Abstract
It remains particularly problematic to define the structures of native macromolecular assemblies, which are often of low abundance. Here we present a strategy for isolating complexes at endogenous levels from GFP-tagged transgenic cell lines. Using cross-linking mass spectrometry, we extracted distance restraints that allowed us to model the complexes' molecular architectures.
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Acknowledgements
We are grateful to A.N. Krutchinsky (The Rockefeller University, New York, New York, USA) for providing the yeast GFP-CDC16 strain; S. Obado for technical assistance; E. Jacobs, J. Lacava, S.J. Kim and B. Webb for discussions and assistance; and Z. Yue (Icahn School of Medicine at Mount Sinai, New York, New York, USA) for sharing Becn1-EGFP/+ mice. Y.S. acknowledges M. Chen (R.G. Roeder lab, The Rockefeller University, New York, New York, USA) for generating a reagent that was not used for this study. This work was funded by the US National Institutes of Health (grants P41 GM103314 (to B.T.C.), R01 GM083960 (to A.S.) and P41 GM109824 (to A.S., M.P.R. and B.T.C.)) and the Ellison Medical Foundation (Q.J.W.).
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Y.S. and B.T.C. conceived the research, with input from M.P.R., J.F.-M., R.P., Q.J.W., A.S. and P.C.F. Y.S., J.F.-M., P.C.F. and M.K.T. cloned and purified the nanobodies. P.C.F. performed the surface plasmon resonance measurements. Y.S. and J.F.-M. carried out the microscopic imaging. Y.S. performed biochemical and mass spectrometric analyses. R.P. performed the modeling analyses. Q.J.W. contributed reagents and discussed the results. P.C.F. and Y.L. discovered the LaG-16 nanobody. Y.S. and B.T.C. wrote the paper with input from M.P.R., J.F.-M., R.P., Q.J.W., A.S. and P.C.F. All authors reviewed the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Protein sequences of the nanobodies 3K1K and LaG-16 and localizations of lysine residues on the crystal structure of PDB# 3K1K.
(a) The protein sequence alignments of 3K1K and LaG-16. The lysine residues are highlighted in cyan. CDRs: complementarity determining regions. CDR1, CDR2 and CDR3 sequences are color coded in blue, pink and orange, respectively.
(b) Localizations of the three lysine residues on the crystal structure of nanobody PDB 3K1K, which were mutated to arginine (R) or glutamine (Q) for the present work. The lysine residues are presented as red spheres.
Supplementary Figure 2 Protein expression levels and affinity (KD) measurements of anti-GFP nanobodies.
(a) The relative expression levels of wild-type and lysineless anti-GFP nanobodies. The protein expression levels are normalized relative to the expression level of wild-type LaG-16. AU, arbitrary unit.
(b) Dissociation constants (KD, in nM) of the different nanobodies indicated as measured by SPR.
(c) SPR KD measurements of LaG16-2K/R. Nanobodies at different concentrations were injected in triplicate for analysis. SPR binding sensorgrams were processed and analyzed using the ProteOn Manager software. Curves fitted by a Langmuir model are shown in black. RU, response units. The x-axis is the dissociation time in seconds.
(d) SPR KD measurements of 3K1K-3K/R.
Supplementary Figure 3 Comparison of the recovery efficacies of cross-linked APC/C complex by in-house-generated Ilama polyclonal antibodies, the LaG-16 and the LaG-16 lysine-less nanobody (2K/R) after on-bead crosslinking followed by denaturing elution.
(a) Silver stain of the on-bead cross-linked APC/C complex that can be eluted by denaturing buffer. Equal amounts of in-house-produced Ilama polyclonal antibodies (Poly), wild-type LaG-16, and lysineless LaG-16-2K/R (2K/R) nanobodies were conjugated in parallel to epoxy magnetic beads. The antibody-conjugated magnetic beads were treated by DSS cross-linker to block residual reactive amines and were subsequently used to affinity isolate APC/C complex (using GFP-tagged Cdc16). The isolated complex was cross-linked on-bead (0.1 mM DSS). After on-bead cross-linking, the cross-linked complex that was non-covalently bound to the nanobody resins was eluted by hot LDS buffer (heated at 85°). The resulting protein complex was subjected to SDS-PAGE. The efficiently cross-linked APC/C complex corresponds to the high-molecular-weight region (>350 kDa) of the gel. A fraction of each sample was visualized by silver staining.
