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Multivalent interactions between CsoS2 and Rubisco mediate α-carboxysome formation

Nature Structural & Molecular Biology volume 27, pages 281–287 (2020)Cite this article

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Abstract

Carboxysomes are bacterial microcompartments that function as the centerpiece of the bacterial CO2-concentrating mechanism by facilitating high CO2 concentrations near the carboxylase Rubisco. The carboxysome self-assembles from thousands of individual proteins into icosahedral-like particles with a dense enzyme cargo encapsulated within a proteinaceous shell. In the case of the α-carboxysome, there is little molecular insight into protein–protein interactions that drive the assembly process. Here, studies on the α-carboxysome from Halothiobacillus neapolitanus demonstrate that Rubisco interacts with the N terminus of CsoS2, a multivalent, intrinsically disordered protein. X-ray structural analysis of the CsoS2 interaction motif bound to Rubisco reveals a series of conserved electrostatic interactions that are only made with properly assembled hexadecameric Rubisco. Although biophysical measurements indicate that this single interaction is weak, its implicit multivalency induces high-affinity binding through avidity. Taken together, our results indicate that CsoS2 acts as an interaction hub to condense Rubisco and enable efficient α-carboxysome formation.

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Fig. 1: CsoS2 sequence characteristics and interaction with Rubisco.
Fig. 2: Identification and energetics of the Rubisco-binding element.
Fig. 3: Structure and features of Rubisco–N* complex.
Fig. 4: Effect of N-peptide repeats on carboxysome formation and NTD–Rubisco phase separation.
Fig. 5: Proposed model for carboxysome assembly.

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Data availability

Atomic coordinates and structure factors for the Rubisco–CsoS2 N-peptide fusion have been deposited in the Protein Data Bank with accession code PDB 6UEW. Plasmids for all protein constructs used are available from Addgene. Raw data for all MST experiments in Fig. 2e and Extended Data Fig. 5 are available as Source Data. All protein sequences used for the binding motif analysis and for Figs. 2c and 3g are available as FASTA files in Supplementary Data 1–3.

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Acknowledgements

We thank C. Blikstad, C.-Y. Lin and M. Hagan for helpful discussions. We also thank P. Huang for his help with the BLI instrumentation and C. Kerfeld for advice on Rubisco crystallization. Y. Bar-On assisted us in gathering the CsoS2 sequences. We acknowledge the staff at the UC Berkeley Electron Microscope Laboratory for training and assistance with TEM. G. Meigs and J. Holton assisted with the X-ray diffraction and we acknowledge their input. We also thank N. Prywes for help with enzyme assays. Whiskers for crystal microseeding were kindly gifted by S.T. Kuhl. Beamline 8.3.1 at the Advanced Light Source is operated by the University of California Office of the President, Multicampus Research Programs and Initiatives grant MR-15-328599, the National Institutes of Health (grant nos. R01 GM124149 and P30 GM124169), Plexxikon Inc. and the Integrated Diffraction Analysis Technologies program of the US Department of Energy Office of Biological and Environmental Research. The work was supported by grants from the US Department of Energy (grant no. DE-SC00016240) and the National Institute of General Medical Sciences (grant no. R01GM129241) to D.F.S. and a grant from the National Institute of General Medical Sciences (grant no. R01GM050945) to S.M.

Author information

Author notes

  1. Thawatchai Chaijarasphong

    Present address: Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok, Thailand

Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA

    Luke M. Oltrogge, Thawatchai Chaijarasphong, Susan Marqusee & David F. Savage

  2. Department of Chemistry, University of California Berkeley, Berkeley, CA, USA

    Allen W. Chen & Susan Marqusee

  3. Biophysics Graduate Program, University of California Berkeley, Berkeley, CA, USA

    Eric R. Bolin

  4. California Institute for Quantitative Biosciences, University of California Berkeley, Berkeley, CA, USA

    Eric R. Bolin & Susan Marqusee

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Contributions

L.M.O., T.C. and D.F.S. designed the research. L.M.O., T.C. and A.W.C. built constructs, purified proteins and conducted binding assays. L.M.O., E.R.B. and S.M. performed and analyzed the HDX experiments. L.M.O. solved the structure. L.M.O. and D.F.S. wrote the manuscript with input and comments from all authors.

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Correspondence to David F. Savage.

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Competing interests

D.F.S. is a co-founder of Scribe Therapeutics and a scientific advisory board member of Scribe Therapeutics and Mammoth Biosciences. All other authors declare no competing interests.

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Peer review information Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Rubisco binding by N-peptides and design of consensus N*-peptide.

a, BLI binding activity toward Rubisco for each of the NTD N-peptides fused to GFP. b, N*-peptide and CsoS2 sequence colored and aligned by repeat peptides. The key conserved N-peptide residues are bolded.

