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- PDB-7mnn: Crystal structure of the N-terminal domain of NUP358/RanBP2 (resi... -

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Basic information

Entry
Database: PDB / ID: 7mnn
TitleCrystal structure of the N-terminal domain of NUP358/RanBP2 (residues 1-752) T653I mutant in complex with Fab fragment
Components
  • Antibody Fab14 Heavy Chain
  • Antibody Fab14 Light Chain
  • E3 SUMO-protein ligase RanBP2
KeywordsTRANSPORT PROTEIN/Immune System / NUCLEAR PORE COMPLEX COMPONENT / NUCLEOCYTOPLASMIC TRANSPORT / TRANSPORT PROTEIN / TRANSPORT PROTEIN-Immune System complex
Function / homology
Function and homology information


cytoplasmic periphery of the nuclear pore complex / SUMO ligase activity / SUMO ligase complex / annulate lamellae / nuclear pore cytoplasmic filaments / Nuclear Pore Complex (NPC) Disassembly / nuclear inclusion body / nuclear pore nuclear basket / Transport of Ribonucleoproteins into the Host Nucleus / Regulation of Glucokinase by Glucokinase Regulatory Protein ...cytoplasmic periphery of the nuclear pore complex / SUMO ligase activity / SUMO ligase complex / annulate lamellae / nuclear pore cytoplasmic filaments / Nuclear Pore Complex (NPC) Disassembly / nuclear inclusion body / nuclear pore nuclear basket / Transport of Ribonucleoproteins into the Host Nucleus / Regulation of Glucokinase by Glucokinase Regulatory Protein / Defective TPR may confer susceptibility towards thyroid papillary carcinoma (TPC) / Transport of the SLBP independent Mature mRNA / Transport of the SLBP Dependant Mature mRNA / NS1 Mediated Effects on Host Pathways / SUMOylation of SUMOylation proteins / Transport of Mature mRNA Derived from an Intronless Transcript / Transferases; Acyltransferases; Aminoacyltransferases / Rev-mediated nuclear export of HIV RNA / SUMOylation of RNA binding proteins / nuclear export / Nuclear import of Rev protein / Transport of Mature mRNA derived from an Intron-Containing Transcript / NEP/NS2 Interacts with the Cellular Export Machinery / tRNA processing in the nucleus / SUMO transferase activity / nucleocytoplasmic transport / centrosome localization / Viral Messenger RNA Synthesis / regulation of gluconeogenesis / NLS-bearing protein import into nucleus / SUMOylation of ubiquitinylation proteins / Vpr-mediated nuclear import of PICs / SUMOylation of DNA replication proteins / protein sumoylation / Regulation of HSF1-mediated heat shock response / mRNA transport / Amplification of signal from unattached kinetochores via a MAD2 inhibitory signal / SUMOylation of DNA damage response and repair proteins / nuclear pore / Mitotic Prometaphase / EML4 and NUDC in mitotic spindle formation / Resolution of Sister Chromatid Cohesion / response to amphetamine / SUMOylation of chromatin organization proteins / GTPase activator activity / HCMV Late Events / RHO GTPases Activate Formins / Transcriptional regulation by small RNAs / Signaling by ALK fusions and activated point mutants / ISG15 antiviral mechanism / small GTPase binding / HCMV Early Events / Separation of Sister Chromatids / protein folding / nuclear envelope / snRNP Assembly / nuclear membrane / intracellular membrane-bounded organelle / protein-containing complex binding / SARS-CoV-2 activates/modulates innate and adaptive immune responses / RNA binding / nucleoplasm / membrane / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Nup358/RanBP2 E3 ligase domain / Nup358/RanBP2 E3 ligase domain / Ran binding protein RanBP1-like / Ran binding domain / RanBP1 domain / Ran binding domain type 1 profile. / Ran-binding domain / Zinc finger domain / Zn-finger in Ran binding protein and others / Zinc finger RanBP2 type profile. ...Nup358/RanBP2 E3 ligase domain / Nup358/RanBP2 E3 ligase domain / Ran binding protein RanBP1-like / Ran binding domain / RanBP1 domain / Ran binding domain type 1 