 5). Moreover, Ala substitutions (2A) and additional Gln or Ala substitutions (5Q or 5A) of F501 and Y503 of hCAP-H positioned at site III′ did not further impair its interaction with hCAP-G (Fig 3C, lanes 4, 6, and 11). This suggests that F501 and Y503 are not directly involved in hCAP-G-H subcomplex formation, but may be required for the stabilization of a closed conformation after dsDNA binding. A Lys substitution of D647 of hCAP-G positioned at site IV (D647K) decreased its interaction with hCAP-H, suggesting that D647-mediated hydrogen bonds with H512 and L513 of hCAP-H are necessary for its interaction with hCAP-G at site IV (Fig 3A and C, lane 7). We also found that deletion of a C-terminal region of hCAP-H (506–514 residues) also reduced its interaction with hCAP-G (Fig 3C, lanes 13 and 14), supporting the idea that hydrophobic interactions formed by site IV are essential for hCAP-G-H subcomplex formation, just like site I. The interaction between hCAP-G and hCAP-H is essential for proper chromosome assembly mediated by condensin I in Xenopus egg extracts To test whether the interaction between hCAP-G and hCAP-H is indeed essential for the function of condensin I, we introduced the motif IV quintuple mutations (F463Q, F469Q, F473Q, F501Q, Y503Q; designated IV-5Q) described above into the context of full-length, holocomplexes (Fig 3B). Using the baculovirus expression system described previously 18, we co-expressed the five subunits of mammalian condensin I containing either the wild-type or mutant form of hCAP-H in insect cells. An equal level of expression of the five subunits in the two samples was confirmed by immunoblotting against total lysates (Fig 4A). Both lysates were then subjected to affinity purification using glutathione-agarose beads (Note that the SMC4 subunit was GST-tagged), follow" @default.
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