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Timestamp: 2019-04-22 04:07:19+00:00

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The design of new protein folds represents a grand challenge for synthetic, chemical and structural biology. Due to the good understanding of the principles governing its pairing specificity, coiled coil (CC) peptide secondary structure elements can be exploited for the construction of modular protein assemblies acting as a proxy for the straightforward complementarity of DNA modules. The prerequisite for the successful translation of the modular assembly strategy pioneered by DNA nanotechnology to protein design is the availability of orthogonal building modules: a collection of peptides that assemble into CCs only with their predetermined partners. Modular CC-based protein structures can self-assemble from multiple polypeptide chains whose pairing is determined by the interaction pattern of the constituent building blocks. Orthogonal CC sets can however also be used for the design of more complex coiled coil protein origami (CCPO) structures. CCPOs are based on multiple CC modules concatenated into a single polypeptide chain that folds into a polyhedral protein cage as the peptide segments assemble into CC dimers. The CCPO strategy has hitherto led to successful de novo design of protein cages in the shape of a tetrahedron, square pyramid and triangular prism. Recent advances in the design of CC modules and design principles have enabled the construction of CCPOs that self-assemble in vivo without any apparent toxicity to human cells or animals, opening the path towards therapeutic applications. The CCPO platform therefore has potential for diverse applications in biomedicine and biotechnology, from drug delivery to molecular cages.
Fabio Lapenta received his MSc in Biotechnology from the University of Bologna, Italy, in 2014 and he is currently enrolled in the Biomedicine doctoral programme at the University of Ljubljana, Slovenia. He joined Prof. Jerala's group in 2015 with an Early Stage Researcher PhD fellowship from the Marie Skłodowska-Curie ITN TOLLerant project. His research is focused on biochemistry, structural biology and protein design.
Jana Aupič obtained her MSc in Chemistry from the University of Ljubljana, Slovenia. As a PhD student, she joined the Department of Synthetic Biology and Immunology at the National Institute of Chemistry, Slovenia, in September 2015, under the supervision of Prof. Jerala. Her research is focused on designing novel coiled coil pairs and developing computational tools for designing and modelling CCPO structures.
Žiga Strmšek obtained his MSc in Industrial Pharmacy and BSc in Biotechnology from the University of Ljubljana, Slovenia. Since July 2015 he has been working as a PhD student at the Department of Synthetic Biology and Immunology at the National Institute of Chemistry, Slovenia. The focus of his research is presentation of protein domains on CCPO structures in order to investigate potential biotechnological applications.
Roman Jerala is head of the Department of Synthetic Biology and Immunology at the National Institute of Chemistry in Ljubljana, Slovenia, and professor at the University of Ljubljana. Within synthetic biology he is investigating designed modular bionanostructures, particularly coiled-coil based protein origami, mammalian cell synthetic biology and medical applications of synthetic biology and within immunology he focuses on the molecular mechanism of signaling in innate immunity and on cancer immunotherapy.
Biomimetic design of nanoscale molecular scaffolds and design of functional molecular machines represent motivation for exploring the space of protein folds. Proteins, due to their intrinsic biocompatibility and structural plasticity, represent an appealing material for both biotechnological and therapeutic applications. Even though proteins can be easily manufactured, the complexity of their folding landscapes hinders the prospect of designing new functional protein assemblies. In contrast, DNA nanotechnology based on the straightforward base-pairing complementarity of polynucleotide chains, while offering a much lower chemical versatility, enables design of complex programmable structures with high predictability and reliability.
The characteristic structural flexibility possessed by nucleic acids has been successfully repurposed to construct complex high-order three-dimensional structures.8–10 In nature, RNA can fold into defined compact structures such as e.g. aptamers or combine with polypeptide chains in the ribosome, which is one of the largest and most complicated molecular machines.11 Due to the much higher chemical variability of amino acid side chains in comparison to nucleic acids, proteins have been selected by evolution as the principal structural and functional material, while polynucleotides have been designated for the conservation and transcription of genetic information due to their straightforward base-pairing complementarity and stability. These two properties, in combination with the possibility to synthesize synthetic polynucleotides of any desired length or sequence, underlined the invention of DNA nanotechnology. Researchers in this field constructed high-order molecular shapes mainly via design of multiple DNA chains that assemble in a highly predictable manner and form defined two- and three-dimensional structures reaching up to micrometer scale.12–14 Moreover, DNA nanotechnology was able to introduce dynamic rearrangement in complex structures to design molecular machines such as molecular walkers or information processing molecular devices.15,16 DNA nanotechnology typically involves either short multiple chains’ self-assembly or a single long chain structured via addition of multiple shorter chains (DNA origami) that are slowly annealed in vitro by temperature ramping or by slow dialysis.17,18 Although it has been recently demonstrated that the design of the folding pathway of DNA nanostructures is also able to encode rapid folding of single chain knotted structures,19 the multichain assembly strategy avoids the problem of kinetic traps due to the formation of knots. However, single chain strategies have important advantages over multichain self-assembly due to independence from concentration, which in turn facilitates technological or in vivo production.
