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- PDB-7mvx: Crystal structure of the Chaetomium thermophilum Nup188-Nic96 com... -

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Entry
Database: PDB / ID: 7mvx
TitleCrystal structure of the Chaetomium thermophilum Nup188-Nic96 complex (Nup188 residues 1-1858; Nic96 residues 240-301)
Components
  • Nucleoporin NIC96
  • Nucleoporin NUP188
KeywordsTRANSPORT PROTEIN / nuclear pore complex / nucleocytoplasmic transport / alpha-helical solenoid / nuclear pore
Function / homology
Function and homology information


structural constituent of nuclear pore / mRNA transport / nuclear pore / protein transport / nuclear membrane
Similarity search - Function
Nup188 SH3-like domain / Nuclear pore protein Nup188, C-terminal / Nuclear pore protein NUP188 C-terminal domain / Nucleoporin Nup188, N-terminal / Nucleoporin Nup188, N-terminal / : / Nucleoporin Nup188, N-terminal subdomain III / Nucleoporin Nup188 / Nucleoporin interacting component Nup93/Nic96 / Nup93/Nic96
Similarity search - Domain/homology
Nucleoporin NIC96 / Nucleoporin NUP188
Similarity search - Component
Biological speciesChaetomium thermophilum (fungus)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 4.35 Å
AuthorsPetrovic, S. / Samanta, D. / Perriches, T. / Bley, C.J. / Thierbach, K. / Brown, B. / Nie, S. / Mobbs, G.W. / Stevens, T.A. / Liu, X. ...Petrovic, S. / Samanta, D. / Perriches, T. / Bley, C.J. / Thierbach, K. / Brown, B. / Nie, S. / Mobbs, G.W. / Stevens, T.A. / Liu, X. / Tomaleri, G.P. / Schaus, L. / Hoelz, A.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM117360 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM111461 United States
Howard Hughes Medical Institute (HHMI)55108534 United States
Heritage Medical Research Institute
CitationJournal: Science / Year: 2022
Title: Architecture of the linker-scaffold in the nuclear pore.
Authors: Stefan Petrovic / Dipanjan Samanta / Thibaud Perriches / Christopher J Bley / Karsten Thierbach / Bonnie Brown / Si Nie / George W Mobbs / Taylor A Stevens / Xiaoyu Liu / Giovani Pinton ...Authors: Stefan Petrovic / Dipanjan Samanta / Thibaud Perriches / Christopher J Bley / Karsten Thierbach / Bonnie Brown / Si Nie / George W Mobbs / Taylor A Stevens / Xiaoyu Liu / Giovani Pinton Tomaleri / Lucas Schaus / André Hoelz /
Abstract: INTRODUCTION In eukaryotic cells, the selective bidirectional transport of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC). Embedded in nuclear envelope ...INTRODUCTION In eukaryotic cells, the selective bidirectional transport of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC). Embedded in nuclear envelope pores, the ~110-MDa human NPC is an ~1200-Å-wide and ~750-Å-tall assembly of ~1000 proteins, collectively termed nucleoporins. Because of the NPC's eightfold rotational symmetry along the nucleocytoplasmic axis, each of the ~34 different nucleoporins occurs in multiples of eight. Architecturally, the NPC's symmetric core is composed of an inner ring encircling the central transport channel and two outer rings anchored on both sides of the nuclear envelope. Because of its central role in the flow of genetic information from DNA to RNA to protein, the NPC is commonly targeted in viral infections and its nucleoporin constituents are associated with a plethora of diseases. RATIONALE Although the arrangement of most scaffold nucleoporins in the NPC's symmetric core was determined by quantitative docking of crystal structures into cryo-electron tomographic (cryo-ET) maps of intact NPCs, the topology and molecular details of their cohesion by multivalent linker nucleoporins have remained elusive. Recently, in situ cryo-ET reconstructions of NPCs from various species have indicated that the NPC's inner ring is capable of reversible constriction and dilation in response to variations in nuclear envelope membrane tension, thereby modulating the diameter of the central transport channel by ~200 Å. We combined biochemical reconstitution, high-resolution crystal and single-particle cryo-electron microscopy (cryo-EM) structure determination, docking into cryo-ET maps, and physiological validation to elucidate the molecular architecture of the linker-scaffold interaction network that not only is essential for the NPC's integrity but also confers the plasticity and robustness necessary to allow and withstand such large-scale conformational changes. RESULTS By biochemically mapping scaffold-binding regions of all fungal and human linker nucleoporins and determining crystal and single-particle cryo-EM structures of linker-scaffold complexes, we completed the characterization of the biochemically tractable linker-scaffold network and established its evolutionary conservation, despite considerable sequence divergence. We determined a series of crystal and single-particle cryo-EM structures of the intact Nup188 and Nup192 scaffold hubs bound to their Nic96, Nup145N, and Nup53 linker nucleoporin binding regions, revealing that both proteins form distinct question mark-shaped keystones of two evolutionarily conserved hetero‑octameric inner ring complexes. Linkers bind to scaffold surface pockets through short defined motifs, with flanking regions commonly forming additional disperse interactions that reinforce the binding. Using a structure‑guided functional analysis in , we confirmed the robustness of linker‑scaffold interactions and established the physiological relevance of our biochemical and structural findings. The near-atomic composite structures resulting from quantitative docking of experimental structures into human and cryo-ET maps of constricted and dilated NPCs structurally disambiguated the positioning of the Nup188 and Nup192 hubs in the intact fungal and human NPC and revealed the topology of the linker-scaffold network. The linker-scaffold gives rise to eight relatively rigid inner ring spokes that are flexibly interconnected to allow for the formation of lateral channels. Unexpectedly, we uncovered that linker‑scaffold interactions play an opposing role in the outer rings by forming tight cross-link staples between the eight nuclear and cytoplasmic outer ring spokes, thereby limiting the dilatory movements to the inner ring. CONCLUSION We have substantially advanced the structural and biochemical characterization of the symmetric core of the and human NPCs and determined near-atomic composite structures. The composite structures uncover the molecular mechanism by which the evolutionarily conserved linker‑scaffold establishes the NPC's integrity while simultaneously allowing for the observed plasticity of the central transport channel. The composite structures are roadmaps for the mechanistic dissection of NPC assembly and disassembly, the etiology of NPC‑associated diseases, the role of NPC dilation in nucleocytoplasmic transport of soluble and integral membrane protein cargos, and the anchoring of asymmetric nucleoporins. [Figure: see text].
History
DepositionMay 15, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 15, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 22, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_id_CSD / _citation.journal_volume ..._citation.journal_id_CSD / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Nucleoporin NUP188
B: Nucleoporin NIC96