profile. / Ran-binding domain / Zinc finger domain / Zn-finger in Ran binding protein and others / Zinc finger RanBP2 type profile. / Zinc finger RanBP2-type signature. / Zinc finger, RanBP2-type superfamily / Zinc finger, RanBP2-type / Cyclophilin-type peptidyl-prolyl cis-trans isomerase, conserved site / Cyclophilin-type peptidyl-prolyl cis-trans isomerase signature. / Cyclophilin-type peptidyl-prolyl cis-trans isomerase domain profile. / Cyclophilin-type peptidyl-prolyl cis-trans isomerase domain / Cyclophilin type peptidyl-prolyl cis-trans isomerase/CLD / Cyclophilin-like domain superfamily / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / Tetratricopeptide-like helical domain superfamily / PH-like domain superfamily
Similarity search - Domain/homology
E3 SUMO-protein ligase RanBP2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 6.7 Å
AuthorsBley, C.J. / Nie, S. / Mobbs, G.W. / Petrovic, S. / Gres, A.T. / Liu, X. / Mukherjee, S. / Harvey, S. / Huber, F.M. / Lin, D.H. ...Bley, C.J. / Nie, S. / Mobbs, G.W. / Petrovic, S. / Gres, A.T. / Liu, X. / Mukherjee, S. / Harvey, S. / Huber, F.M. / Lin, D.H. / Brown, B. / Tang, A.W. / Rundlet, E.J. / Correia, A.R. / Chen, S. / Regmi, S.G. / Stevens, T.A. / Jette, C.A. / Dasso, M. / Patke, A. / Palazzo, A.F. / Kossiakoff, A.A. / Hoelz, A.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM117360 United States
Howard Hughes Medical Institute (HHMI)55108534 United States
Heritage Medical Research Institute United States
CitationJournal: Science / Year: 2022
Title: Architecture of the cytoplasmic face of the nuclear pore.
Authors: Christopher J Bley / Si Nie / George W Mobbs / Stefan Petrovic / Anna T Gres / Xiaoyu Liu / Somnath Mukherjee / Sho Harvey / Ferdinand M Huber / Daniel H Lin / Bonnie Brown / Aaron W Tang / ...Authors: Christopher J Bley / Si Nie / George W Mobbs / Stefan Petrovic / Anna T Gres / Xiaoyu Liu / Somnath Mukherjee / Sho Harvey / Ferdinand M Huber / Daniel H Lin / Bonnie Brown / Aaron W Tang / Emily J Rundlet / Ana R Correia / Shane Chen / Saroj G Regmi / Taylor A Stevens / Claudia A Jette / Mary Dasso / Alina Patke / Alexander F Palazzo / Anthony A Kossiakoff / André Hoelz /
Abstract: INTRODUCTION The subcellular compartmentalization of eukaryotic cells requires selective transport of folded proteins and protein-nucleic acid complexes. Embedded in nuclear envelope pores, which are ...INTRODUCTION The subcellular compartmentalization of eukaryotic cells requires selective transport of folded proteins and protein-nucleic acid complexes. Embedded in nuclear envelope pores, which are generated by the circumscribed fusion of the inner and outer nuclear membranes, nuclear pore complexes (NPCs) are the sole bidirectional gateways for nucleocytoplasmic transport. The ~110-MDa human NPC is an ~1000-protein assembly that comprises multiple copies of ~34 different proteins, collectively termed nucleoporins. The symmetric core of the NPC is composed of an inner ring encircling the central transport channel and outer rings formed by Y‑shaped coat nucleoporin complexes (CNCs) anchored atop both sides of the nuclear envelope. The outer rings are decorated with compartment‑specific asymmetric nuclear basket and cytoplasmic filament nucleoporins, which establish transport directionality and provide docking sites for transport factors and the small guanosine triphosphatase Ran. The cytoplasmic filament nucleoporins also play an essential role in the irreversible remodeling of messenger ribonucleoprotein particles (mRNPs) as they exit the central transport channel. Unsurprisingly, the NPC's cytoplasmic face represents a hotspot for disease‑associated mutations and is commonly targeted by viral virulence factors. RATIONALE Previous studies established a near-atomic composite structure of the human NPC's symmetric core by combining (i) biochemical reconstitution to elucidate the