The purpose of this review is to illustrate the field of modular protein design relying on the orthogonally interacting CC as the basic structural unit. First, the special properties and designability of the CC motif are discussed, followed by a review of reported designed orthogonal CC sets. Then we describe the successful designs of protein nanostructures using orthogonal CC sets with an emphasis on CCPO structures and potential applications.
Coiled coils represent a highly suitable building block for building modular protein structures due to relatively well-understood rules governing their folding and specificity. Coiled coils are one of the most widespread protein structure elements in nature, estimated to be present in as much as 10% of the eukaryotic proteome,22 where they perform both structural and functional roles, acting as protein–protein interaction domains and DNA-binding domains.23 Coiled coils are described by the interaction between two or more alpha helices that in a canonical form assume a twisted left-handed supercoiled structure with a seven residue periodicity (7/2) and a pitch angle of 20° (Fig. 1).24 Those structural parameters, initially proposed by Francis Crick, impose a regular, tight side chain packing interface termed as knobs into holes, which is permitted only by a distortion of the number of residues per turn from 3.6 in normal helices to 3.5 in CCs.25–27 The seven-amino-acids periodicity that confers structural regularity to the CC motif is typically called the heptad repeat, where each residue is commonly represented as a letter in the string abcdefg (Fig. 1). The helices that compose CCs are usually highly amphipathic and exhibit a strong affinity conferred by both hydrophobic and electrostatic interactions. In canonical dimeric CCs, hydrophobic residues occupy positions a and d, and polar residues occupy positions e and g. The former are important in establishing the tight knobs into holes packing while the latter determine the formation of salt bridges between the two helices (Fig. 1).28,29 Such regularity, in addition to specificity of binding, made CCs a malleable and versatile tool in the hands of protein engineers and they have been used in multiple ways in the last few decades.
The ability to construct complex modular protein assemblies depends on the availability of required building blocks. Whereas nature offers large sets of specific CCs,46 the design of toolsets of CC elements that bind their target with high specificity remains a challenge. Sets of CC elements that bind solely to their designated partner peptide and do not cross-interact, also called orthogonal sets, designed so far possess only a limited size. This is mostly due to the small free energy differences between the desired and off-target associations. To facilitate the design of CC nanostructures several orthogonal CC sets have been developed in the last decade, differing in size, length, and orientation of constituent peptides.
One of the first examples were the α-helical tectons designed by Bromley et al.47 The set was composed of 6 three-heptad-long peptides that specifically formed 3 parallel CC heterodimers. Gradišar et al.48 reported the design of a set of 4 parallel CC heterodimers based on the combinations of patterns of charge interactions and a pattern of asparagine residues at heptad position a and evaluated by the energy scoring function introduced by Hagemann.49 The peptides comprised 4 heptads and contained a N-terminal capping sequence intended to stabilize the α-helical sequence. Orthogonal sets have been constructed from subgroups of synthetic peptides called SYNZIP.50 It was discovered that these peptides, initially designed to specifically bind the leucine zipper region of bZIP transcription factors,51 also exhibit strong hetero-association within the set. In vitro biophysical characterization of 14 SNYZIP peptides revealed that they were capable of assembling into 22 different heterodimeric CCs with groups of up to 4 CC pairs in each orthogonal set. Here, the peptides were of varying length, spanning 5–7 heptad repeats, which was reflected in the high stability of CC dimers, with the majority of the measured KD values in the nM range. Recently, Crooks et al. developed the largest CC set so far.52 Using the bCIPA algorithm53 a set of 8 parallel heterodimers was constructed comprising 4 heptad repeats. However, Tm measurements revealed that only 7 pairs behaved as designed with Tm > 70 °C and at least a 10 °C gap before the most stable off-target interaction.
Negron et al.54 reported the design of the only orthogonal set of antiparallel CC dimers found in the literature. Two sets of three homodimeric antiparallel CCs consisting of 6 heptads were designed. Three of the designed peptides preferentially formed antiparallel homodimers and were furthermore orthogonal to a previously designed antiparallel CC dimer,36 while higher order structures were observed for two of the designs. The availability of antiparallel orthogonal CCs is of high significance in the design of CC based protein origami, since it was shown that certain CCPO structures can be achieved only by inclusion of both parallel and antiparallel CC pairs.
