Diversification of Protein Cage Structure Using Circularly Permuted

Dec 19, 2017 - Self-assembling protein cages are useful as nanoscale molecular containers for diverse applications in biotechnology and medicine. To e...
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Diversification of Protein Cage Structure Using Circularly Permuted Subunits Yusuke Azuma, Michael Herger, and Donald Hilvert J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b10513 • Publication Date (Web): 19 Dec 2017 Downloaded from http://pubs.acs.org on December 19, 2017

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Diversification of Protein Cage Structure Using Circularly Permuted Subunits Yusuke Azuma, Michael Herger† and Donald Hilvert* Laboratory of Organic Chemistry, ETH Zurich, 8093 Zurich, Switzerland

Supporting Information Placeholder ABSTRACT: Self-assembling protein cages are useful as nanoscale molecular containers for diverse applications in biotechnology and medicine. To expand the utility of such systems, there is considerable interest in customizing the structures of natural cage-forming proteins and designing new ones. Here we report that a circularly permuted variant of lumazine synthase, a cageforming enzyme from Aquifex aeolicus, AaLS, affords versatile building blocks for the construction of nanocompartments that can be easily produced, tailored, and diversified. The topologically altered protein, cpAaLS, self-assembles into spherical and tubular cage structures with morphologies that can be controlled by the length of the linker connecting the native termini. Moreover, cpAaLS proteins integrate into wild-type and other engineered AaLS assemblies by co-production in Escherichia coli to form patchwork cages. This co-assembly strategy enables encapsulation of guest proteins in the lumen, modification of the exterior through genetic fusion, and tuning of the size and electrostatics of the compartments. This addition to the family of AaLS cages broadens the scope of this system for further applications and highlights the utility of circular permutation as a potentially general strategy for tailoring the properties of cage-forming proteins.

Biological systems utilize self-assembling polyhedral protein shells to form spatially segregated compartments for diverse tasks, ranging from storage and transport of guest molecules to catalysis of short metabolic sequences.1 Such supramolecular structures also lend themselves to applications in synthetic biology. For example, natural protein cages, including virus-like particles, ferritins and other protein assemblies, have been widely exploited as nanoscale reaction chambers,2 bioimaging agents,3 and display or delivery vehicles.4 Customizing the size, shape, topology and charge properties of these self-assembling protein scaffolds has the potential to expand these opportunities considerably. De novo design is a powerful but demanding approach for generating proteins that spontaneously assemble into defined quaternary structures. Recently, symmetry-based strategies have been successfully employed to construct single and multi-component hollow cage/cube architectures as well as other nanostructures.5 In a complementary approach, natural cage-forming proteins can be (re-)engineered to create molecular containers having tailored properties.6 The cage-forming enzyme lumazine synthase, for instance, is an attractive candidate for the development of molecular loading, imaging and display systems because of its thermal stability,7a tolerance to modification,7b-d and morphological plasticity. 8,9 We have engineered variants of Aquifex aeolicus lumazine synthase (AaLS) that possess negatively supercharged

Figure 1. Design and assembly of circularly permuted AaLS. (a) Structure of an AaLS-wt T = 1 capsid (PDB: 1HQK). Each pentameric subunit is shown in different colors. (b) Ribbon diagram of a representative pentameric capsomer, with a monomer unit highlighted in color: residues 1-119, orange; residues 120-156, blue. (c,d) Topology diagrams of the AaLS-wt monomer and the circularly permuted cpAaLS(L8) variant. (e) TEM images of AaLS-wt. (f) TEM images of the tubular and spherical assemblies formed by cpAaLS(L8) following separation by SEC. Scale bar = 100 nm. interiors and efficiently encapsulate a wide variety of positively charged cargo molecules at diffusion limited rates.10 The resulting complexes enable templated synthesis of polymers11a and mimicry of bacterial microcompartments such as the carboxysome,11b among other applications.7b-d,12 Here we report that AaLS is amenable to even more radical redesign by topological rearrangement, providing access to a suite of tailorable molecular compartments. Circular permutation is a widely used strategy to change the connectivity of secondary structure elements in a protein while maintaining overall three-dimensional shape.13 This type of topological rearrangement has been observed for natural cage-forming proteins,14a,b and has also been employed for engineering purposes, permitting alteration of cage morphology14c and relocation of the N and C chain termini of capsid subunits.14d To create a circularly permuted protein in the laboratory, the native N and C termini are connected via a short peptidic linker, and new termini

