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Atomic insights into the genesis of cellular filaments by globular proteins

Abstract

Self-assembly of proteins into filaments, such as actin and tubulin filaments, underlies essential cellular processes in all three domains of life. The early emergence of filaments in evolutionary history suggests that filament genesis might be a robust process. Here we describe the fortuitous construction of GFP fusion proteins that self-assemble as fluorescent polar filaments in Escherichia coli. Filament formation is achieved by appending as few as 12 residues to GFP. Crystal structures reveal that each protomer donates an appendage to fill a groove between the two following protomers along the filament. This exchange of appendages resembles runaway domain swapping but is distinguished by higher efficiency because monomers cannot competitively bind their own appendages. Ample evidence for this ‘runaway domain coupling’ mechanism in nature suggests it could facilitate the evolutionary pathway from globular protein to polar filament, requiring a minimal extension of protein sequence and no substantial refolding.

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Fig. 1: Fluorescence images of E. coli cells containing GFP-RNase A fusion proteins.
Fig. 2: Structure of the GFP-RNase(1–8) protofilament determined by X-ray crystallography.
Fig. 3: Protofilament architecture and bundling in vivo.
Fig. 4: Four basic protofilament-forming mechanisms and their observed combinations.

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Acknowledgements

We thank M. Collazo and D. Cascio of the UCLA-DOE X-ray Crystallization and Crystallography Core Facilities for their assistance with crystallization and data collection, J. Abraham for discussion, and S. Dove for critical reading of the manuscript. We thank the Howard Hughes Medical Institute and National Science Foundation (grant 1616265) for support to D.S.E. and National Institutes of Health (grants OD003806 and GM115941) for support to A.H. Diffraction data were collected at the Northeastern Collaborative Access Team beamlines 24-ID-E and C, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P41 GM103403). The Pilatus 6 M detector on 24-ID-C beam line is funded by an NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357.

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Contributions

L.M. designed the study, performed cell biological and mutagenesis experiments and contributed to writing the manuscript. D.M.H. designed the study, performed cell biological experiments and protein purifications, and contributed to writing the manuscript. D.S.E. discussed plans and results with A.H. and M.R.S. A.H. designed the study and contributed to writing the manuscript. M.R.S. determined and analyzed the crystal structures, performed in cellulo diffraction experiments and contributed to writing the manuscript.

Corresponding authors

Correspondence to Ann Hochschild or Michael R. Sawaya.

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Integrated supplementary information

Supplementary Figure 1 Amyloid-forming propensities for each of the eight indicated GFP-RNase fusion linkers, as predicted by ZipperDB.

Orange bars represent hexapeptides predicted to form the spines of amyloid fibrils (energies below –23 kcal/mol). Most notably, the glutamine variant, which was predicted to have the strongest amyloid-forming propensity, produced no filaments, suggesting that the filaments are not amyloid-like.

Supplementary Figure 2 Crystals of GFP-RNase(1–8) invariably display a needle morphology.

This needle morphology recapitulates on a larger scale the rod-like shape of GFP-RNase assemblies observed in cells. (a) A representative sampling of the many conditions producing GFP-RNase(1-8) crystals. Only a small percentage of these conditions were tested for diffraction. Each subpanel reports the crystallization kit and composition of the reservoir that produced the crystal. The scale bar at the top left applies to all subpanels. (b) The four crystals used for structure determination, each with distinct crystal packing and unit cell parameters. The scale bar at the left applies to all subpanels.

Supplementary Figure 3 Structural effects of point mutations on a major interface of the GFP-RNase protofilament assembly.

The donor helix (residues 233-247) is colored green and the acceptor barrel is colored brown. The top panel illustrates the crystal structure; the remaining panels are hypothetical models illustrating the compatibility or incompatibility of the designated mutation with the filament structure. The only variant of M240 that is structurally and physically compatible with the filament structure is L240. The A240 mutation creates a destabilizing gap. The E240 and Q240 mutations place polar atoms in a hydrophobic environment. Nitrogen, oxygen, and sulfur atoms are colored blue, red, and yellow, respectively. The yellow starbursts and red disks indicate steric clash.

Supplementary Figure 4 Protomer interfaces observed in the GFP-RNase(1–8) protofilament crystal structure: dimensions and organization.

Three distinct interface regions are marked with dashed lines. The amount of surface area buried in each interface is indicated. The filament axis is vertical in this view.

Supplementary Figure 5 Immunoblot showing that the GFP-V219E fusion protein variant and parent fusion protein are produced at comparable levels.

The blot was probed with anti-His antibody to detect the His6-tagged GFP fusion constructs, GFP-AMA-RNaseA(1-8)-His6 and GFP-V219E-AMA-RNaseA(1-8)His6, and. A separate blot prepared in tandem with identical samples was probed with an antibody to detect the RpoA protein of E. coli, which served as a loading control.

Supplementary Figure 6 Bragg spacings observed in the diffraction pattern of rod-containing E. coli match prominent molecular spacings in the GFP-RNase protofilament crystals in space group C2.

(a) The diffraction pattern from dried and aligned E. coli cells containing GFP-RNaseA(1-8) rods. The numbers in parentheses correspond to resolution (Bragg spacing) of the reflection in angstroms. The integers specify the Miller indices for the reflections based on correspondence with the C2 crystal form of GFP-RNase(1-8). Reflections related by symmetry share the same symbol. (b-g) Illustrations of the Bragg planes associated with six of the strongest reflections. The structure of GFP-RNase(1-8) filaments in the C2 crystal form is shown in cartoon ribbons. Individual barrels of GFP are highlighted by cylinders. A single reference protofilament is colored in light and dark green. Bragg planes are shown in blue. The Miller index is labeled above each panel. The unit cell is outlined in red. Unit cell directions (a, b and c) are labeled. (h) A clear outline of the filament architecture can be seen from the sum of the six Fourier waves illustrated in the previous panels.

Supplementary Figure 7 A mechanism for generating new protofilaments by circular permutation.

By this mechanism, components of preexisting intramolecular interfaces are split over distant surfaces, requiring protomers to assemble into a filament in order to reconstitute the interface. This mechanism of generating a protofilament-competent interface might occur faster than an alternative mechanism in which individual residues are mutated over generations to form a stable interface de novo.

Supplementary Figure 8 A plot of solvation free energy versus area buried in the interfaces of 28 surveyed filaments.

Values were calculated using the structures indicated. PDB accession codes are reported in parentheses. The plot indicates that GFP-RNase ranks lower in stability compared to most filaments surveyed.

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Supplementary Figures 1–8, Supplementary Notes 1 and 2 and Supplementary Table 1

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McPartland, L., Heller, D.M., Eisenberg, D.S. et al. Atomic insights into the genesis of cellular filaments by globular proteins. Nat Struct Mol Biol 25, 705–714 (2018). https://doi.org/10.1038/s41594-018-0096-7

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