Huang G et al. (2022), Cryo-EM structure of the inner ring from the Xe...

XB-ART-58931

Cell Res 2022 May 01;325:451-460. doi: 10.1038/s41422-022-00633-x.

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Cryo-EM structure of the inner ring from the Xenopus laevis nuclear pore complex.

Huang G , Zhan X , Zeng C , Liang K , Zhu X , Zhao Y , Wang P , Wang Q , Zhou Q , Tao Q , Liu M , Lei J , Yan C , Shi Y .

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Nuclear pore complex (NPC) mediates nucleocytoplasmic shuttling. Here we present single-particle cryo-electron microscopy structure of the inner ring (IR) subunit from the Xenopus laevis NPC at an average resolution of 4.2 Å. A homo-dimer of Nup205 resides at the center of the IR subunit, flanked by two molecules of Nup188. Four molecules of Nup93 each places an extended helix into the axial groove of Nup205 or Nup188, together constituting the central scaffold. The channel nucleoporin hetero-trimer of Nup62/58/54 is anchored on the central scaffold. Six Nup155 molecules interact with the central scaffold and together with the NDC1-ALADIN hetero-dimers anchor the IR subunit to the nuclear envelope and to outer rings. The scarce inter-subunit contacts may allow sufficient latitude in conformation and diameter of the IR. Our structure reveals the molecular basis for the IR subunit assembly of a vertebrate NPC.

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Species referenced: Xenopus laevis
Genes referenced: aaas ndc1 nup155 nup160 nup188 nup205 nup54 nup58 nup62 nup93
GO keywords: nuclear envelope [+]

