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Constructing protein polyhedra via orthogonal chemical interactions

Abstract

Many proteins exist naturally as symmetrical homooligomers or homopolymers1. The emergent structural and functional properties of such protein assemblies have inspired extensive efforts in biomolecular design2,3,4,5. As synthesized by ribosomes, proteins are inherently asymmetric. Thus, they must acquire multiple surface patches that selectively associate to generate the different symmetry elements needed to form higher-order architectures1,6—a daunting task for protein design. Here we address this problem using an inorganic chemical approach, whereby multiple modes of protein–protein interactions and symmetry are simultaneously achieved by selective, ‘one-pot’ coordination of soft and hard metal ions. We show that a monomeric protein (protomer) appropriately modified with biologically inspired hydroxamate groups and zinc-binding motifs assembles through concurrent Fe3+ and Zn2+ coordination into discrete dodecameric and hexameric cages. Our cages closely resemble natural polyhedral protein architectures7,8 and are, to our knowledge, unique among designed systems9,10,11,12,13 in that they possess tightly packed shells devoid of large apertures. At the same time, they can assemble and disassemble in response to diverse stimuli, owing to their heterobimetallic construction on minimal interprotein-bonding footprints. With stoichiometries ranging from [2 Fe:9 Zn:6 protomers] to [8 Fe:21 Zn:12 protomers], these protein cages represent some of the compositionally most complex protein assemblies—or inorganic coordination complexes—obtained by design.

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Fig. 1: Design of protein cages.
Fig. 2: Characterization of BMC2 cages.
Fig. 3: Characterization of BMC3 cages.
Fig. 4: Characterization of BMC4 cages.

Data availability

The principal data supporting the findings of this work are available within the figures and the Supplementary Information. Additional data that support the findings of this study are available from the corresponding author on request. Structural data obtained by X-ray crystallography and cryo-EM have been deposited into the RCSB PDB and EMDB data banks with the following accession codes: 6OT4 (BMC2), 6OT7 (BMC3), 6OT8 (BMC4), 6OT9 (BMC1) and 6OVH (BMC3 cryo-EM) in the PDB or EMD-20212 at The Electron Microscopy Data Bank.

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Acknowledgements

This work was supported by the US Department of Energy (Division of Materials Sciences, Office of Basic Energy Sciences; DE-SC0003844; for the design strategy, EM imaging and analysis, and biochemical analysis) and by the National Science Foundation (Division of Materials Research; DMR-1602537; for crystallographic analysis). E.G. acknowledges support by an EMBO Long-Term Postdoctoral Fellowship (ALTF 1336-2015). J.E. acknowledges support by a DFG Research Fellowship (DFG 393131496). R.H.S. was supported by the National Institute of Health Chemical Biology Interfaces Training Grant UC San Diego (T32GM112584). We acknowledge the use of the UCSD Cryo-EM Facility, which is supported by NIH grants to T.S.B. and a gift from the Agouron Institute to UCSD. Crystallographic data were collected either at Stanford Synchrotron Radiation Lightsource (SSRL) or at the Lawrence Berkeley Natural Laboratory on behalf of the Department of Energy.

Author information

Affiliations

Authors

Contributions

E.G. conceived the project, and designed and performed most experiments. R.H.S. and R.G.A. performed and processed the ns-TEM data and performed structural modelling and analysis. R.H.S. conducted encapsulation experiments. J.E. performed crystallographic analysis. J.B.B. and J.A.C. synthesized the IHA ligand. X.Y., T.B. and T.S.B. performed the cryo-EM data collection and processing. F.A.T. conceived and directed the project and wrote the manuscript with contributions from all authors.

Corresponding author

Correspondence to F. Akif Tezcan.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Jack Johnson, Todd Yeates and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Characterization of the IHA ligand and the BMC constructs.

a, b, NMR spectra of N-hydroxy-2-iodoacetamide in DMSO-d6 for 1H (a) and 13C (b). cf, ESI-MS of as-isolated and HA-functionalized BMC constructs, and AUC profiles of HA-functionalized protomers for BMC1 (c), BMC2 (d), BMC3 (e) and BMC4 (f). The calculated masses for each unlabelled protein are determined by summing the mass of the polypeptide sequence and the c-type haem (618 Da) covalently linked to the cytochrome.

Extended Data Fig. 2 Structural comparison of CFMC1 and BMC1 cages.

a, The symmetric substructures of the CFMC1 dodecameric unit and its per-protomer SASA and BSA values. Associative surfaces on the protomers are coloured red for homologous interactions and red/orange or blue/cyan for heterologous interactions (right). b, Summary of engineered metal-coordination motifs for BMC constructs (see Supplementary Table 1 for all mutations). c, d, Comparison of C2 and C3 symmetric interfaces and corresponding metal binding sites for CFMC1 (c) and BMC1 (d). Full cages are shown as surfaces; insets show details of each interface. Fe and Zn ions are represented as orange and teal spheres, respectively. e, Cartoon representation of a full-size BMC1 cage with all metal ions shown as spheres. PDB ID: 3M4B (CFMC1), 6OT9 (BMC1).

