Coil-to-α-helix transition at the Nup358-BicD2 interface activates BicD2 for dynein recruitment

  1. James M Gibson
  2. Heying Cui
  3. M Yusuf Ali
  4. Xiaoxin Zhao
  5. Erik W Debler
  6. Jing Zhao
  7. Kathleen M Trybus  Is a corresponding author
  8. Sozanne R Solmaz  Is a corresponding author
  9. Chunyu Wang  Is a corresponding author
  1. Department of Biological Sciences, Department of Chemistry and Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, United States
  2. Department of Chemistry, Binghamton University, United States
  3. Department of Molecular Physiology and Biophysics, Larner College of Medicine, University of Vermont, United States
  4. Laboratory of Cell Biology, The Rockefeller University, United States
9 figures, 7 tables and 1 additional file

Figures

Figure 1 with 1 supplement
A minimal Nup358 domain interacts with BicD2 with micromolar affinity.

(a) Nup358 interacts with both BicD2/dynein/dynactin and kinesin-1 (via kinesin-1 light chain 2, KLC2) to mediate bidirectional nuclear positioning in G2 phase of the cell cycle (Splinter et al., …

Figure 1—source data 1

ITC thermogram of BicD2-CTD and Nup358-min.

https://cdn.elifesciences.org/articles/74714/elife-74714-fig1-data1-v4.zip
Figure 1—source data 2

ITC thermogram of BicD2-CTD and Nup358-min-GST (i.e., with the GST-tag intact).

https://cdn.elifesciences.org/articles/74714/elife-74714-fig1-data2-v4.zip
Figure 1—source data 3

ITC thermogram of Nup358-min into buffer.

https://cdn.elifesciences.org/articles/74714/elife-74714-fig1-data3-v4.zip
Figure 1—source data 4

ITC thermogram of Nup358-min-GST into buffer.

https://cdn.elifesciences.org/articles/74714/elife-74714-fig1-data4-v4.zip
Figure 1—figure supplement 1
The GST-fusion has a neglible effect on the interaction between BicD2-CTD and Nup358-min.

(a) The isothermal titration calorimetry (ITC) thermogram of BicD2-CTD with Nup358-min-GST (i.e., with the GST-tag intact) is shown, from which the affinity was determined to be 1.6 ± 1.0 μM, …

Figure 2 with 16 supplements
Nup358min-zip is capable of forming a dynein-dynactin-BicD2-Nup358min-zip complex (DDBNmin-zip) that is activated for processive motility.

(a) Schematic of a DDBNmin-zip complex bound to a microtubule. BicD2 and Nup358 are shown labeled with two different color quantum dots (Qdot; stars). (b) Representative kymograph of the DDBNmin-zip

Figure 2—source data 1

The number of processive events of DDBNmin-zip compared with DDBCC1, DDB, and DDNmin-zip.

https://cdn.elifesciences.org/articles/74714/elife-74714-fig2-data1-v4.xlsx
Figure 2—source data 2

Speed of DDBNmin-zip compared with the constitutively active complex DDBCC1.

https://cdn.elifesciences.org/articles/74714/elife-74714-fig2-data2-v4.xlsx
Figure 2—source data 3

Run length of DDBNmin-zip compared with the constitutively active complex DDBCC1.

https://cdn.elifesciences.org/articles/74714/elife-74714-fig2-data3-v4.xlsx
Figure 2—figure supplement 1
Dimerization of Nup358min increases the formation of DDBNmin-zip complexes.

(a) A leucine-zippered Nup358 (Nup358min-zip) forms DDBN complex (yellow) at a higher frequency than that of Nup358 monomer (Nup358min) (gray). The percent formation of DDBNmin-zip complex was 35% …

Figure 2—video 1
Dynein-dynactin-BicD2-Nup358min-zip (DDBNmin-zip) is indicated by dual-color quantum dot (Qdot) (yellow) moving on microtubule tracks (not always seen due to photobleaching) at 2 mM MgATP.

Nup358 min-zip is labeled with a 655 nm Qdot (red), BicD2 is labeled with a 525 nm Qdot (green), and dynein-dynactin is unlabeled. Some single-color Qdots are moving long distances, presumably …

Figure 2—video 2
Fewer dynein-dynactin-BicD2 (DDB) complexes (green) show processive events compared to DDBNmin-zip (see Figure 2f).