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	Fig. 1 Cryo-EM structure of the IR of the X. laevis NPC. a Cryo-EM reconstruction of the IR of X. laevis NPC at 22 Å resolution. Two perpendicular views are shown. The inner diameter of the IR is ~45 nm. b The final EM reconstruction for the IR subunit displays an average resolution of 4.2 Å. Shown here is a curve of the Fourier Shell Correlation (FSC) over resolution. c Distribution of the local resolution for the EM reconstruction of the IR subunit. The color-coded resolution bar is shown below.
	Fig. 2 Overall structure of the IR subunit from the X. laevis NPC. a Overall EM map of the IR subunit from X. laevis NPC. Two perpendicular views are shown. The IR subunit displays a 2-fold symmetry, with a cytoplasmic half and a nuclear half. The EM maps for individual nucleoporins in each half are color-coded identically. The NE is shown in gray, the two layer-like density representing lipid bilayer of NE is apparent in the right panel. b Structure of the IR subunit from the X. laevis NPC. Two views are shown. In the left panel, individual nucleoporins are color-coded. In the right panel, nucleoporins of the nuclear half are colored gray; different copies of the same protein in the cytoplasmic half are differentially colored. c Overall structure of the IR subunit from X. laevis NPC. Top and bottom: the overall structure of the IR subunit is displayed in two perpendicular views (first column from left). For each view, the IR subunit is disseminated into three layers: ten molecules of Nup155/ALADIN/NDC1 close to the NE (second column from left), the central scaffold of eight molecules of Nup188/Nup205/Nup93 (third column from left), and four CNTs (fourth column from left).
	Fig. 3 Two molecules of Nup205 and two molecules of Nup188 constitute the central components of the IR subunit. a A homo-dimer of Nup205 at the center of the IR subunit is flanked by two molecules of Nup188. The two Nup205 molecules contact each other via their C-terminal helices. b A side-by-side comparison of the structures of Nup188 and Nup205. The overall appearance and size are similar between Nup188 (left panel) and Nup205 (right panel). c A close-up view on the dimeric interface between Nup205 and Nup205′. The C-terminal helices α75/α78 from Nup205 stack against the corresponding helices from Nup205′. d A close-up view on the interface between Nup205 and Nup188. Three inter-helical surface loops from Nup188 and Nup205 are involved in the interactions.
	Fig. 4 Four molecules of Nup93 interact with Nup188 and Nup205 to form the central scaffold. a Overall distribution of four Nup93 molecules in the IR subunit. On the cytoplasmic side, Nup93-1 and Nup93-2 interact with Nup188 and Nup205, respectively. b Structure of Nup93. Shown here is a cartoon representation of Nup93-1. The short N-terminal helix is known to interact with CNT.13 The extended N-terminal helix α5 binds Nup188 or Nup205. c A close-up view on the asymmetric interface between Nup93-1 and Nup93-2. d A close-up view on the interface between Nup93-1 and Nup188. Notably, helix α5 of Nup93-1 is placed in the axial groove of the Nup188 α-solenoid and interacts with the C-terminal helices of Nup188. e A close-up view on the interface between Nup93-2 and Nup205. In striking analogy to the Nup93-1/Nup188 interface, helix α5 of Nup93-2 is placed in the axial groove of the Nup205 α-solenoid and interacts with the C-terminal helices of Nup205. f A side-by-side comparison of the interaction between helix α5 of Nup93 and Nup188 (upper panel) or Nup205 (lower panel). Nup188 and Nup205 are shown in surface representation.
	Fig. 5 Nup155 links the central scaffold. a An overall view of six Nup155 molecules in each IR subunit. Of the six Nup155 molecules, Nup155-2 and Nup155-3 form a dimer on the cytoplasmic side; Nup155-2′ and Nup155-3′ form a homo-dimer on the nuclear side. b Nup155 connects the IR subunit to the CR subunit. The C-terminal helices of Nup155 on the cytoplasmic side of the IR subunit are sandwiched by inner Nup160 and inner Nup205 of the CR subunit. c A close-up view on the Nup155 homo-dimer. The helical domains of Nup155-2 and Nup155-3 interact with each other in a head-to-tail fashion, generating an elongated Nup155 homo-dimer of ~290 Å in length. d Close-up views on the interface between Nup155-2 and Nup155-3. Two views are shown. e A close-up view on the interface between Nup155-2 and Nup93-1. f A close-up view on the interface between Nup155-2 and Nup188. g A close-up view on the interface between Nup155-3 and Nup93-2. This interface is analogous to that between Nup155-2 and Nup93-1. h A close-up view on the interface between Nup155-3 and Nup205. This interface is analogous to that between Nup155-2 and Nup188.