Theoretical massNumber of molelcules
Total (without water)213,1982
Polymers213,1982
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, SEC-MALS
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3560 Å2
ΔGint-31 kcal/mol
Surface area78660 Å2
MethodPISA
Unit cell
Length a, b, c (Å)302.648, 302.648, 152.675
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number155
Space group name H-MH32
Symmetry operation#1: x,y,z
#2: -y,x-y,z
#3: -x+y,-x,z
#4: x-y,-y,-z
#5: -x,-x+y,-z
#6: y,x,-z
#7: x+1/3,y+2/3,z+2/3
#8: -y+1/3,x-y+2/3,z+2/3
#9: -x+y+1/3,-x+2/3,z+2/3
#10: x-y+1/3,-y+2/3,-z+2/3
#11: -x+1/3,-x+y+2/3,-z+2/3
#12: y+1/3,x+2/3,-z+2/3
#13: x+2/3,y+1/3,z+1/3
#14: -y+2/3,x-y+1/3,z+1/3
#15: -x+y+2/3,-x+1/3,z+1/3
#16: x-y+2/3,-y+1/3,-z+1/3
#17: -x+2/3,-x+y+1/3,-z+1/3
#18: y+2/3,x+1/3,-z+1/3

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Components

#1: Protein Nucleoporin NUP188 / Nuclear pore protein NUP188


Mass: 205977.188 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719 / Gene: NUP188, CTHT_0070850 / Production host: Escherichia coli (E. coli) / References: UniProt: G0SFH5
#2: Protein Nucleoporin NIC96 / Nuclear pore protein NIC96