(b) Comparison of the recoveries of the cross-linked APC/C protein complex that could be eluted from GFP antibody-conjugated magnetic resins. Gel regions corresponding to the GFP-tagged protein Cdc16 (~70–160 kDa for non-cross-linked APC/C complex or >350 kDa for cross-linked APC/C complex) were in-gel digested by trypsin and analyzed by LC-MS. The experiments were independently repeated three times using freshly prepared antibody-conjugated beads (biological repeats), and for each biological repeat three analytical repeats were performed. The relative intensities of the GFP-tagged protein Cdc16 (which represents the relative amount of the complex) from three different anti-GFP reagents were quantified by MaxQuant (version 1.5) using label-free quantification (LFQ). Relative intensities of cross-linked APC/C are normalized to the sum of the input intensities of 2K/R.
Supplementary Figure 4 SDS-PAGE gels of the Rrp6-GFP- and Ski7-GFP-associated exosome complexes that were affinity captured by lysineless nanobodies
Protein bands on each SDS PAGE gel were sliced and in-gel digested. The digested peptides from different bands were pooled, desalted and analyzed by LC-MS. The major proteins corresponding to the bands are labeled
Supplementary Figure 5 Subcellular localization of Rrp6-GFP and Ski7-GFP.
Rrp6-GFP and Ski7-GFP yeast strains were grown to mid-log phase and harvested. Cells were stained with 2.5 μg/ml DAPI for 20 min and washed with PBS buffer, and the GFP signals were visualized using a fluorescence microscope as described in the Online Methods. The images were processed with Adobe Photoshop. DIC, differential interference contrast.
Supplementary Figure 6 Overview of cross-links that can be measured on the crystal structure of exo11 (PDB 4IFD).
Blue lines represent DSS cross-links connecting two amino acid residues within the reach of 30 Å, and red lines are DSS cross-links that are longer than 44 Å. The figure was prepared using UCSF Chimera software.
Supplementary Figure 7 Euclidean Cα-Cα distances of the cross-links on the crystal structures PDB 4IFD and PDB 2WP8 and a representative high-resolution MS/MS cross-link spectrum.
a) Euclidean Cα-Cα distances of the cross-links on the crystal structures PDB 4IFD and PDB 2WP8. The blue bars represent the cross-link distances measured on the 4IFD structure, and the pink bar represents the cross-link distances measured on the 2WP8 structure. Asterisks indicate the cross-links that are longer than 44 Å. b) A high-resolution MS/MS spectrum of the DSS cross-link between lysine 497 of Rrp44 and lysine 292 of Rrp45. The Cα-Cα distance between the cross-linked residues is 60.6 Å on the crystal structure. A ladder of HCD fragmentations occurs along the peptide bonds of both peptide chains, and fragmentations are labeled as b-ions and y-ions. The underlined methionine residue is oxidized. The charge state of this peptide ion is +5. The asterisk designates an immonium ion, which is commonly observed in HCD fragmentation spectra.
Supplementary Figure 8 Residue-specific RMSFs (root-mean-square fluctuations) of the three exosome subunits of Lrp, Rrp6 and Ski7.
Residue-specific RMSFs of the three exosome subunits of (a) Lrp1, (b) Rrp6 and (c) Ski7. The x-axis of the plots is each individual residue number of the protein sequence, and the y-axis is the calculated RMSF in Å.
Supplementary Figure 9 A high-resolution MS/MS spectrum of the “same residue” cross-link on lysine 565 of the APC/C subunit Cdc27.
A ladder of HCD fragmentations occur along the peptide bonds of both peptide chains and fragments are labeled as b-ions and y-ions. The charge state of the selected peptide ion is +7. Asterisks as in Supplementary Figure 8.
Supplementary Figure 10 Cross-linking titrations of affinity-purified (a) exosome, (b) APC/C and (c) Beclin 1 complexes.
The protein complexes were affinity-captured and cross-linked on-bead at various concentrations of DSS cross-linker. The cross-linked complexes were eluted and separated on SDS-PAGE gels, and the cross-linked complexes were silver-stained for visualization.