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introduced at a secondary site elsewhere in the polypeptide sequence.13 We used this approach to move the N and C termini of AaLS from the exterior of the protein shell to its interior. We envisaged that such constructs would be able to internalize cargo molecules by genetic fusion of peptides or proteins to individual capsomer subunits and thus complement and extend current encapsulation strategies.10,15 The circularly permuted AaLS was designed based on the primary sequence and crystal structure of the wild-type T = 1 dodecahedral capsid, which is constructed from 60 identical subunits (Figure 1a).7a The AaLS monomer adopts a flavodoxin-like fold in which helix and sheet motifs span the shell wall (Figure 1b). The native N and C termini, located roughly 19 Å apart on the exterior of the structure, were linked via a flexible GTGGSGSS octapeptide. New chain termini were introduced in a loop that faces the lumenal cavity, between residues 119 and 120 (Figure 1c). The resulting construct, cpAaLS(L8), rearranges the primary sequence but should retain the secondary and tertiary structure of AaLS-wt (Figure 1d).16 Given the morphological plasticity of AaLS,8,9 it was expected that this engineered protein would still self-assemble into cage-like structures. Like its parent, cpAaLS(L8) is readily produced in recombinant form in E. coli cells. Following purification by ammonium sulfate precipitation and anion exchange chromatography, the morphology of the isolated protein was analyzed by size-exclusion chromatography (SEC) and transmission electron microscopy (TEM). In contrast to the ~16 nm diameter AaLS-wt assemblies (Figure 1a,e), cpAaLS(L8) exists as a mixture of unassembled cage fragments, ~24 nm and ~28 nm spheres, and hollow ~24-nm wide rod-shaped structures of variable length (70 to 2,000 nm) (Figure 1f and Figure S2a). While 24-nm particles and tubular assemblies have not been seen before, the 28-nm cpAaLS(L8) assemblies resemble AaLS-neg,10a an AaLS variant that forms unusual tetrahedrally symmetric 180-subunit cages with large keyhole-shaped pores in the shell wall.9 The cpAaLS(L8) assemblies are surprisingly dynamic. When fractions containing purified 24- or 28-nm spherical cages were incubated for a week at room temperature, almost all particles converted to the tubular structures, which appear to be favored over other morphologies (Figure S2). In analogous experiments with cage fragments, ~70% of the protein remained disassembled, indicating that transformation of assembled spherical cages into tubular structures is more facile than self-assembly of purified fragments in vitro. That cpAaLS(L8) tiles both spherical and rod-like shells is likely related to the flexibility of its circularly permuted fold (Figure 1b). As for all structurally characterized AaLS assemblies to date,7a,9 the supramolecular structures formed by cpAaLS(L8) are probably constructed from wedge-shaped pentameric capsomers.17 The AaLS-wt pentamer resembles a truncated cone that forms a closed-shell dodecahedron. In previous studies, introduction of multiple negatively charged residues in close proximity widened the lumenal surface, and thus converted these capsomers into more cylindrical structures, affording expanded shells with increased radii of curvature.9 Circular permutation may exert a similar effect (Figure 2a). In this case, the linker connecting the native termini would be expected to constrain the exterior face of the individual subunits. At the same time, cleavage between residues 119 and 120 would allow regions of the protein closer to the lumen to move apart, giving rise to a more cylindrically shaped capsomer, thus favoring assembly of larger shells and tubular structures. Conformational changes seen in naturally permuted bacterial shell proteins support this hypothesis.14a,b The circular dichroism (CD) spectrum of cpAaLS(L8) is qualitatively similar to that of AaLS-wt (Figure S3a,b). Nevertheless,

