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. 2020 Feb 25;117(8):4088-4098.
doi: 10.1073/pnas.1915542117. Epub 2020 Feb 7.

Structural dynamics of the human COP9 signalosome revealed by cross-linking mass spectrometry and integrative modeling

Craig Gutierrez  1 , Ilan E Chemmama  2 , Haibin Mao  3 , Clinton Yu  1 , Ignacia Echeverria  2 , Sarah A Block  4 , Scott D Rychnovsky  4 , Ning Zheng  3   5 , Andrej Sali  2   6 , Lan Huang  7
Affiliations

Structural dynamics of the human COP9 signalosome revealed by cross-linking mass spectrometry and integrative modeling

Craig Gutierrez et al. Proc Natl Acad Sci U S A. .

Abstract

The COP9 signalosome (CSN) is an evolutionarily conserved eight-subunit (CSN1-8) protein complex that controls protein ubiquitination by deneddylating Cullin-RING E3 ligases (CRLs). The activation and function of CSN hinges on its structural dynamics, which has been challenging to decipher by conventional tools. Here, we have developed a multichemistry cross-linking mass spectrometry approach enabled by three mass spectometry-cleavable cross-linkers to generate highly reliable cross-link data. We applied this approach with integrative structure modeling to determine the interaction and structural dynamics of CSN with the recently discovered ninth subunit, CSN9, in solution. Our results determined the localization of CSN9 binding sites and revealed CSN9-dependent structural changes of CSN. Together with biochemical analysis, we propose a structural model in which CSN9 binding triggers CSN to adopt a configuration that facilitates CSN-CRL interactions, thereby augmenting CSN deneddylase activity. Our integrative structure analysis workflow can be generalized to define in-solution architectures of dynamic protein complexes that remain inaccessible to other approaches.