Extended Data Fig. 3 ns-TEM characterization of BMC constructs.

a, b, Dissolved Fe:Zn:BMC1 (a) and Fe:Zn:BMC2 crystals (b) in a buffer containing 100 mM HEPES (pH 7.5), 200 mM MgCl2 and 800 μM ZnCl2. c, Self-assembled Fe:Zn:BMC3 cages in a buffer containing 20 mM Tris (pH 8.5), 20 μM FeSO4 and 60 μM ZnCl2. Histograms in b, c reflect the size distributions of Fe:Zn:BMC2 and Fe:Zn:BMC3 cage diameters as measured from ns-TEM images. Gaussian fits to both distributions are drawn as solid lines along with their centres and standard deviations reported. Scale bars, 50 nm.

Extended Data Fig. 4 Cavity volumes of BMC cages.

Solvent-accessible cavity volumes within BMC cages as calculated by a 1.4 Å rolling probe are shown visually as blue meshes and reported numerically below. Spherical cavities, shown as yellow spheres in Figs. 2, 4, are reproduced for comparison to the calculated volumes. BMC proteins are represented as transparent cylinders.

Extended Data Fig. 5 Anomalous densities of engineered metal binding sites and conformational flexibility of Cys82–HA site.

aj, Cartoon and stick representations of the symmetric interfaces of BMC1 (a, b), BMC2 (ce), BMC3 (fh) and BMC4 (i, j) showing the engineered metal binding sites with the C63–HA ligands (a, c, f), C82–HA ligands (d, g, i) and Zn binding sites (b, e, h, j). The difference in the anomalous signal between pairs of datasets above and below the K-shell energy of Zn and Fe, respectively, are depicted as blue or orange meshes. A strong signal illustrates a strong change in anomalous signal across the respective edge, in turn suggesting the presence of the respective metal. The top right corner of each panel indicates the energies of the datasets used for the map of the respective colour. All anomalous difference maps are contoured at 3σ. As datasets around the Fe-edge were not available for BMC1 and BMC3 (necessitating calculations using anomalous difference density of singular datasets), the calculated f″ values for Zn at 7.3 and 9.3 keV are 0.82 and 0.52 (that is, non-zero) and thus some residual anomalous signal of the lower energy maps around the Zn atoms is expected to result even from strictly selective Zn loading. For a more quantitative analysis of the nature of the bound metal, ratios of the anomalous signal to the expected values (bottom left corner of each panel) were calculated as described in the Methods. k, Stick representation of the BMC2 Cys82–HA binding site in both alternative conformations with the anomalous difference density over the Fe-edge shown as orange mesh and a simulated annealing omit map (omitting all C82–HA atoms and Fe) of the normal electron density as light blue mesh contoured at 2σ. For all Cys–HA binding sites, arrows indicate the handedness of the binding site as Δ (right handed) or Λ (left handed). The reversion of handedness in k with the respective view angle is indicated by arrows. Colour code for atoms in all panels: Fe in orange, Zn in blue, S in yellow, O in red and N in dark blue.

Extended Data Fig. 6 Solution characterization of self-assembled BMC3 and BMC4 cages.

ac, The oligomerization state of BMC3 cages as monitored by AUC measurements following incubation with various first-row transition metal ions (a), incubation with Zn2+ and Fe3+ (Fe(acetylacetonate)3) (b) and disassembly via sequestration of metal ions by EDTA (c). d, AUC profiles of BMC variants after equilibration for 2 h at the indicated temperatures (top). Thermal unfolding of BMC variants as measured by circular dichroism spectroscopy at 222 nm (bottom). e, AUC profiles of BMC following treatment with chemical reductants of different reduction potentials (left). ns-TEM micrographs (middle and right) are shown for cage samples incubated with the corresponding chemical reductants.

Extended Data Fig. 7 Cryo-EM analysis of BMC3 cages.

a, Representative cryo-EM micrograph and 2D class averages. b, Flowchart detailing image processing from collected movie stacks to final map. Additional details can be found in the Methods. c, FSC curves calculated between the half-maps (black line), atomic model to the unmasked full map (purple line) and atomic model to the masked full map (blue line). Resolution values are indicated at the gold-standard FSC 0.143 criterion. d, Local resolution estimates of the final reconstruction calculated using ResMap. e, Electron density shown at BMC3 C3 interfaces highlighting poorly resolved density (reflecting high flexibility) at hydroxamate sites and multiple conformations of W66.

Extended Data Fig. 8 Encapsulation of rhodamine inside BMC3 cages.

a, Fluorescence characterization of BMC3 samples incubated with rhodamine. Cages encapsulating rhodamine were treated with EDTA and washed before measuring fluorescence intensity. b, AUC profiles of cages encapsulating rhodamine monitored at the haem Soret absorption maximum (λmax = 415 nm) and rhodamine absorption maximum (λmax = 555 nm). c, UV-vis characterization of BMC3 samples incubated with rhodamine. d, Difference spectra of BMC3 samples and BMC3 protomer shown in c. Free rhodamine dissolved in buffer is shown as dark-red dashes. e, Repeated fluorescence characterization of a solution containing BMC3 cages encapsulating rhodamine over several days. The sample was washed three times before each fluorescence measurement.

Extended Data Table 1 X-ray data collection, processing and refinement statistics
Extended Data Table 2 Cryo-EM data collection, processing, and refinement statistics

Supplementary information

Supplementary Information

This file contains Supplementary Methods and Supplementary Tables 1-6. Supplementary Tables include the sequences, crystallization conditions and crystallographic quantification of metal content for the different proteins employed in this research.

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Golub, E., Subramanian, R.H., Esselborn, J. et al. Constructing protein polyhedra via orthogonal chemical interactions. Nature 578, 172–176 (2020). https://doi.org/10.1038/s41586-019-1928-2

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