BicD2 was labeled with a 525nm Qdot (green), and dynein-dynactin was unlabeled. Movie is displayed 12X real speed. Scale bar 5µm.

Figure 2—video 3
Dynein-dynactin-BicD2CC1 (DDBCC1) complex (green) moving on microtubule tracks at 2mM MgATP at 2mM MgATP.

BicD2 was labeled with a 525nm Qdot (green) and dynein-dynactin was unlabeled. Movie is displayed 12X real speed. Scale bar 5µm.

Figure 2—video 4
Source video (replicate of Figure 2—video 1).

DDBNmin-zip (yellow) moving on microtubule tracks at 6X real speed.

Figure 2—video 5
Source video (replicate of Figure 2—video 1).

DDBNmin-zip (yellow) moving on microtubule tracks at 6X real speed.

Figure 2—video 6
Source video (replicate of Figure 2—video 1).

DDBNmin-zip (yellow) moving on microtubule tracks at 6X real speed.

Figure 2—video 7
Source video (replicate of Figure 2—video 2).

Fewer dynein-dynactin-BicD2 (DDB) complexes (green) show processive events compared to DDBNmin-zip (displayed at 6X real speed).

Figure 2—video 8
Source video (replicate of Figure 2—video 2).

Fewer dynein-dynactin-BicD2 (DDB) complexes (green) show processive events compared to DDBNmin-zip (displayed at 6X real speed).

Figure 2—video 9
Source video (replicate of Figure 2—video 2).

Fewer dynein-dynactin-BicD2 (DDB) complexes (green) show processive events compared to DDBNmin-zip (displayed at 6X real speed).

Figure 2—video 10
Source video (replicate of Figure 2—video 3).

Dynein-dynactin-BicD2CC1 (DDBCC1) complex moving on microtubule tracks, displayed at 6X real speed.

Figure 2—video 11
Source video (replicate of Figure 2—video 3).

Dynein-dynactin-BicD2CC1 (DDBCC1) complex moving on microtubule tracks, displayed at 6X real speed.

Figure 2—video 12
Source video (replicate of Figure 2—video 3).

Dynein-dynactin-BicD2CC1 (DDBCC1) complex moving on microtubule tracks, displayed at 6X real speed.

Figure 2—video 13
Source video.

Dynein-dynactin-Nup358min-zip (DDNmin-zip) complexes (red) do not show processive motion on microtubule tracks. Nup358min-zip was labeled with a 655nm Qdot (red), and dynein-dynactin was unlabeled. …

Figure 2—video 14
Source video (replicate of Figure 2—video 13).

Dynein-dynactin-Nup358min-zip (DDNmin-zip) complexes do not show processive motion on microtubule tracks. Movie displayed at 6X real speed.

Figure 2—video 15
Source video (replicate of Figure 2—video 13).

Dynein-dynactin-Nup358min-zip (DDNmin-zip) complexes do not show processive motion on microtubule tracks. Movie displayed at 6X real speed.

Figure 3 with 3 supplements
Nuclear magnetic resonance (NMR) titration mapped the BicD2-binding site to the N-terminal half of Nup358-min.

NMR mapping of Nup358 regions involved in BicD2-CTD binding was performed by titration of 15N-labeled Nup358-min with BicD2-CTD. (a) The HSQC spectrum of a 1:1 Nup358-min/BicD2-CTD complex (blue) is …

Figure 3—figure supplement 1
Fully assigned 15 N-1H HSQC nuclear magnetic resonance (NMR) spectrum for Nup358-min.

Inset shows the assignment of peaks in a crowded region.

Figure 3—figure supplement 2
Three points of Nup358:BicD2 titration.

The titration clearly shows a consistent drop in intensity of the peaks in Nup358 that are in the BicD2-binding region. The Nup358 concentration was held constant at 1, and unlabeled BicD2 was …

Figure 3—figure supplement 3
TALOS-N demonstrates random coil for Nup358-min.

TALOS-N, a highly accurate program for determining secondary structure based on chemical shifts, was used to determine the secondary structure of the apo protein. The majority of the protein heavily …

Figure 4 with 4 supplements
Chemical exchange saturation transfer (CEST) maps chemical shifts of nuclear magnetic resonance (NMR)-invisible, BicD2-bound state of Nup358.

