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. 2018 Jun 1;293(22):8554-8568.
doi: 10.1074/jbc.RA117.001568. Epub 2018 Apr 12.

Conformational flexibility within the nascent polypeptide-associated complex enables its interactions with structurally diverse client proteins

Esther M Martin  1 , Matthew P Jackson  1 , Martin Gamerdinger  2 , Karina Gense  2 , Theodoros K Karamonos  1 , Julia R Humes  1 , Elke Deuerling  2 , Alison E Ashcroft  1 , Sheena E Radford  3
Affiliations

Conformational flexibility within the nascent polypeptide-associated complex enables its interactions with structurally diverse client proteins

Esther M Martin et al. J Biol Chem. .

Abstract

As newly synthesized polypeptides emerge from the ribosome, it is crucial that they fold correctly. To prevent premature aggregation, nascent chains interact with chaperones that facilitate folding or prevent misfolding until protein synthesis is complete. Nascent polypeptide-associated complex (NAC) is a ribosome-associated chaperone that is important for protein homeostasis. However, how NAC binds its substrates remains unclear. Using native electrospray ionization MS (ESI-MS), limited proteolysis, NMR, and cross-linking, we analyzed the conformational properties of NAC from Caenorhabditis elegans and studied its ability to bind proteins in different conformational states. Our results revealed that NAC adopts an array of compact and expanded conformations and binds weakly to client proteins that are unfolded, folded, or intrinsically disordered, suggestive of broad substrate compatibility. Of note, we found that this weak binding retards aggregation of the intrinsically disordered protein α-synuclein both in vitro and in vivo These findings provide critical insights into the structure and function of NAC. Specifically, they reveal the ability of NAC to exploit its conformational plasticity to bind a repertoire of substrates with unrelated sequences and structures, independently of actively translating ribosomes.

Keywords: NAC native mass spectrometry (MS); chaperone; molecular chaperone; nuclear magnetic resonance (NMR); protein cross-linking; protein folding; protein misfolding.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Schematic representation of NAC. a, domain structure of human NAC highlighting the UBA domain on α-NAC and the location of the ribosome-binding motif (RRK(X)n)KK) on β-NAC. b, dimerization of the NAC domains, with α-NAC shown in blue and β-NAC shown in purple (PDB 3LKX) (18).
Figure 2.
Figure 2.
Comparison of native ESI mass spectra of WT-NAC and ΔUBA-NAC. a and b, native ESI mass spectra of WT-NAC (a) and ΔUBA-NAC (b). Each spectrum shows two charge-state distributions for each heterodimer (green and red for WT-NAC and ΔUBA-NAC, respectively), with low populations of α2β2-NAC dimers as indicated. In addition, low populations of dissociated α-NAC and β-NAC subunits are observed (blue). The insets show the proteins analyzed by native PAGE, which reveal a single band of the heterodimer and no evidence of dissociation. c, estimated collision cross-sections (CCS) from ESI-IMS-MS experiments for WT-NAC (green circles) and ΔUBA-NAC (red triangles) show that compact and extended forms co-exist for both species (see also Table S1). d, far-UV CD spectra of WT-NAC (green) and ΔUBA-NAC (red). The secondary structure content obtained using CONTIN (58) is given in Table S2.
Figure 3.
Figure 3.
Collision-induced unfolding (CIU)-IMS-MS for the 12+ charge state ions of WT-NAC and ΔUBA-NAC. The quadrupole selected 12+ charge state ions are shown at a trap collision energy of 25 V for WT-NAC (a) andΔUBA-NAC (left) (b). Extracted ATDs for these charge states at 10 V, 25 V and 35 V are shown alongside.
Figure 4.
Figure 4.
Limited proteolysis of WT-NAC and ΔUBA-NAC followed by ESI-MS analysis. Each protein was treated with 1:500 (w/w) trypsin/substrate for 15 min at room temperature. a, native ESI-MS shows a truncated complex with a reduced mass of 2462 Da (blue peaks) and, which remains assembled, is the first cleavage product for both WT-NAC and ΔUBA-NAC. b, tryptic fragments observed in the low m/z range reveal a range of peptides released from the N-terminal domain of α-NAC (blue) and from the C-terminal domain of β-NAC (orange). The same fragments are observed for WT-NAC and ΔUBA-NAC. Numbers denote the fragments within each NAC domain. Charge states resulting from the intact (undigested) NAC are shown in black.
Figure 5.
Figure 5.
Interactions of α-synuclein with WT-NAC and ΔUBA-NAC. a, Coomassie Blue-stained native polyacrylamide gel of α-synuclein alone and mixed with an equimolar concentration of WT-NAC or ΔUBA-NAC, showing that a stable complex is not detected. b, kinetic aggregation assays of α-synuclein (125 μm, black), a 1:1 molar ratio of α-synuclein with WT-NAC (purple), and WT-NAC alone (green) measured using ThT fluorescence. An equimolar concentration of WT-NAC to α-synuclein inhibits its aggregation over a 70-h time scale. Images alongside show negative stain transmission electron micrographs of the reaction end point (at 70 h) for each sample. Scale bar, 500 nm. c, SDS-PAGE analysis of cross-linked WT-NAC and ΔUBA-NAC mixed with equimolar α-synuclein (a 50-fold excess of BS3 was used (“Experimental procedures”)). The arrows highlight the covalent complex between NAC and α-synuclein. d, map of cross-links between WT-NAC and α-synuclein identified following in-gel tryptic digestion of the band arrowed in c. Intra-NAC or intra-α-synuclein cross-links (purple), inter-NAC cross-links (green), and NAC–α-synuclein cross-links (red) are shown. Peptides identified are listed in Tables S3–S5. e, RNAi-mediated NAC depletion leads to increased α-synuclein puncta formation in vivo. Fluorescence microscope images of transgenic worms (head regions are shown) expressing α-synuclein::YFP in body-wall muscle cells. Worms were grown on empty vector control (ev) or αβ-NAC RNAi, respectively. Images were taken at days 1 and 3 of adulthood. Scale bar, 50 μm. Inset, Western blotting shows NAC protein expression levels, at indicated time points, by immunoblotting. Immunoblot against actin served as loading control.
Figure 6.
Figure 6.
1H–15N HSQC spectra of α-synuclein following addition of WT-NAC. a, spectra are overlaid of 50 μm 15N-labeled α-synuclein alone (black) and in the presence of 1 molar eq of WT-NAC (red). Resonances that shift upon the addition of NAC are indicated with an arrow. b, chemical shift perturbations in α-synuclein upon NAC binding. Residues exhibiting a significant chemical shift difference (>1 S.D. over the mean (dashed line)) are highlighted in red.
Figure 7.
Figure 7.
NAC binds WT Im7 and TM Im7. a, far-UV CD spectra of WT Im7 (blue) and TM Im7 (orange) under the conditions used for the cross-linking experiments (see “Experimental procedures”). b, SDS-PAGE analysis of NAC cross-linked alone (green) or to WT Im7 (blue) or TM Im7 (orange) using BS3. Lanes show the addition of a 20 or 50× excess of BS3. The red arrow highlights the putative complex between NAC and the protein substrates. c, map of the cross-links identified following in-gel tryptic digest of the putative NAC–Im7 complex: intra-NAC cross-links (purple), inter-NAC cross-links (green), and NAC-WT Im7 cross-links (red). d, map of the cross-links identified following in-gel tryptic digest of the putative NAC–TM Im7 complex band colored as in c. Peptides identified are listed in Tables S6–S10.
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