The design of the above described orthogonal sets was achieved by a mixture of rational and computational approaches. Although different computational algorithms were used, they share similar core features. Firstly, the explored sequence space is restrained in accordance with previous rules discovered to govern CC oligomerization and pairing specificity. In the previously mentioned examples concerning the design of parallel CC sets,47,48,52 only lysine and glutamic acid were allowed at e:g positions, while the a heptad positions could be occupied either by asparagine or isoleucine and at d positions only leucine was allowed. Only a, d, e and g heptad positions were subjected to design, while b, c, and f positions were occupied by helicity promoting amino acids. Secondly, the interaction energy between peptide pairs is evaluated using sequence-based scoring functions as a weighted sum of terms for hydrophobic and electrostatic interactions. The scoring functions differ in weights used and inclusion of certain terms (e.g. helicity53).
First successes of modular protein design exploiting orthogonally interacting CC sets highlighted the importance of such sets for the field of protein design. Strategically linking non-associating α-helical tectons via glycine–glycine linkers resulted in 6–9 nm long helical nanorods as revealed by CD, DLS and AUC measurements.47 Using the same strategy that yielded α-helical tectons an additional set of 3 heterodimeric CC was designed with two pairs interacting in a parallel fashion, while helix orientation in the third pair was antiparallel.56 The latter comprised 3 heptads, while parallel dimers were composed of 4 heptad repeats. The developed set allowed successful construction of a three-stranded chassis intended to function as a hub in synthetic molecular motors (Fig. 2a and c). The resulting assembly was verified via CD, AUC and MALDI-TOF measurements (Fig. 2a). SYNZIP peptides served as the basis for the design of a two-dimensional nano-triangle composed of three polypeptide chains. Similarly to nanorods, the triangle shape was specified by cleverly linking non-interacting peptide pairs by a 10 amino acid linker (Fig. 2b and d).57 A combination of structure characterization techniques (DLS, SAXS, AFM) confirmed the fusion proteins assembled into a triangular shape as intended with a characteristic particle dimension of 10 nm (Fig. 2b).
Fig. 2 Models of nanostructures based on orthogonally interacting CC peptides. (a) Tripartite chassis formed by linking three CC dimers through disulfide bonds; the cysteine residues are visible as spheres.56 (b) Nano-triangle composed from three interacting peptide chains confirmed by SAXS analysis.57 (c and d) Respective schemes; CC segments are represented as colored arrows.
In the above described studies, the final assemblies were achieved as a result of correct pairing between multiple polypeptide chains, which depended on concentration and equilibrium determined by the affinity of CC segments. However, discrete protein structures could be realized more accurately by connecting orthogonally interacting CC dimers into a single polypeptide chain.58 This strategy was utilized to design the CCPO structures, i.e. polyhedron-shaped protein cages (Fig. 3),20,21 from building blocks provided by the CC set introduced by Gradišar et al.,45,48 the APH toolkit54 and modified naturally occurring CC dimers.59,60 Since finding the right sequential order of peptide modules for more complex polyhedral shapes becomes quickly intractable using back-of-the-envelope approaches, this design strategy relies on the foundations established by the mathematical graph theory.
Fig. 3 Model structure of representative CCPO cages in the shape of a (a) tetrahedron, (b) square pyramid and (c) trigonal prism, respectively composed of 12, 16 or 18 CC dimers, obtained from SAXS analysis and superimposed to volumes obtained by EM negative stain single-particle reconstruction.21 (d–f) Respective schemes; CC segments are represented as colored arrows.
At its core, the design of CCPO structures consists of connecting orthogonal CC peptides into a single-chain that will guide the polypeptide chain to fold into a polyhedron-shaped protein cage as the peptide modules self-assemble into intramolecular CC dimers forming the edges of the polyhedron. The task of finding the right arrangements of peptide segments is equivalent to the mathematical problem of finding a strong trace, a subset of double Eulerian paths, i.e. an oriented path that traverses each edge of the graph object exactly twice and interlocks the path into a stable structure, which means that all edges are connected with others in vertices.61,62 While the principles of designing CCPO structures have been described in detail,21 here we provide a brief overview and underline some important considerations.