In the CEST profile curve, E2194 and I2211 have only a single dip in 15N-CEST (a) and 13C′-CEST (b), respectively, due to little chemical shift perturbation upon BicD2-CTD binding. This suggests …

Figure 4—figure supplement 1
15N chemical exchange saturation transfer (CEST) profiles for Nup358 residues showing exchange between random coil state and an α-helical state.

These CEST profiles have a double-dip appearance, showing the bound state, represented by the minor dip as well as the free state, represented by the major dip. This experiment was repeated under …

Figure 4—figure supplement 2
13C′ chemical exchange saturation transfer (CEST) profiles for Nup358 residues showing exchange between random coil state and an α-helical state, showing the minor dip as well as the major dip, representing the chemical shift of the bound state.

This experiment was performed using a 20:1 molar ratio of Nup358-min to BicD2-CTD. The following residues, already identified as α-helical residues, using the 15N CEST experiments, were confirmed …

Figure 4—figure supplement 3
15N chemical exchange saturation transfer (CEST) profiles for Nup358 residues showing NO exchange between the random coil state and an α-helical state.

These 15N CEST curves show only a single dip at the major chemical shift, indicating that these sites do not undergo chemical exchange to a bound state. Note that these sites include residues around …

Figure 4—figure supplement 4
13C chemical exchange saturation transfer (CEST) profiles for Nup358 residues showing NO exchange between a random coil state and an α-helical state, showing only a single dip at the major chemical shift.

Note that these sites include residues around the LEWD motif as well as sites at the C-terminus half of Nup358-min (see Figure 4).

Figure 5 with 1 supplement
Circular dichroism (CD) spectroscopy confirms formation of an α-helix in the Nup358/BicD2 complex.

CD wavelength scans of BicD2-CTD (red), Nup358-min (blue), and the Nup358-min/BicD2 complex (green) at 4°C are shown. The sum of the individual wavelength scans of Nup358-min and BicD2-CTD is shown …

Figure 5—figure supplement 1
Calibration curve for the circular dichroism (CD) signal, based on a published melting curve of BicD2-CTD.

We previously published the thermal unfolding curve of wild-type BicD2-CTD that was recorded by CD spectroscopy at 222 nm (Figure 4E in reference Noell et al., 2019). Here, this curve was replotted …

Figure 6 with 4 supplements
Low-resolution structures determined by small-angle X-ray scattering (SAXS) confirm that the complex has a rod-like shape that is more compact than the individual proteins.

(a) Dimensionless Kratky plots of the SAXS data collected from the minimal Nup358/BicD2 complex, from Nup358-min and BicD2-CTD (q: scattering vector; RG: radius of gyration; I(q): scattering …

Figure 6—figure supplement 1
SAXS plots for Nup358-min/BicD2-CTD complex.

(a) The scattering intensity profile I(q) is shown as a function of the scattering vector (q). (b) The Guinier plot (top panel) is shown together with the normalized residual of the Guinier fit …

Figure 6—figure supplement 2
SAXS plots for Nup358-min.

(a) The scattering intensity profile I(q) is shown as a function of the scattering vector (q). (b) The Guinier plot (top panel) is shown together with the normalized residual of the Guinier fit …

Figure 6—figure supplement 3
SAXS plots for BicD2-CTD.

(a) The scattering intensity profile I(q) is shown as a function of the scattering vector (q). (b) The Guinier plot (top panel) is shown together with the normalized residual of the Guinier fit …

Figure 6—figure supplement 4
The pair distance distribution p(r) functions of the Nup358-min/BicD2-CTD complex, Nup358-min, and BicD2-CTD.

The p(r) functions were derived from the SAXS profiles (Figure 6—figure supplements 13) and normalized towards the highest signal.

Figure 7 with 1 supplement
Mutagenesis of the Nup358 cargo recognition helix.

All residues of the Nup358 α-helix were mutated to alanine, and binding was assessed by GST-pull-down assays with purified Nup358-min-GST followed and BicD2-CTD. The elution fractions were analyzed …

Figure 7—figure supplement 1
Interface residues of BicD2 and Nup358 were identified by mutagenesis.