Figure 2. Linker length controls the assembly state of circularly permuted AaLS. (a) Scheme illustrating the hypothetical relationship between linker length, capsomer shape, and assembly of higher-order structures. (b-d) TEM images of the assemblies produced by different cpAaLS(LxHy) variants. The rod-shaped cpAaLS(L8H4) structures were obtained by self-assembly of isolated capsid fragments. Scale bar = 100 nm. the protein exhibits a somewhat smaller negative Cotton effect than AaLS-wt, perhaps due to partial fraying of the short helix adjacent to the new N-terminus. Such local perturbations would be consistent with the proposed widening of the lumenal surface and, if several conformations were energetically accessible, might also explain the observed polymorphism. They do not appear to impact stability, however, since cpAaLS(L8), like AaLS-wt, undergoes thermal denaturation only above 90 °C (Figure S3a,b). To investigate the relationship between linker length and assembly state, the original GTGGSGSS sequence in cpAaLS(L8) was replaced by 8, 12, or 16 amino acid-long peptides containing an embedded polyhistidine segment to facilitate purification (Figure 2b-d). The resulting cpAaLS(LxHy) variants, where x and y refer to total linker length and number of histidine residues, respectively, were analyzed by SEC and TEM following Ni-NTA affinity chromatography. The cpAaLS(L8H4) variant, which has an octapeptide linker like cpAaLS(L8), mainly afforded unassembled cage fragments (Figure S4a), which, upon standing, spontaneously self-assembled into tubular structures and a few 24- and 28-nm spherical particles (Figure 2b and Figure S4a). The variants possessing longer linkers preferentially formed spherical cages that showed little tendency toward rearrangement into tubular structures (Figure 2c,d and Figure S4c,d). Moreover, the length of the cpAaLS(L16H6) and cpAaLS(L12H6) linkers directly affected the size distribution of the assemblies. The former yielded predominantly 24-nm cages (~90% of all particles on a representative TEM grid) and the latter 28-nm cages (~75%). Both variants are highly thermostable (Tm > 90 °C) and, in contrast to cpAaLS(L8), have CD spectra essentially identical to that of AaLS-wt (Figure S3). These findings thus support the hypothesis that linker length can be used to alter the cone-angle of the capsomers, with longer linkers promoting formation of smaller spherical structures and shorter linkers favoring larger spheres and tubular assemblies. This approach to controlling higher-order assembly via subtle but straightforward modulation of capsomer shape contrasts with computational approaches that focus on designing chemically complementary capsomer-capsomer interfaces.5 Despite their morphological plasticity, native-like T = 1 structures were not observed for any of the circularly permuted AaLS variants. Nevertheless, as the subunit interfaces were not altered upon circular permutation, we surmised that cpAaLS(L8) might