Fig. 4 Steps involved in the design of CCPO structures. (a) Selection of the target shape. The target shape dictates the number of necessary orthogonal segments, a potential limiting factor for designing higher-order structures. (b) Construction and selection of a double Eulerian path. Different paths require the same total number of CC dimers; however the number of parallel vs. antiparallel pairs differs between paths. (c) Linearization of the double chain. TCO is the guiding parameter for selecting the best circular permutation. (d) Selection of needed CC building blocks. The heatmap shows the protein–protein interaction pattern for the orthogonal peptide set reported in ref. 21. (e) Placing CC-forming peptides into a single polypeptide chain according to the selected circular permutation. (f) Construction of the atomistic model for the designed polyhedral protein cage.
(i) Selection of the target polyhedral structure. From graph theory it follows that any arbitrary polyhedron-like cage based on a single polypeptide chain composed of concatenated segments that form intramolecular CC dimers (or any other dimer building module) serving as edges can be uniquely constructed.58 In reality, there are certain limitations – most importantly the number of available orthogonal building blocks.
(iii) Selection and placement of the CC building blocks at appropriate positions in the sequence. Next, an amino acid sequence is generated by connecting orthogonal CC building blocks from the toolbox of orthogonal CC dimers via flexible linkers in a manner defined by the selected circular permutation. CC dimers in the CCPO CC toolbox differ in stability, charge, length and helical propensity; however natural or other designed CCs can be used as well. Experimental testing of approx. 20 CCPO designs revealed several design rules such as e.g. to avoid positioning of less stable CC pairs at the C- or N-terminus as that can lead to fraying,21 or at positions that are far apart in the polypeptide chain. Regarding the choice of linkers, current experimental results suggest that their sequence does not play a key role in determining design success or stability of the CCPO structures as long as they comprise helix-breaking, small, polar residues, enabling flexible connection between rigid modules that define the fold. To facilitate the design of CCPO structures, a freely available computational design platform, CoCoPOD, was developed, allowing the above described design steps to be performed in a semi-automated manner.21 In addition to facilitating amino acid sequence design, CoCoPOD also permits construction of atomistic model structures for designed CCPO cages. CoCoPOD can be accessed at github.com/NIC-SBI/CC_protein_origami, and comes with three tutorial videos intended as a quick start for users.
The CCPO folds are based on a highly modular design strategy, based on long range, designable native contacts defined at the level of dimeric CC units to form the edges and guide the assembly of the cage. This strategy therefore bypasses the complexity of the design of cooperative protein core interactions. The affinity and specificity of CC segments to their partner modules underlies the formation of the CCPO protein fold. Therefore, the abovementioned importance of developing orthogonal CC sets assumes particular relevance for the construction of high order CCPO structures. CCPO cages form an internal cavity, whose shape and volume are determined by the geometry of the chosen polyhedron and the length of the edges. In a recent publication,21 the boundaries of CCPO design have been further extended to high order polyhedra with experimentally confirmed construction of cages possessing the shape of a square pyramid and a triangular prism in addition to alternative tetrahedral topologies. The first generation CCPO structures had to be refolded in vitro from the produced protein, as in most designs of DNA nanostructures, which limited the potential technological and therapeutic applications. One of the elements pivotal for the success of the second generation CCPOs was the design and usage of supercharged CC elements that ushered the correct in vivo self-assembly of CCPO under the physiological conditions, without the requirement of in vitro refolding steps.20 The design platform CoCoPOD provided a suitable environment for the design of different polyhedra, showcasing the utility of the developed software.
Three representative structures, a tetrahedron, a square pyramid and a triangular prism, formed by 12, 16 and 18 CC segments respectively, were confirmed by both small angle X-ray scattering (SAXS) and single particle TEM reconstruction (Fig. 3). As an indication of the flexibility of these nanostructures, SAXS experiments revealed that the trigonal prism is present in solution in both rectangular and oblique conformation. Furthermore, Kratky plots of other CCPO structures also indicated partial flexibility. Since CC elements represent rigid modules, conformational changes are due to the flexibility of the loops, which allows angles of non-constrained faces a certain degree of freedom, resulting in cages with a limited conformational variability. In addition, the tetrahedron TET12SN was structurally characterized by application of chemical cross-linking coupled with proteolytic digestion and mass spectrometry,21 which can be employed to investigate the fold of modular CCPOs. Crosslinking was performed with three different reagents, DSS, BS(PEG)5, and BS(PEG)9, that can bridge Cα–Cα distances up to 2.4, 3.4 and 4.8 nm respectively, covering the range of distances relevant for the tetrahedral protein cage. After cross-linking, the protein was subjected to proteolytic cleavage resulting in crosslinked peptide fragments. The latter were analyzed using mass spectrometry. In the case of the shorter cross-linker, several connections between pairing CC segments were detected, confirming that in the context of TET12SN peptides assembled into CC dimers as expected. With the longer cross-linkers, BS(PEG)5 and BS(PEG)9, long-range crosslinks between non-neighboring pairs of peptides (in terms of sequence) were detected. These connections were consistent with distances observed for the corresponding peptide fragments during MD simulations of the model TET12SN cage, indicating that the polypeptide chain folded according to the design.