Extended Data Fig. 2 BLI of select CsoS2 peptides with Rubisco.

a, Primary sequence of CsoS2 highlighting each of the repeated and/or conserved elements. b, Schematic representation of a set of BLI experiments testing the specificity of the Rubisco - CsoS2 interaction. Each of a series of CsoS2 elements and control sequences was fused to polyproline II helices that were surface immobilized to a Ni-NTA functionalized biosensor surface via an N-terminal hexahistidine tag. c, BLI traces of the constructs from (b) when incubated with 100 nM Rubisco. The trace colors match the dots in (b). Only the N*-peptide demonstrates any specific binding activity.

Extended Data Fig. 3 BLI of NTD and Rubisco mutants.

a, BLI response towards 100 nM Rubisco with bait of either the NTD or the NTD with R3A, R10A mutations made within all four of the N-peptide repeats. Removing these conserved arginines entirely eliminates the binding. b, Size exclusion chromatograms of wild-type H. neapolitanus Rubisco (wtRubisco), a mutant with all cation-π aromatics mutated to alanines (CbbL: Y72A, F346A; CbbS: Y96A), and a salt bridge disrupting mutation (CbbL: Y72A). All species eluted at a volume consistent with the CbbL8S8 structure. c, Each Rubisco species was tested for binding activity by BLI to the polyproline helix / N*-peptide fusion construct, N*-polyPro, (solid lines) and the randomized N*-polyPro negative control (dashed lines). Only the wild-type Rubisco had specific binding activity to N*-polyPro over the randomized N*-peptide control. The aromatic removal mutant (yellow) had some non-specific binding to both baits but showed no preference for the real N*-peptide sequence. d, Differential BLI binding signal of each Rubisco species to N*-polyPro relative to random N*-polyPro. Both Rubisco binding site mutants clearly possess no specific association.

Extended Data Fig. 4 Enrichment of binding motif from existing peptide array data.

Cai et al19. performed a fluorescent peptide array experiment assaying the binding of Rubisco to every 8-mer of CsoS2 tiled residue-by-residue and found broadly scattered activity, precluding the specific identification of the interaction sequence. We reexamined this dataset (generously provided in their Supplementary Material) in light of our new biochemical evidence to look for a statistical enrichment of binding activity for those peptides containing two positive residues or, more specifically, containing at least two arginines matching the RxxxxxRR motif. a, Cumulative distributions of Rubisco binding fluorescence response for CsoS2 array peptides including the full dataset (n = 1070), those with more than two basic residues (n = 319), and those matching the N*-peptide arginine motif (n = 91). b, Distributions of bootstrap results. 91 peptides were taken at random (with replacement) from either the full dataset or those with two or more basic residues and the median fluorescence response calculated. 10,000 trials were conducted with each set and none exceeded the motif matching median implying a strong statistical enrichment (p < 10−4).

Extended Data Fig. 5 MST salt dependence and single N-peptide response.

a, MST responses for [N1-N2]-GFP association to Rubisco. The concentration of the target, [N1-N2]-GFP, was 50 nM. The abscissa represents the concentration of effective binding sites and is four times the Rubisco CbbL8S8 concentration since each target will engage two of the eight possible sites. Binding experiments were performed at 20, 60, and 160 mM NaCl. At 20 mM NaCl three replicates were performed across 16 Rubisco concentrations. Black lines indicate the means while the gray whiskers show + /- one standard deviation. At 60 mM NaCl the experiment was performed twice with slightly varying concentrations. At 160 mM NaCl data from one representative experiment is shown. The fits to the 20 mM and 60 mM NaCl data are according to Eq. S2 and represent the mean fit parameters from bootstrap sampling of the data. For 160 mM NaCl no binding could be determined over this concentration range and the dashed orange line is drawn at zero response as a visual guide. b, Comparison between a double N-peptide, [N1-N2]-GFP, and single N-peptide, [N1]-GFP, species by MST. Both had 50 nM target. The Rubisco binding site concentration is specific to the two different targets. For [N1-N2]-GFP it is the concentration of CbbL8S8 multiplied by 4 and for [N1]-GFP it is the concentration of CbbL8S8 multiplied by 8 since the former has four potential binding sites on the Rubisco holoenzyme while the latter has eight. The [N1-N2]-GFP data points are the mean values from (a). The [N1]-GFP data points are from one representative experiment and indicate no conclusive binding over the concentration range. The dashed red line is at zero response as a visual guide.

Extended Data Fig. 6 Rubisco / N*-peptide fusion characterization.

a, Size exclusion analysis of wild-type Rubisco and the N*-peptide fusion construct. Both elute at volumes commensurate with compact CbbL8S8 complexes. A run with the Bio-Rad Gel Filtration Standard is included for comparison. Standard masses are indicated. b, BLI responses of wtRubisco and the N*-peptide fusion Rubisco at 100 nM with N*-polyPro as the surface bait. The fusion showed no binding. c, Proposed cartoon model of differential BLI binding activities. N*-peptide fusion Rubisco is apparently self-passivated by saturating the binding sites from stable association of the fused N*-peptides.