Keywords: COP9 signalosome; architectures of protein complexes; cross-linking mass spectrometry; integrative structure modeling; structural dynamics.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
PPI maps of the CSN complexes based on cross-link data from all three linkers (DSSO, DHSO, BMSO). (A) CSN (CSN1–8). (B) CSNn (CSN1–9). Each CSN subunit is represented by colored nodes. The edges between two connected nodes are color-coded to describe PPIs resulted from individual or combinations of cross-linkers: That is, blue, DSSO; red, DHSO; purple, BMSO; lime, DSSO+BMSO; magenta, DHSO+BMSO; gold, DSSO+DHSO; black, DSSO+DHSO+BMSO. Edge thickness was determined by the total number of unique cross-links identified between the interactors.
Fig. 2.
Fig. 2.
Integrative structures of CSN. (A) The integrative structure of CSN determined at 16-Å precision when all three cross-link datasets (DSSO+DHSO+BMSO) were used for modeling. For each subunit, the localization probability density of the ensemble of models is shown with a representative structure (the centroid) from the ensemble embedded within it. (B) Integrative modeling of CSN determined using DHSO or DHSO+DSSO datasets yielded models determined at 29- and 24-Å precision, respectively. (C) Graphical representation of determined model precisions with seven combinations of our three cross-link datasets, illustrating that increasing the number of cross-linking chemistries (abscissa axis) for integrative structure modeling leads to increased model precision (ordinate axis). CSN subunit was color-coded as illustrated.
Fig. 3.
Fig. 3.
Comparison of integrative and X-ray structures of the CSN complexes. (A) Overall architectures of CSN: X-ray structure (PBD ID code 4D10) (Top), CSN integrative structure (Middle), and CSNn integrative structure (Bottom). For each subunit in the integrative structures, the localization probability density of the ensemble of models is shown with a representative structure (the centroid) from the ensemble embedded within it. The CSN and CSNn structures show that the models adopt a more condensed state as compared to the X-ray structure, but they generally retain the overall architecture with only the helical bundle being constrained during modeling. (B) The arrangement of the CSN5–CSN6 (MPN domain containing subunits) dimer was an emerging feature in integrative structures; however, a slight shift in the interface was observed in the CSNn model. (C) Models indicate that the arrangement of CSN1, CSN2, and CSN3 was altered in the presence of CSN9; CSN2 moved from a state interacting with CSN3 in CSN to an opened state in the CSNn model, resembling the overall architecture of the CSN X-ray structure. (D) Respective binary subunit–subunit comparison of the CSN integrative structure with the CSN X-ray structure (Upper) and the CSNn integrative structure (Lower), respectively. The structures were compared by calculating their ensemble overlap; the overlap was quantified by the ratio of the distance between ensemble centroids to three times the sum of the ensemble precisions. Differences are shown in red. The CSN subunit was color-coded as illustrated.
Fig. 4.
Fig. 4.
Binding of CSN9 in the CSNn integrative structure. (A) The integrative structure of CSNn determined at 22-Å precision using all three cross-link datasets (DSSO+DHSO+BMSO). For each subunit, the localization probability density of the ensemble of models is shown with a representative structure (the centroid) from the ensemble embedded within it. The higher probable localization of CSN9, corresponding to its C terminal, on the CSNn model is represented by the orange localization probability density, and a representative structure from the ensemble is shown with spheres corresponding to two residues per beads connected by an extrapolated trace of the backbone. CSN9 primarily interacts with the main body of CSN3 (red) while its C-terminal tail also falls into the cavity between CSN1 (purple), CSN3 (red), and CSN8 (green). The Inset displays a closer view of CSN9 interaction. Green lines represent CSN9-containing DHSO cross-links. (B) Two-dimensional DHSO cross-link map linking CSN9 to CSN1 and CSN3 at specific residues.
Fig. 5.
Fig. 5.
PRM-based targeted quantitation of DHSO cross-linked peptides to validate CSN9-induced structural changes in CSN. (A) Skyline outputs for PRM quantitation of a representative DHSO intrasubunit (CSN4:E306–CSN4:E345) (Upper) and an intersubunit (CSN2:E63–CSN3:E333) (Lower) cross-linked peptides to compare their relative abundance in the CSN and CSNn complexes. Based on peak areas, the relative abundance ratio (CSN/CSNn) of the intrasubunit cross-link was determined as 1.11 (Upper), indicating no significant change. In contrast, the relative abundance of the intersubunit cross-link (CSN/CSNn) was determined as 30.15 (Lower), suggesting a significant change. (B) The distribution of cross-link ratios (CSN/CSNn) of 229 DHSO cross-linked peptides (represented as log2 values) determined by PRM quantitation, in which only 22 cross-linked peptides displayed significant changes (>2.5-fold, greater than 3σ), including 4 with decreased ratios (red dots) and 18 with increased ratios (blue dots). The cross-link ratios (CSN/CSNn) describe the relative abundance of cross-linked peptides in the two compared complexes. (C) Abundance of five quantifiable CSN2–CSN3 cross-links (CSN2:D45–CSN3:E333, CSN2:E59–CSN3:E284, CSN2E59–CSN3:E333, CSN2:E63–CSN3:E333, and CSN2:E161–CSN3:E284) detected in the CSN and CSNn complexes. The underlined numbers shown represent relative abundance ratios (CSN/CSNn) of the selected cross-linked peptides between the two complexes, indicating that these interactions are favored in CSN. (D) The five cross-links shown in (C) were mapped on CSN and CSNn integrative structures. The linkages in the CSN model (green) are satisfied within the expected distance (<30 Å), which are not satisfied in the CSNn model (magenta). Details on PRM quantitation of the cross-linked peptides are listed in Dataset S26.
Fig. 6.
Fig. 6.
The proposed structural model of CSN9 binding to facilitate CSN interaction with neddylated CRLs. CSN and neddylated CRL subunits were color-coded as illustrated. (I) CSN9-free CSN needs to undergo substantial conformational changes upon binding to a neddylated CRL. In comparison, (II) CSN9-bound CSN adopts a configuration better suited for CRL binding.
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