Supplementary Figure 11 SDS-PAGE analysis of exosome complexes purified from whole-cell lysates treated with various concentrations of DSS cross-linker.
Exosome components with a molecular weight greater than 50 kDa are labeled. Affinity-captured exosome protein composites from 0 and 5 mM DSS treatment were analyzed by LC-MS. The proteins were then identified by X! Tandem, and their relative abundance was quantified by spectra counting.
Supplementary Figure 12 Representation of the exosome subunits for integrative modeling.
All subunits are represented by a set of beads arranged into either rigid bodies corresponding to structured parts (blue segments in the bars) or flexible strings corresponding to unknown/disordered parts (yellow segments in the bars). The entire exo10 complex was kept rigid.
Supplementary Figure 13 The relative stoichiometry of the cell compartment–specific exosome complexes.
The SDS-PAGE gels of the Rrp6- and Ski7-associated exosome complexes were stained with Sypro Ruby. The proteins were visualized using an LAS-3000 Fujifilm system, and the corresponding intensities of the four protein bands were measured by ImageJ to calculate their stoichiometries relative to Rrp44. The error bars represent standard deviations from four independent experiments. The gel band at 20 kDa corresponds to both Lrp1 and Mpp6. However, because Lrp1 is the dominant protein in the band, the protein intensity of Mpp6 was neglected for the present stochiometric analysis of Rrp6-associated, nucleus-specific exosome complex.
Supplementary Figure 14 Cross-link satisfaction and normalized contact frequency maps of (a) Rrp6-Lrp1-exo10 and (b) Ski7-exo10 exosome complexes.
Each box is a two-dimensional matrix whose x- and y-axes are the residue indexes of two exosome subunit sequences. Satisfied cross-links and violated cross-links are represented by green and orange circles, respectively. The normalized contact frequency map is represented by gray shades, where darker gray represents residue-residue contacts that occur more frequently in the cluster of solutions.
Supplementary Figure 15 Mapping of the Beclin 1 cross-links on the available crystal structure (PDB 3Q8T) of the coil-coil domain (residues 172–265).
Lysine residues are represented as red spheres, and the cross-links between lysine residues are represented by black lines. The figure was prepared using UCSF Chimera.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–15 and Supplementary Note (PDF 2202 kb)
Supplementary Table 1
MS identification of the Saccharomyces cerevisiae Rrp46-associated exosome complexes. (XLSX 15 kb)
Supplementary Table 2
MS identifications of the Rrp6-associated exosome complex. (XLSX 16 kb)
Supplementary Table 3
MS identifications of the Ski7-associated exosome complex. (XLSX 17 kb)
Supplementary Table 4
A data set of cross-linked peptides from exosome complexes. Intersubunit cross-links are highlighted in orange. (XLSX 29 kb)
Supplementary Table 5
MS identifications of the Saccharomyces cerevisiae APC/C complex. (XLSX 18 kb)
Supplementary Table 6
A data set of cross-linked peptides from the APC/C complex. Intersubunit cross-links are highlighted in orange. (XLSX 30 kb)
Supplementary Table 7
MS identifications of the Beclin 1 complexes from mouse (Mus musculus) liver. (XLSX 16 kb)
Supplementary Table 8
A data set of cross-linked peptides from the Beclin 1 complex. Intersubunit cross-links are highlighted in orange. (XLSX 18 kb)
Supplementary Table 9
Clustering analysis of the exosome protein complexes (composites) that are affinity-isolated from the whole-cell lysates that are treated with 0 mM and 5 mM DSS cross-linker. (XLSX 52 kb)
Supplementary Table 10
Comparison of cross-link data sets. Intersubunit cross-links are highlighted in orange. (XLSX 40 kb)
Supplementary Data
Annotated, high-resolution HCD-MS/MS cross-link spectra for the APC/C complex, the exosome complex, and the Beclin 1 complex. (ZIP 7728 kb)
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Shi, Y., Pellarin, R., Fridy, P. et al. A strategy for dissecting the architectures of native macromolecular assemblies. Nat Methods 12, 1135–1138 (2015). https://doi.org/10.1038/nmeth.3617
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DOI: https://doi.org/10.1038/nmeth.3617
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