SDS-PAGE analyses of elution fractions of the pull-down assays (see Figure 7) to probe the effect of Nup358 mutants on the interaction with BicD2. The molecular weights and band position of standard …

Figure 7—figure supplement 1—source data 1

BicD2-CTD calibration curve for quantification.

https://cdn.elifesciences.org/articles/74714/elife-74714-fig7-figsupp1-data1-v4.xlsx
Figure 8 with 1 supplement
Nup358 point mutations that diminish the interaction with BicD2-CTD also diminish the formation of the DDBN complex.

(a) Bar graph of the processive events of DDBNmin-zip complexes per min per micrometer MT length. The number of processive events of DDBNmin-zip complexes formed with WT-Nup358min-zip is …

Figure 8—source data 1

The number of processive events of DDBNmin-zip complexes formed with WT-NUP358min-zip compared with DDBNmin-zip formed with NUP 358min-zip mutants I2167A, M2173A, F2180A, and L2184A.

https://cdn.elifesciences.org/articles/74714/elife-74714-fig8-data1-v4.xlsx
Figure 8—source data 2

The presence of dynein-dynactin increases the formation of BicD2-NUP358min-zip complexes.

https://cdn.elifesciences.org/articles/74714/elife-74714-fig8-data2-v4.xlsx
Figure 8—source data 3

Comparison of the speeds of DDBN complexes formed with WT-NUP358min-zip and mutant NUP358min-zip constructs.

https://cdn.elifesciences.org/articles/74714/elife-74714-fig8-data3-v4.xlsx
Figure 8—source data 4

Comparison of the run lengths of DDBN complexes formed with WT-NUP358min-zip and mutant NUP358min-zip.

https://cdn.elifesciences.org/articles/74714/elife-74714-fig8-data4-v4.xlsx
Figure 8—figure supplement 1
Nup358 point mutations that diminish the interaction with BicD2-CTD also diminish the formation of the DDBNmin-zip complex.

(a–e) Visualization of complex formation of DDBNmin-zip, formed with WT Nup358min-zip and Nup358min-zip mutants I2167A, M2173A, L2177A, and L2184. DDBNmin-zip formed with mutant F2180A is not shown …

We propose that BicD2 recognizes its cargo through a short ‘cargo recognition α-helix,’ which may also be a structural feature that stabilizes the activated state of BicD2 for the recruitment of dynein and dynactin.

(a) Cutaway view of half of an NPC. Each of the eight spokes of the NPC contains four molecules of Nup358 on the cytoplasmic side (i.e., 32 in total), which provide binding sites for dynein (via …

Tables

Table 1
Chemical shift differences from chemical exchange saturation transfer (CEST) of Nup358/BicD2 and apo-Nup358 (Δδbound-apo) match closely to Δδ for coil-to-α-helix transition (Δδhelix-coil).
ResidueΔδbound-apoΔδhelix-coil
A2163*–3.6–2.2
A2164*–3.6–2.2
K2165*–0.8–1.3
L2166*–3.8–1.9
I2167*–5.7–1.2
K2178*–3.6–1.3
L2184*–5.5–1.9
R21621.72.3
A21642.81.7
K21652.72.1
L21662.61.6
R21692.92.3
E21712.22.2
E21723.02.2
L21770.91.6
K21813.02.1
F21833.31.5
L21841.01.6
  1. CD spectroscopy confirms formation of an α-helix in Nup358 upon binding to BicD2.

  2. *

    Change in chemical shifts of amide 15N.