interaction network between symmetric nucleoporins, (ii) crystal and single-particle cryo-electron microscopy structure determination of nucleoporins and nucleoporin complexes to reveal their three-dimensional shape and the molecular details of their interactions, (iii) quantitative docking in cryo-electron tomography (cryo-ET) maps of the intact human NPC to uncover nucleoporin stoichiometry and positioning, and (iv) cell‑based assays to validate the physiological relevance of the biochemical and structural findings. In this work, we extended our approach to the cytoplasmic filament nucleoporins to reveal the near-atomic architecture of the cytoplasmic face of the human NPC. RESULTS Using biochemical reconstitution, we elucidated the protein-protein and protein-RNA interaction networks of the human and cytoplasmic filament nucleoporins, establishing an evolutionarily conserved heterohexameric cytoplasmic filament nucleoporin complex (CFNC) held together by a central heterotrimeric coiled‑coil hub that tethers two separate mRNP‑remodeling complexes. Further biochemical analysis and determination of a series of crystal structures revealed that the metazoan‑specific cytoplasmic filament nucleoporin NUP358 is composed of 16 distinct domains, including an N‑terminal S‑shaped α‑helical solenoid followed by a coiled‑coil oligomerization element, numerous Ran‑interacting domains, an E3 ligase domain, and a C‑terminal prolyl‑isomerase domain. Physiologically validated quantitative docking into cryo-ET maps of the intact human NPC revealed that pentameric NUP358 bundles, conjoined by the oligomerization element, are anchored through their N‑terminal domains to the central stalk regions of the CNC, projecting flexibly attached domains as far as ~600 Å into the cytoplasm. Using cell‑based assays, we demonstrated that NUP358 is dispensable for the architectural integrity of the assembled interphase NPC and RNA export but is required for efficient translation. After NUP358 assignment, the remaining 4-shaped cryo‑ET density matched the dimensions of the CFNC coiled‑coil hub, in close proximity to an outer-ring NUP93. Whereas the N-terminal NUP93 assembly sensor motif anchors the properly assembled related coiled‑coil channel nucleoporin heterotrimer to the inner ring, biochemical reconstitution confirmed that the NUP93 assembly sensor is reused in anchoring the CFNC to the cytoplasmic face of the human NPC. By contrast, two CFNCs are anchored by a divergent mechanism that involves assembly sensors located in unstructured portions of two CNC nucleoporins. Whereas unassigned cryo‑ET density occupies the NUP358 and CFNC binding sites on the nuclear face, docking of the nuclear basket component ELYS established that the equivalent position on the cytoplasmic face is unoccupied, suggesting that mechanisms other than steric competition promote asymmetric distribution of nucleoporins. CONCLUSION We have substantially advanced the biochemical and structural characterization of the asymmetric nucleoporins' architecture and attachment at the cytoplasmic and nuclear faces of the NPC. Our near‑atomic composite structure of the human NPC's cytoplasmic face provides a biochemical and structural framework for elucidating the molecular basis of mRNP remodeling, viral virulence factor interference with NPC function, and the underlying mechanisms of nucleoporin diseases at the cytoplasmic face of the NPC. [Figure: see text].
History
DepositionMay 1, 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_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Oct 18, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id ..._struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: E3 SUMO-protein ligase RanBP2
B: E3 SUMO-protein ligase RanBP2
H: Antibody Fab14 Heavy Chain
L: Antibody Fab14 Light Chain
K: Antibody Fab14 Heavy Chain
M: Antibody Fab14 Light Chain