	Fig. 6 The ALADIN–NDC1 hetero-dimer helps to anchor the IR subunit to the NE. a An overall view of two ALADIN–NDC1 hetero-dimers in the IR subunit. b A close-up view on the ALADIN–NDC1 hetero-dimer. Blade-3 and blade-4 of the ALADIN β-propeller are in close proximity to helices α11/α14/α16 of the PD of NDC1. c An overall view on the cytoplasmic ALADIN–NDC1 hetero-dimer relative to its neighboring nucleoporins Nup155 and Nup93. d A close-up view on the interface between ALADIN and Nup155-1. e A close-up view on the interface between NDC1 and Nup93-2.
	Fig. 7 Structural features of the IR scaffold. a An overall view of the IR scaffold. b An overall view on inter-subunit interface between subunit 1 (S1) and subunit 2 (S2). c A close-up view on the cytoplasmic side of the interface between S1 and S2. Given the spatial gap in between, Nup205 of S1 may not directly interact with Nup93-1 or Nup188 from S2. d The IR subunit is slightly tilted toward the cytoplasmic side. e The IR subunit is linked to the outer rings through the linker nucleoporins Nup93, NDC1, ALADIN, and Nup155-1. The IR subunit is connected in opposite directions to the outer rings. Such connection depends on the ALADIN–NDC1 hetero-dimer to fill up the vacancy between Nup155-1 and Nup93-2.
	Fig. S1 Cryo-EM analysis of the IR from the X. laevis NPC. a, Flowchart of data analysis for reconstruction of the IR from the X. laevis NPC at 22 Å resolution. b, Structural comparison of the IR from the X. laevis NPC determined using single particle cryo-EM with that of the human NPC obtained through cryo-ET. The EMDB codes for the 3D maps, including the one for this study, are shown on top.
	Fig. S2 Cryo-EM data processing for the IR subunit from X. laevis NPC. a, Flowchart of data processing for reconstruction of the IR subunit to an overall resolution of 5.6 Å. This part of the data analysis only involves the bin-4 (pixel size 5.548 Å) and bin-2 levels (pixel size 2.774 Å). b, Flowchart of data processing for the IR subunit to an overall resolution of 4.2 Å. This part of the data analysis involves the bin-1 level (pixel size 1.387 Å). Please refer to the section “Data processing and reconstruction of the IR subunit” in the MATERIALS AND METHODS for details.
	Fig. S3 Cryo-EM analysis of the IR subunit. a, Angular distribution of the single-particle cryo-EM reconstruction for the IR subunit. Each cylinder represents one view and the height of the cylinder is proportional to the number of particles for that view. b, Directional Fourier Shell correlation (FSC) curves and directional FSC histograms for cryo-EM reconstruction of the IR subunit. The directional FSC curve was calculated using the website https://3dfsc.salk.edu1.
	Fig. S4 EM maps and model fitting for Nup188 and Nup205. a, Resolution maps for the two copies of Nup188 and Nup205. Nup188 and Nup205 on the cytoplasmic side (left) and on the nuclear side (right) are nearly identical. The local resolution maps are calculated in Relion 3.0 and presented in Chimera. b, EM maps for the two copies of Nup188, Nup205, and the extended helices α5 from Nup93 that respectively bind to them. The same views are shown as in panel a. c, Model building of Nup188 and Nup205 into their EM corresponding densities. The EM maps, shown as semitransparent surface in ChimeraX, are color coded for different proteins or domains. Nup205 specific TAIL-C and tower helix are highlighted in magenta (top right) and blue (bottom right), respectively. All EM maps in this figure were prepared using the 4.2-Å reconstruction of the IR subunit with a contour level of 5-7 σ.
	Fig. S5 EM maps and model fitting for Nup93. a, Resolution maps for the two copies of Nup93 pairs. Nup93 pairs on the cytoplasmic side (left) and on the nuclear side (right) are nearly identical. The local resolution maps are calculated in Relion 3.0 and presented in Chimera. b, EM maps for the two copies of Nup93 pairs. The views are identical to the corresponding ones in panel a. c,d, Model building of Nup93-1 and -2 into their corresponding densities. The EM maps, shown as semitransparent surface, were prepared using the 4.2-Å reconstruction of the IR subunit with a contour level of 5-7 σ in ChimeraX.
	Fig. S6 EM maps and model fitting for Nup155. a, Resolution maps for the two copies of the Nup155 trimer. Nup155 trimers on the cytoplasmic side (top) and nuclear side (bottom) are nearly identical. The local resolution maps are calculated in Relion 3.0 and presented in Chimera. b, Color-coded EM maps for the two copies of the Nup155 trimer. c, Model building of the NTD of Nup155 into their EM densities. d, Model building of the CTD of Nup155 into their EM densities. The EM maps, shown as semitransparent surface in ChimeraX, are color coded based on the protein identity. All EM maps in this figure were prepared using the 4.2-Å reconstruction of the IR subunit with a contour level of 4-6 σ.