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Figure 3. Incorporation of cpAaLS into other AaLS assemblies. (a) Scheme illustrating the formation of patchwork assemblies in E. coli. Ptet, tetracycline promoter; tetO, tetracycline operon; PT7, T7 promoter; lacO, lactose operon. (b) Size-exclusion chromatogram of cpAaLS(L8)-GFP co-assembled with AaLS-wt (black), AaLS-neg (blue), and AaLS-13 (red). Continuous and dashed lines respectively indicate absorbance at 280 nm (A280) and fluorescence (F500) (ex, 470 nm; em, 500 nm) for each fraction. (c) TEM images of cpAaLS(L8)-GFP co-assembled with AaLS-wt, AaLS-neg and AaLS-13. Scale bar = 100 nm. fold into wild-type-like structures when co-assembled with excess AaLS-wt.18 The resulting patchwork cages would possess distinct genetically modifiable chain termini on both their interior and exterior surfaces, further extending the structural complexity of AaLS assemblies. To test this possibility, we fused green fluorescent protein to the C-terminus of cpAaLS(L8), and co-produced the cpAaLS(L8)-GFP construct lacking a His-tag with His-tagged AaLS-wt in E. coli cells (Figure 3a). The intracellular concentration of the two proteins was regulated separately by tetracycline and isopropyl-β-D-thiogalactopyranosid (IPTG), which control the expression of their respective genes. If the two proteins coassemble, the resulting cage structures should possess externalized His-tags for purification, and internalized GFP fluorophores for detection. Particles isolated by Ni-NTA affinity chromatography, and subsequently purified by SEC, exhibited characteristic absorbance at 474 nm (Figure S5), indicating successful integration of cpAaLS(L8)-GFP into the AaLS-wt host. Analysis by SEC, SDSPAGE, and TEM confirmed that the co-assemblies contained both proteins and adopted sizes and shapes similar to AaLS-wt (Figure. 3b,c and Figure S6). The number of cpAaLS-GFP subunits integrated into AaLS-wt cages was calculated from the 280/474 nm absorbance ratio.10e,18 At 100 ng/mL tetracycline and 0.1 mM IPTG, the isolated 60mer cages contained on average three cpAaLS-GFPs. This loading efficiency is substantially higher than the previously achieved ~0.5 GFPs per cage using a native sorting tag to direct encapsulation.15 Additionally, the number of guests per cage could be controlled by varying the tetracycline concentration (Figure S7a).2a,d However, if a maximum of approximately four GFP molecules per cage was exceeded, an increasingly large fraction of the sample consisted of incomplete assemblies (Figure S7b), in accord with the expected maximal loading capacity of AaLS-wt.15 The steric bulk of GFP hinders cage formation by cpAaLS(L8)GFP in the absence of AaLS-wt. Equipping the fusion protein with a C-terminal His-tag enabled its facile purification by Ni-

NTA affinity chromatography, suggesting that the tag is exposed rather than encapsulated. Indeed, only smaller capsid fragments were detected when the samples were analyzed by SEC (Figure S8). Importantly, patchwork assemblies were not obtained when this cpAaLS(L8)-GFP variant was mixed with pre-assembled AaLS-wt cages in vitro (Figure S7), demonstrating that incorporation of the cpAaLS variants into AaLS cages only occurs during cage assembly in cells. The flexible structure of cpAaLS(L8) facilitates co-assembly not only with AaLS-wt but also with the more capacious negatively supercharged AaLS-neg and AaLS-13 variants.10a,b When the latter cages were co-produced with cpAaLS(L8)-GFP in vivo, patchwork assemblies were likewise obtained. Since no fluorescence was detected in the flow-through of the Ni-NTA affinity columns, association efficiencies of cpAaLS(L8)-GFP with these AaLS scaffolds, as well as with AaLS-wt, appears to be nearly quantitative (Figure S9). SEC and TEM data for the isolated particles showed them to be similar in size to the host AaLS-neg (~28 nm) and AaLS-13 (~40 nm) cages, with guest GFP internalized within the lumenal space (Figure 3b,c and Figure S10).19 Reflecting their larger lumenal volumes, AaLS-neg and AaLS-13 accommodate more GFP molecules than AaLS-wt (at least 11 and 24 guests per cage, respectively). Employing different AaLS variants as host scaffolds for cpAaLS(L8) imparts control over the size and electrostatics of the patchwork assemblies, providing a simple means of tuning these proteinaceous compartments for specific functions. In summary, circularly permuted AaLS variants can serve as flexible building blocks for the construction of novel single and multi-component protein nanocompartments. They self-assemble into hollow spherical and tubular structures of variable dimension. Their morphology can be controlled either by the length of the polypeptide linker connecting native termini or co-assembly with other AaLS scaffolds. Notably, such structures are readily produced in E. coli, and their assembly does not depend on additional engineering of specific interactions between individual capsomers. In the case of patchwork assemblies, each capsid component can be modified independently, enabling covalent functionalization of both the interior and exterior surfaces of the particles via genetic fusion. Because such assemblies are easy to prepare, tailor and diversify, this design flexibility will lend itself to diverse applications in catalysis, sensing, storage, and delivery.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental procedures, CD spectra, TEM images, UV-Vis absorbance spectra, SEC charts (PDF)

