Kobayashi W et al. (2019), Structural and biochemical analyses of the nucl...

XB-ART-56919

Commun Biol 2019 Jan 01;2:163. doi: 10.1038/s42003-019-0385-7.

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Structural and biochemical analyses of the nuclear pore complex component ELYS identify residues responsible for nucleosome binding.

Kobayashi W , Takizawa Y , Aihara M , Negishi L , Ishii H , Kurumizaka H .

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The nuclear pore complex embedded within the nuclear envelope is the essential architecture for trafficking macromolecules, such as proteins and RNAs, between the cytoplasm and nucleus. The nuclear pore complex assembly occurs on chromatin in the post-mitotic phase of the cell cycle. ELYS (MEL-28/AHCTF1) binds to the nucleosome, which is the basic chromatin unit, and promotes assembly of the complex around the chromosomes in cells. Here we show that the Arg-Arg-Lys (RRK) stretch of the C-terminal ELYS region plays an essential role in the nucleosome binding. The cryo-EM structure and the crosslinking mass spectrometry reveal that the ELYS C-terminal region directly binds to the acidic patch of the nucleosome. These results provide mechanistic insight into the ELYS-nucleosome interaction, which promotes the post-mitotic nuclear pore complex formation around chromosomes in cells.

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Species referenced: Xenopus laevis
Genes referenced: ahctf1 h2ac21 h2bc21 mtnr1al pmp22

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	Fig. 1. Nucleosome binding by ELYSC. a The Xenopus laevis ELYSC fragments used in this study. The purple and orange boxes represent the regions corresponding to the AT-hook DNA-binding domain and the RRK stretch, respectively. In the ELYSC 2R-A mutant, the Arg2332 and Arg2334 residues in the AT-hook DNA binding domain are replaced by Ala. The amino acid residues are numbered. In the ELYSC Δ10 mutant, the C-terminal ten residues including the RRK stretch are deleted. The amino acid residues are numbered. b Gel-filtration analysis of the ELYSC–nucleosome complex. ELYSC and the nucleosome containing the 145 bp Widom 601 DNA were mixed in a 2.5:1 molar ratio, at room temperature for 20 min. The red line indicates the elution profile of the ELYSC–nucleosome complex. Eluted fractions were analyzed by 18% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie Brilliant Blue staining. The uncropped gel image is shown in Supplementary Fig. 4. c Experimental scheme for the pull-down assay with GS4B beads. d The pull-down assay for the ELYSC–nucleosome interaction. Lane 1 represents molecular mass markers. Lane 2 indicates input nucleosome (50%). Lane 3 indicates a negative control experiment with glutathione S-transferase (GST). Lanes 4–6 indicate the experiments with GST-ELYSC, GST-ELYSC 2R-A, and GST-ELYSC Δ10. The samples were analyzed by 18% SDS-PAGE with Coomassie Brilliant Blue staining. Experiments were independently repeated four times, and consistent results were obtained. The uncropped gel image is shown in Supplementary Fig. 4
	Fig. 2. The RRK stretch at the C-terminus of ELYS is important for nucleosome binding. a Sequence alignment of the Xenopus laevis, Homo sapiens, Mus musculus, Gallus gallus, and Bos taurus ELYS proteins. The RRK stretch is colored red. The amino acid residues are numbered. b The pull-down assay for the nucleosome with GST, GST-ELYSC, or GST-ELYSC mutants. Lane 1 represents molecular mass markers. Lane 2 indicates input nucleosome (50%). Lane 3 indicates a negative control experiment with GST. Lanes 4–10 represent the experiments with GST-ELYSC and GST-ELYSC mutants (R2404A, R2405A, K2406A, R2408A, and R2404A-R2405A-K2406A), respectively. Experiments were independently repeated three times, and consistent results were obtained. The uncropped gel image is shown in Supplementary Fig. 4
	Fig. 3. The cryo-electron microscopic structure of the ELYSC–nucleosome complex. a Digital micrograph of the ELYSC–nucleosome complexes. Scale bar indicates 100 nm. b Selected two-dimensional class averages from single particle images of the ELYSC–nucleosome complex. The box size is 21 nm2. c Fourier shell correlation (FSC) curve after gold-standard map refinement. The overall resolution of the ELYSC–nucleosome complex is 4.3 Å at an FSC = 0.143. d Local resolution map of the ELYSC–nucleosome complex, showing the resolution range across the map from 3.8 to 7 Å. e Enlarged view of the ELYS density with a scale bar
	Fig. 4. ELYSC interacts with the acidic patch of the nucleosome. a Cryo-electron microscopic structure of the ELYSC–nucleosome complexes at 4.3 Å, contoured at 3.7 sigma above mean density. ELYSC, H2A, and H2B are colored cyan, magenta, and yellow, respectively. Enlarged views around the acidic patches encircled with rectangles are presented. The acidic (red) and basic (blue) regions of the nucleosome surface are colored according to the Coulombic surface charge. The extra density corresponding to part of ELYSC is colored cyan. b The acidic amino acid residues of the nucleosome surface around the ELYSC region are presented. The H2A Glu56, Glu61, Glu64, Asp90, Glu91, and Glu92 residues are colored magenta. The H2B Glu105 and Glu113 residues are colored yellow
	Fig. 5. The ELYS–nucleosome interaction determined by crosslinking mass spectrometry. a Schematic representation of the crosslinking mass spectrometric analysis. The interlinks between histone core regions (H2A, H2B, and H3.1) and ELYSC are depicted with lines. The amino acid residues involved in the interlinks are indicated with numbers. The purple and gray boxes represent the regions corresponding to the AT-hook DNA-binding domain and the C-terminal ten amino acid residues, respectively. H3.1, H2A, H2B, and ELYSC are colored blue, magenta, yellow, and green, respectively. b Possible location of the ELYS Lys2400 residue, predicted by the crosslinking mass spectrometric analysis. The black dashed lines with numbers show the distances between the Cα atoms of the H2A Lys95, H2B Lys120, and H3 Lys56 residues. The red circle represents the 20 Å radius, which indicates the possible crosslinking area of the H2A Lys95 residue by DSS-H12/D12 (the central point is the Cα atom). The yellow circle represents the 20 Å radius, which indicates the possible crosslinking area of the H2B Lys120 residue by DSS-H12/D12 (the central point is the Cα atom). The blue circle represents the 20 Å radius, which indicates the possible crosslinking area of the H3.1 Lys56 residue by DSS-H12/D12 (the central point is the Cα atom). The possible location of the ELYS Lys2400 residue is indicated as a green sphere. The crystal structure of the nucleosome containing a 145 -bp Widom 601 DNA (PDB ID: 3lZ0) was used39. The black arrow indicates the dyad axis of the nucleosome. c The cryo-electron microscopic structure of the ELYSC–nucleosome complex. The predicted location of ELYS Lys2400 is superimposed on the structure

References [+] :

Antonin, Nuclear pore complex assembly through the cell cycle: regulation and membrane organization. 2008, Pubmed