	Fig. S7 EM maps and model fitting for β-propeller of Nup155, ALADIN, and NDC1. a, Resolution maps for the two copies of the Nup155-ALADIN-NDC1 trimer. The Nup155-ALADIN-NDC1 trimers on the cytoplasmic side (top) and on the nuclear side (bottom) are nearly identical. The local resolution maps are calculated in Relion 3.0 and presented in Chimera. b,c, Model building of Nup155-ALADIN-NDC1 trimers into their corresponding EM maps. The color-coded EM maps, shown as semitransparent surface in ChimeraX, were prepared using the 4.2-Å reconstruction of the IR subunit with a contour level of 4-6 σ.
	Fig. S8 Model fitting for CNTs. Semitransparent contour of the EM maps docked with structural models of CNTs are shown in Chimera. The same views are shown with low and high thresholds on the top and bottom, respectively. CNT and the N-terminal helix from Nup93 are colored orange and pink, respectively.
	Fig. S15 The two halves of the IR subunit are nearly identical. The two halves of the IR subunit can be superimposed with a root-mean-squared deviation (RMSD) of ~ 1.34 Å over 8,911 aligned Cα atoms. Two perpendicular views are shown.
	Fig. S16 Structural comparison of corresponding IR nucleoporins on the cytoplasmic and the nuclear sides. Structures of the indicated components on the cytoplasmic side are color coded, and the corresponding ones on the nuclear side are colored grey and denoted with an apostrophe.
	Fig. S17 Structural comparison of Nup93 molecules within the IR subunit. Structures of the indicated components on the cytoplasmic side are color coded, and the corresponding ones on the nuclear side, except Nup93-1’ and Nup93-2’ in panel c, are colored grey and denoted with an apostrophe.
	Fig. S18 Structural mapping of phosphorylation sites on select components of the IR subunit. a, Mapping of the phosphorylation sites Ser307 and Ser675 onto Nup155. Based on our structure, both Ser307 and Ser675 face the NE. Phosphorylation may alter their interaction with the membrane, thus potentially affecting membrane localization of Nup155. b, Mapping of the phosphorylation sites Ser117, Thr368, Thr537, and Ser655 onto Nup93. Phosphorylation of Ser117 may affect the interaction between the extended helix α5 and Nup205 or Nup188. Phosphorylation of Thr368 or Ser655 may alter the interface between Nup93-1 and Nup93-2. Phosphorylation of Thr537 may change the local conformation. c, Mapping of phosphorylation sites onto Nup188, Nup205, and CNT. The phosphorylated residues include Ser794/Ser992/Ser1686/Ser1687 in Nup188, Thr328/Ser421/Thr739 in Nup205, Thr250/Thr314/Thr444 in Nup54, and Thr433 in Nup62. Their phosphorylation likely affects their mutual interactions or local conformations.
	Fig. S19 Structural mapping of phosphorylation sites and disease mutation sites on ALADIN. a, Mapping of three phosphorylation sites (Ser186, Thr295, and Ser300) on ALADIN. Phosphorylation of these sites may alter the local conformation of ALADIN. b, Mapping of 15 residues in ALADIN that are mutated in the triple A syndrome (AAAS)1,2. Each of these mutations is likely to affect the local conformation and/or stability of ALADIN.
	Fig. S20 Structural mapping of disease-derived mutations onto components of the IR subunit. a, Mapping of four residues that are targeted for mutations in steroid-resistant nephrotic syndrome (SRNS)1. These four residues are Arg389/Gly592/Tyr630 in Nup93 and Phe1994 in Nup205. The mutations, R389W, Y630C, G592V and F1994S, may alter the local conformation. b, Mapping of the disease autosomal recessive infantile bilateral striatal necrosis (IBSN) mutation Q416P in Nup622. The mutation of Gln416 to a helix-breaking residue Pro in the middle of an extended helix may destabilize the helical conformation.
	Fig. S21 A conserved interaction between Nup93 and Nup205 in the CR and the IR. Structural comparison of the X. laevis Nup205/Nup93 pairs from the IR subunit with those from the CR subunit are shown. Structures of the Nup205/Nup93 pairs on the indicated positions, cytoplasmic side vs nuclear side or inner ring vs outer ring, are superimposed to the corresponding ones in the CR subunit.
	Fig. S22 Structural comparison of six Nup155 molecules from the IR subunit. a, Structures of the three Nup155 molecules on the cytoplasmic side are nearly identical with those on the nuclear side. b, Nup155-2 and Nup155-3 on the cytoplasmic side show distinct conformations. c, Structural differences between Nup155-2’ and Nup155-3’. Similar to the comparison between Nup155-2 and Nup155-3, their counterparts on the nuclear side also display different local conformations.

References [+] :

Akey, Comprehensive structure and functional adaptations of the yeast nuclear pore complex. 2022, Pubmed