Extended Data Fig. 7 N-peptide electron density and interchain heterogeneity.

a, Views of the electron density at each of the N*-peptide binding sites within the asymmetric unit. The Fo–Fc maps (3.0 σ) are displayed as green (positive) and red (negative) mesh, and the 2Fo–Fc maps (1.0 σ) are shown as semi-transparent blue surfaces. All maps were calculated with the omission of the modeled N*-peptide atoms; the displayed N*-peptide sticks are present simply as a visual guide. b, All of the N*-peptides within the asymmetric unit superposed using only the adjacent Rubisco subunits (that is not using the N*-peptide coordinates). 2Fo–Fc maps are shown as mesh contoured at 1.0 σ. c–f, Zoomed in views of each of the conserved interaction sites. While some of the sites, such as N* R9 (c), are highly uniform in conformation and occupancy, others demonstrate a range of possible conformations and/or poor occupancy. N* R3 (f) in particular has a triad of possible salt bridges with CbbL D26, CbbL E344, or N* D4, respectively.

Extended Data Fig. 8 Structural comparison to CcmM/Rubisco interaction.

a, Surface representation of the Rubisco / N*-peptide complex with aligned CcmM-SSUL from the model of Wang et al17. in semi-transparent green. b, Detailed comparative view of the scaffold/Rubisco interaction interface. The inset table pairs equivalent Rubisco positions from alignment and the dashed lines indicate select specific interactions to the corresponding scaffold element shown with salt bridges in black and cation-π interactions in green. “Hnea” is the α-carboxysomal Form IA Rubisco from Halothiobacillus neapolitanus with CbbL (in orange/yellow) and CbbS (in cyan). The N*-peptide-bound structure is from the current study with PDB ID: 6UEW. “Selon” is the β-carboxysomal Form IB Rubisco from Synechococcus elongatus PCC 7942 with large subunit, RbcL, and small subunit, RbcS, both in grey. The bound small subunit-like repeat, CcmM-SSUL1, is shown in green. The atomic model was determined from cryo-electron microscopy single particle analysis and has PDB ID: 6HBC.

Extended Data Fig. 9 Hydrogen / deuterium exchange of Rubisco inside and outside carboxysomes.

The structure displayed contains two CbbLs and two CbbS and shows the CbbL2 dimer interface across which the N*-peptide (in magenta) binds. The Rubisco cartoon is colored according to the differential protection to amide hydrogen exchange. Those residues in blue experience greater protection within purified carboxysomes and those in red experience greater protection as free Rubisco. The comparison between these states was carried out with HDExaminer (Sierra Analytics) using moderate smoothing. Four specific peptides outlined i.n black highlight some of the diversity of HDX behavior. Most peptides that were observed from both states had essentially identical exchange kinetics as exemplified by the top right subpanel for CbbS: 57–67. Less common were peptides with different exchange profiles between encapsulated and unencapsulated Rubisco. CbbL: 34–44 (lower left subpanel) had slightly more protection in free Rubisco. CbbL: 328–341 (upper left subpanel) and CbbL: 262–267 (lower right subpanel) both had greater protection inside carboxysomes. The lack of a clear HDX protection footprint from the N peptide binding site may point toward a dynamic and liquid carboxysome interior in which the weak N-peptide / Rubisco interactions are rapidly exchanged.

Extended Data Fig. 10 Phase separation microscopy.

Phase contrast and fluorescence images of Rubisco, NTD-GFP, and the mixture (all at 1 μM) at 20 mM and 150 mM NaCl. At low salt Rubisco + NTD-GFP demixes into round liquid droplets. NTD-GFP alone at low salt shows a number of small fluorescent puncta (potentially small aggregates) but does not form large droplets. No droplets or aggregates are observed for any sample at high salt. All figures have the same scale with the common 10 μm scalebar in the bottom right image.

Supplementary information

Supplementary Information

Supplementary Notes 1 and 2, Figs. 1 and 2 and Tables 1 and 2.

Reporting Summary

Supplementary Data 1

FASTA protein sequences of CsoS2 used for Fig. 2c and Fig. 4b.

Supplementary Data 2

FASTA protein sequences of Rubisco small subunit used for Fig. 3g.

Supplementary Data 3

FASTA protein sequences of Rubisco large subunit used for Fig. 3g.

Source data

Source Data Fig. 1

Full-length image of gel from Fig. 1c.

Source Data Fig. 2

MST data for Fig. 2d and Extended Data Fig. 5.

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Oltrogge, L.M., Chaijarasphong, T., Chen, A.W. et al. Multivalent interactions between CsoS2 and Rubisco mediate α-carboxysome formation. Nat Struct Mol Biol 27, 281–287 (2020). https://doi.org/10.1038/s41594-020-0387-7

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