  3. Change in chemical shifts of carbonyl 13C.

  4. Values taken from Wishart, 2011.

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Escherichia coli)Rosetta 2(DE3)-pLysSFisher ScientificCat# 714033
Strain, strain background (E. coli)BL21-DE3-CodonPlus-RILFisher scientificCat#50-125-350
Recombinant DNA reagentHuman BicD2-CTD pet28aGenScript Reference
Noell et al., 2019
doi:10.1021/acs.jpclett.9b01865
Sequence encoding residues 715–804 of human BicD2 cloned into a pet28a vector via the NdeI and XhoI restriction sitesFor the protein sequence expressed from this vector, see ‘Supplementary methods’
Recombinant DNA reagentHuman Nup358-min pGEX-6P-1GenScript Reference
Noell et al., 2019doi:10.1021/acs.jpclett.9b01865
Sequence encoding residues 2148–2240 of human Nup358 cloned into a pGEX-6P-1 vector via the BamHI and XhoI restriction sitesFor the protein sequence expressed from this vector, see ‘Supplementary methods’
Recombinant DNA reagentNup358-min-pGEX-6P-1GenScriptThis paperModified Nup358-min pGEX-6P-1 vector that includes a SNAP-tag at its C-terminal domainFor the protein sequence expressed from this vector, see ‘Supplementary methods’This vector can be obtained from Dr. Solmaz’s lab
Recombinant DNA reagentNup358-min-zip pGEX-6P-1GenScriptThis paperModified Nup358-min-pGEX-6P-1 vector that includes a leucine zipper that was added at the C-terminus before the snap-tagFor the protein sequence expressed from this vector, see ‘Supplementary methods’This vector can be obtained from Dr. Solmaz’s lab
Recombinant DNA reagentHuman Nup358 (residues 2158–2199) pGEX-6P-1GenScriptThis paperSequence encoding residues 2158–2199 of human Nup358 cloned into a pGEX-6P-1 vector via the BamHI and XhoI restriction sitesProtein overexpression plasmid, which can be obtained from Dr. Solmaz’s lab
Peptide, recombinant proteinPreScission proteaseCytivaCat# 27084301
Peptide, Recombinant proteinThrombin, human plasmaFisher ScientificCat# 6051951000U
Chemical compound, drug13C D-Glucose (U-13C6)Cambridge Isotope LaboratoriesCat# CLM-1396-PK
Chemical compound, drug15NH4ClCambridge Isotope LaboratoriesCat# NLM-467-10
Chemical compound, drugcOmplete EDTA-free Protease Inhibitor Cocktail tabletsRocheCat# 45-5056489001-EA
Software, algorithmOrigin Student 2018OriginLabCD spectroscopy
Software, algorithmOrigin GE Microcal ITC200OriginLabITC
Software, algorithmBioXTAS RAW software suite (version 2.0.3).Reference Hopkins et al., 2017
Software, algorithmImageJ 1.52vReference Schneider et al., 2012
Software, algorithmUCSF Chimera (version 1.14)Resource for Biocomputing, Visualization, and Informatics at the University of California, San FranciscoReference Pettersen et al., 2004
Chemical compound, drugCoomassie brilliant blue R-250VWRCat# VWRV0472-25G
Chemical compound, drugRNase InhibitorPromegaN261B
Chemical compound, drugQ-dot 525 streptavidin conjugateInvitrogenQ10141MP
Chemical compound, drugQ-dot 565 streptavidin conjugateInvitrogenQ10131MP
Chemical compound, drugQ-dot 655 streptavidin conjugateInvitrogenQ10121MP
Chemical compound, drugSNAP-BiotinNew England BioLabsS9110S
Chemical compound, drugTubulin protein (X-rhodamine): bovine brainCytoskeleton, IncTL620M-A
Chemical compound, drugPaclitaxelCytoskeleton, IncTXD01
Recombinant DNA reagentBicaudal D homolog 2 isoform 2 (Homo sapiens)This paperNCBI:NP_056065.1Protein overexpression plasmid, which can be obtained from Dr. Solmaz’s lab
Biological sample (Bos taurus)Dynein-dynactinBovine brain
Biological sample(B. taurus)TubulinBovine brain
Software, algorithmNikon ECLIPSE Ti microscopeNikon
Software, algorithmNikon NIS ElementsNikon
Software, algorithmAndor EMCCD CameraAndor Technology USA
Software, algorithmPrismGraphPadv7; RRID:SCR_002798
Chemical compound, drug2-[Methoxy(polyethyleneoxy)propyl]trimethoxysilaneJ&K Scientific967192
Chemical compound, drugn-ButylamineAcros OrganicsA0344582
Software, algorithmImageJ FijiNIH1.53c
Software, algorithmNMRPipeNIH, reference Delaglio et al., 1995
Software, algorithmNMRFAM_SPARKYReference Lee et al., 2015
Appendix 1—table 1
Summary of the 15N CEST Curve Fits.
(a) Summary of 15N CEST curve fits at 20:1 molar ratio of BicD2:15N-Nup358
Residuekex (s–1)PbΔδ (ppm)
A2164704 ± 2110.07 ± 0.02–4.3 ± 0.4
K2165139 ± 1620.25 ± 0.08–0.8 ± 0.3
L2166381 ± 1820.07 ± 0.03–3.8 ± 0.3
I2167975 ± 6240.02 ± 0.04–5.7 ± 0.2
K217831 ± 350.01 ± 0.01–3.6 ± 0.2
L2184160 ± 920.07 ± 0.03–5.5 ± 0.2
(b) Summary of 15N CEST curve fits at 10:1 molar ratio of BicD2:15N-Nup358
Residuekex (s–1)PbΔδ (ppm)
A216312 ± 730.07 ± 0.05–3.6 ± 0.2
A2164228 ± 720.09 ± 0.02–3.6 ± 0.2
K2165757 ± 2730.13 ± 0.07–3.6 ± 0.2
L2166297 ± 1950.05 ± 0.02–3.6 ± 0.2
  1. The change in chemical shift in Table 1 was taken from Table S1b for the alanine residues due to a clearer minor peak. For the others, the change in chemical shift in Table 1 was taken from Table S1a. The values used in the weighted averages for kex and Pb were taken from Table S1a, excluding the K2165 and K2178 with abnormal Pb and significant noise in their CEST curves. Including the K2165 and K2178 values, the weighted average kex value would be 80 ± 30 s–1, and the Pb value would be 0.03 ± 0.01.