AUTHOR INFORMATION Corresponding Author *[email protected]

Present Addresses † Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge, CB2 1GA UK

Notes The authors declare no competing financial interests.

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ACKNOWLEDGMENT We thank Peter Tittmann (Scientific Center for Optical and Electron Microscopy (ScopeM), ETH Zurich) and Dr. Christoph Giese (Department of Biology, ETH Zurich) for help with the electron microscopy and CD experiments, respectively. This work was supported by the ETH Zurich and the European Research Council (Advanced ERC Grant ERC-AdG-2012-321295 to D.H.). Y.A. is grateful for an Uehara Memorial Foundation Research Fellowship and an ETH Zurich Postdoctoral Fellowship (co-funded by the Marie Curie Actions program).

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(11) (a) Frey, R.; Hayashi, T.; Hilvert, D. Chem. Commun. 2016, 52, 10423–10426. (b) Frey, R.; Mantri, S.; Rocca, M.; Hilvert, D. J. Am. Chem. Soc. 2016, 138, 10072–10075. (12) Shenton, W.; Mann, S.; Cölfen, H.; Bacher, A.; Fischer, M. Angew. Chem. Int. Ed. 2001, 40, 442–445. (13) Yu, Y.; Lutz, S. Trends Biotechnol. 2011, 29, 18–25. (14) (a) Crowley, C. S.; Sawaya, M. R.; Bobik, T. A.; Yeates, T. O. Structure 2008, 16, 1324–1332. (b) Tanaka, S.; Sawaya, M. R.; Yeates, T. O. Science 2010, 327, 81–84. (c) Jorda, J.; Leibly, D. J.; Thompson, M. C.; Yeates, T. O. Chem. Commun. 2016, 52, 5041–5044. (d) Dedeo, M. T.; Duderstadt, K. E.; Berger, J. M.; Francis, M. B. Nano Lett. 2010, 10, 181–186. (15) Azuma, Y.; Zschoche, R.; Hilvert, D. J. Biol. Chem. 2017, 292, 10321–10327. (16) Domain swapped structures are also conceivable, but the distances between other pairs of termini, for example from different subunits, are considerably longer (>26 Å, see Supporting Information Figure S1), making maintenance of the original tertiary structure more likely. (17) In analogy to the quaternary structural changes induced by circular permutation of the PduA protein,14c it is possible the permutation converted native AaLS pentamers into hexamers, which would enable formation of quasi-equivalent assemblies with triangulation numbers T  3. However, hexamers have never been observed for any AaLS variant. Definitive resolution of this issue will require detailed structural information on the cpAaLS(L8) variant. (18) The capsids of Cowpea chlorotic mottle virus have been similarly shown to form patchwork assemblies in vitro, providing some control over loading of guest proteins fused to the capsid subunits. Rurup, W. F.; Verbij, F.; Koay, M. S. T.; Blum, C.; Subramaniam, V.; Cornelissen, J. J. L. M. Biomacromolecules 2014, 15, 558–563. (19) We recently designed additional cpAaLS variants containing Nterminal polyarginine tags that form patchwork assemblies with AaLS in vivo and capture cellular RNA. Protection of the encapsulated RNA guests from RNase digestion provides strong evidence that both the tag and the cargo molecules are localized within the interior of the cage shell. Azuma, Y.; Edwardson, T. G. W.; Terasaka, N.; Hilvert, D. submitted.

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