Mass: 7220.833 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719 / Gene: NIC96, CTHT_0008480 / Production host: Escherichia coli (E. coli) / References: UniProt: G0S024
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.16 Å3/Da / Density % sol: 61.06 %
Crystal growTemperature: 294 K / Method: vapor diffusion, hanging drop / pH: 7.5 / Details: 0.05 M HEPES pH 7.5, 6.5 % (w/v) PEG 20000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL12-2 / Wavelength: 0.97944 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jun 22, 2017
RadiationMonochromator: Liquid nitrogen-cooled double crystal Si(111)
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97944 Å / Relative weight: 1
ReflectionResolution: 4.35→25 Å / Num. obs: 17578 / % possible obs: 99.5 % / Redundancy: 19.6 % / Biso Wilson estimate: 221 Å2 / CC1/2: 1 / CC star: 1 / Rmerge(I) obs: 0.247 / Rpim(I) all: 0.057 / Rrim(I) all: 0.253 / Net I/σ(I): 11.3
Reflection shellResolution: 4.35→4.86 Å / Redundancy: 19.7 % / Rmerge(I) obs: 2.429 / Num. unique obs: 4981 / CC1/2: 0.777 / CC star: 0.935 / Rpim(I) all: 0.559 / Rrim(I) all: 2.493 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
Blu-Icedata collection
DIALSdata reduction
DIALSdata scaling
Aimlessdata scaling
pointlessdata scaling
PHASERphasing
Cootmodel building
RefinementMethod to determine structure: SAD / Resolution: 4.35→24.93 Å / SU ML: 0.78 / Cross valid method: FREE R-VALUE / σ(F): 1.41 / Phase error: 37.38 / Stereochemistry target values: MLHL
RfactorNum. reflection% reflection
Rfree0.3167 895 5.09 %
Rwork0.2723 16672 -
obs0.2745 17567 99.9 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 413.2 Å2 / Biso mean: 255.6425 Å2 / Biso min: 144.94 Å2
Refinement stepCycle: final / Resolution: 4.35→24.93 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms13265 0 0 0 13265
Num. residues----1699
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 6 / % reflection obs: 100 %

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all
4.35-4.620.36861720.350327372909
4.62-4.970.36931390.335127572896
4.98-5.470.36991480.333927692917
5.47-6.250.38731420.351427702912
6.25-7.840.3531500.31627842934
7.84-24.930.25161440.207428552999
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.3243-0.2828-0.48870.02880.05890.09860.4081-0.4846-1.11680.9022-0.1993-0.80050.00070.5124-0.14555.4137-0.0831-0.70133.21330.68872.7503209.3059297.016380.8019
22.71350.57640.01922.30541.7742.81490.1657-0.6361-1.7137-0.4318-1.4850.17930.6992-0.60021.66452.637-0.28260.06322.79820.21733.2624222.7022284.139865.9552
33.7513-2.779-1.06453.617-0.08067.17440.923-0.7566-0.8172-0.73971.76440.8210.64220.6401-1.85574.41690.9372-0.51293.1093-0.16272.4175217.0108257.507848.5143
42.78910.2414-1.10361.7904-0.23912.5824-0.1714-0.45550.61680.91680.48380.69040.0424-1.6852-0.30382.0240.51120.1942.61520.12791.8828220.6081340.438584.2584
50.59171.0511-0.05362.60010.39981.802-0.0829-0.20980.03060.59540.1531-0.7144-0.68090.0026-0.05652.12830.23-0.16732.21210.06921.9194254.7797350.446977.2946
61.82811.3693-0.22543.58521.02670.37840.20450.42590.31280.275-0.14910.12050.0486-0.098-0.05652.29430.1507-0.17152.48160.19262.5692228.3691311.752562.6029
71.47130.08470.06753.43711.580.6468-0.148-0.2988-0.00670.26970.0222-0.0793-0.2155-0.1250.08582.7770.03240.03372.68070.15612.2845232.0809267.815450.6332
82.32320.1414-0.4670.8504-0.70992.61990.06021.1630.2182-0.318-0.9851.1424-0.1163-1.4739-0.45111.9487-0.41130.40561.9967-0.36412.2966214.6871248.439960.1739
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain B and resid 246:255B246 - 255
2X-RAY DIFFRACTION2chain B and resid 256:282B256 - 282
3X-RAY DIFFRACTION3chain B and resid 283:299B283 - 299
4X-RAY DIFFRACTION4chain A and resid 3:205A3 - 205
5X-RAY DIFFRACTION5chain A and resid 206:1091A206 - 1091
6X-RAY DIFFRACTION6chain A and resid 1092:1399A1092 - 1399
7X-RAY DIFFRACTION7chain A and resid 1400:1720A1400 - 1720
8X-RAY DIFFRACTION8chain A and resid 1745:1850A1745 - 1850

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