Theoretical massNumber of molelcules
Total (without water)270,1406
Polymers270,1406
Non-polymers00
Water0
1
A: E3 SUMO-protein ligase RanBP2
H: Antibody Fab14 Heavy Chain
L: Antibody Fab14 Light Chain


Theoretical massNumber of molelcules
Total (without water)135,0703
Polymers135,0703
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: E3 SUMO-protein ligase RanBP2
K: Antibody Fab14 Heavy Chain
M: Antibody Fab14 Light Chain


Theoretical massNumber of molelcules
Total (without water)135,0703
Polymers135,0703
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)161.537, 161.537, 647.375
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number179
Space group name H-MP6522
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11(chain A and (resid 145 through 506 or (resid 511...
21(chain B and resid 145 through 673)
12(chain A and resid 674 through 752)
22(chain B and resid 674 through 752)
13chain H
23chain K
14(chain L and resid 2 through 213)
24chain M

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
111ASPASPHISHIS(chain A and (resid 145 through 506 or (resid 511...AA145 - 506146 - 507
121GLNGLNGLNGLN(chain A and (resid 145 through 506 or (resid 511...AA511512
211ASPASPSERSER(chain B and resid 145 through 673)BB145 - 673146 - 674
112ILEILEGLUGLU(chain A and resid 674 through 752)AA674 - 752675 - 753
212ILEILEGLUGLU(chain B and resid 674 through 752)BB674 - 752675 - 753
113GLUGLUVALVALchain HHC4 - 2304 - 230
213GLUGLUVALVALchain KKE4 - 2304 - 230
114ASPASPGLYGLY(chain L and resid 2 through 213)LD2 - 2132 - 213
214ASPASPGLYGLYchain MMF2 - 2132 - 213

NCS ensembles :
ID
1
2
3
4

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Components

#1: Protein E3 SUMO-protein ligase RanBP2 / 358 kDa nucleoporin / Nuclear pore complex protein Nup358 / Nucleoporin Nup358 / Ran-binding ...358 kDa nucleoporin / Nuclear pore complex protein Nup358 / Nucleoporin Nup358 / Ran-binding protein 2 / RanBP2 / p270


Mass: 86334.789 Da / Num. of mol.: 2 / Mutation: I599M, T653I
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RANBP2, NUP358 / Production host: Escherichia coli (E. coli)
References: UniProt: P49792, Transferases; Acyltransferases; Aminoacyltransferases
#2: Antibody Antibody Fab14 Heavy Chain


Mass: 25476.312 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#3: Antibody Antibody Fab14 Light Chain


Mass: 23258.783 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)

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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: 4.51 Å3/Da / Density % sol: 72.75 %
Crystal growTemperature: 294 K / Method: vapor diffusion, hanging drop / pH: 4.5
Details: 4 (w/v) % PEG 4,000; 0.15 M sodium acetate; 0.1 M sodium citrate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL12-2 / Wavelength: 0.9795 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Mar 26, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 6.7→30 Å / Num. obs: 9674 / % possible obs: 98.9 % / Redundancy: 98.7 % / CC1/2: 0.996 / Rmerge(I) obs: 0.779 / Rpim(I) all: 0.077 / Rrim(I) all: 0.783 / Net I/σ(I): 11 / Num. measured all: 955034 / Scaling rejects: 1
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
6.7-7.49102.86.37827523426770.6750.6216.4091.5100
14.98-3086.70.184757298730.9990.0190.18530.687.5

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Processing

Software
NameVersionClassification
Aimless0.7.4data scaling
PHENIX1.17.1refinement
PDB_EXTRACT3.27data extraction
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4GA0, 5CWS

4ga0

5cws


Resolution: 6.7→30 Å / SU ML: 0.81 / Cross valid method: THROUGHOUT / σ(F): 1.33 / Phase error: 25.21 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2634 456 4.75 %
Rwork0.2175 9135 -
obs0.2197 9591 99.82 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 509.28 Å2 / Biso mean: 363.7518 Å2 / Biso min: 286.95 Å2
Refinement stepCycle: final / Resolution: 6.7→30 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17499 0 0 0 17499
Num. residues----2233
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A4832X-RAY DIFFRACTION6.441TORSIONAL
12B4832X-RAY DIFFRACTION6.441TORSIONAL
21A712X-RAY DIFFRACTION6.441TORSIONAL
22B712X-RAY DIFFRACTION6.441TORSIONAL
31H2012X-RAY DIFFRACTION6.441TORSIONAL
32K2012X-RAY DIFFRACTION6.441TORSIONAL
41L1914X-RAY DIFFRACTION6.441TORSIONAL
42M1914X-RAY DIFFRACTION6.441TORSIONAL
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 3 / % reflection obs: 100 %

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all
6.7-7.660.29491370.285229473084
7.66-9.590.26221530.217330043157
9.59-300.25681660.199931843350

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