  2. CEST: chemical exchange saturation transfer.

Appendix 1—table 2
Summary of 13C′ CEST curve fits at 20:1 molar ratio of [BicD2]:[Nup358].
Residuekex (s–1)PbΔδ (ppm)
R2162313 ± 3280.05 ± 0.031.7 ± 0.3
A2164474 ± 3040.07 ± 0.032.8 ± 0.2
K2165208 ± 1790.05 ± 0.072.7 ± 0.1
L2166391 ± 1700.04 ± 0.032.6 ± 0.2
R2169140 ± 1250.06 ± 0.012.9 ± 0.2
E2171359 ± 1750.07 ± 0.032.2 ± 0.2
E21728 ± 980.22 ± 0.073.0 ± 0.1
L2177540 ± 2410.03 ± 0.020.9 ± 0.3
K2181316 ± 1550.06 ± 0.023.0 ± 0.1
F2183464 ± 980.06 ± 0.013.3 ± 0.3
L2184238 ± 1650.08 ± 0.041.0 ± 0.2
Appendix 2—table 1
Summary of SAXS data.
SampleRG (Å)Guinier plotRG (Å) p(r) functionI(0) p(r) function/I(0) Guinier plotTE /χ2p(r) functionDMax p(r) functionMW* (kDa)
Nup358/BicD248.1 ± 0.655.5 ± 0.20.086/0.0860.61/1.0419047.7/46.0/47.6
Nup358-min28.4 ± 0.330.6 ± 0.40.035/0.0360.69/1.0512012.7/12.3/na
BicD2-CTD42.6 ± 1.049.7 ± 0.90.066/0.0700.67/1.10190na/na/38.7
  1. Note that not all methods can be applied to all samples (na). The error of molar masses determined by SAXS is 10%. The calculated MWs are 10.9 kDa for BicD2-CTD and 10.6 kDa for Nup358-min.

  2. RG: radius of gyration; I(0): scattering intensity at zero angle; TE: total estimate; DMAX: maximum particle diameter; MW: molar mass; SAXS: small-angle X-ray scattering.

  3. *

    MWs were determined from I(0), using glucose isomerase as a standard (Konarev et al., 2003).

  4. MWs were determined from I(0) without a molar mass standard (Konarev et al., 2003).

  5. MWs were determined from the volume of correlation as described by Rambo and Tainer, 2013.

Appendix 2—table 2
Statistics of SAXS bead model 3D reconstruction.
SampleMean normalized spatial discrepancy score (NSD)χ2 (refined model)/χ2 (representative model)Resolution (Å)Ambimeter score
Nup358-min/BicD2-CTD0.70 ± 0.051.08/1.0541 ± 31.23
Author response table 1
Binding ratioStandard deviation3x Standard deviationInterface residue?
WT1.00n/an/an/a
21690.860.030.09Yes
21720.930.010.03No
21810.870.030.09Yes

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