The increase in the complexity of CCPO structures is also reflected in the increase of TCO values (4.3, 5, and 5.6 for the CCPO tetrahedron, square pyramid and trigonal prism). Although CCPO structures are defined by long-range interactions between CC modules and not a tightly packed hydrophobic core, the kinetics of folding for the CCPO tetrahedron, square pyramid and trigonal prism, obtained via stopped-flow CD and stopped-flow FRET experiments,21 were discovered to be comparable to that of natural proteins of similar length.63 The experimentally determined secondary structure folding rates (17 s−1, 14 s−1 and 7.7 s−1, respectively for the tetrahedron, square pyramid and trigonal prism) were in agreement with the overall folding rates observed via the FRET effect of fluorescently labeled N- and C-terminal ends (31 s−1, 15 s−1 and 10 s−1 respectively for the tetrahedron, square pyramid and trigonal prism). Interestingly, the increase in TCO values was correlated with a decrease in folding rates as expected from theoretical considerations.64 However, this correlation, as well as the complete characterization of the folding pathways of CCPO cages (e.g. potential kinetic traps, folding rates after annealing), still requires additional studies that would offer a better understanding and a means of controlling the CCPO folding process. A further increase of the CC module length will likely introduce knotted CCPOs, which will represent strong kinetic barriers that will need to be considered but also exploited as demonstrated before for single chain DNA nanostructures.19 The current state of the art CCPO cages undergo a reversible unfolding process, retaining their monomeric state after temperature unfolding followed by rapid cooling, while at 4 °C they are stable for weeks. Successful testing of approx. 20 CCPO cages reflected the robustness of this strategy. Additionally, biophysical characterization and SAXS analysis of 10 tetrahedral variants and another four-squared pyramid confirmed the applicability of this strategy to differently composed polyhedra and helped to understand the rules governing the formation of these non-natural folds.
The novelty of this modular design strategy is in the atypical fold assumed by these cages, whose robust designability and formation of an internal cavity can, in turn, be used for different applications. Besides self-assembly in an in vitro transcription–translation reaction and bacterial production, CCPO structures also self-assembled in mammalian cells as well as in living animals. The correct folding was confirmed by reconstitution of protein reporters, fluorescent proteins and luciferase catalytic activity fused to the termini of the CCPO structures. Biocompatibility of the designed CCPO structures with mammalian cells was proved by monitoring inflammasome activation and unfolded protein response. In addition, the absence of inflammation and liver damage markers confirmed that the tested CCPO structures are not sensed by mammalian cells as foreign and adopt the correct native structure in vivo. Therefore, due to the lack of observable adverse effects in vivo, CCPO cages show considerable promise for biological applications.
The ability to accurately manipulate objects at the nano-scale level is advantageous for various applications from biomedicine, materials science to chemical technology and beyond. While a wide spectrum of different nanomaterials is already available,65–67 polypeptide-based materials own a specific combination of features such as programmability, ability to accommodate functional chemical moieties with nano-scale accuracy, self-assembly, biocompatibility, biodegradability and sustainable technological production that make them a highly suitable material for biomedicine and other technological applications.
Cages can be constructed by employing a wide range of different building blocks, from bulky protein domains to smaller, rigid units, as in the case of CC elements. In particular, CCs were used as modular units either for direct self-assembly of multimeric hollow ∼100 nm large spheres109,110 or for the design of monomeric protein cages as in the case of CCPO structures. In comparison to other de novo cages, CCPO structures are the only example of assemblies that accommodate cavities within a single polypeptide chain.
Monomeric natural proteins possess internal cavities with an average volume of 0.25 nm3; larger cavities reach around 2.5 nm3 but often assume irregular shapes and typically occur at the protein–protein interaction surfaces (Fig. 5a).111 In contrast, multimeric protein cages either natural or de novo designed can accommodate large internal cavities, measuring from 40 nm3 to more than 4000 nm3 (Fig. 5b–d and f–g). Due to their peculiar fold, CCPO structures exhibit a large and hydrophilic cavity of approximately 40 nm3 (in the case of a tetrahedral fold) within a single polypeptide chain (Fig. 5e). In comparison to symmetry-based protein self-assemblies, where the cavity is tightly enclosed, CCPO cages offer a much more exposed cavity. The formation of such extensive cavities in a single polypeptide assembly makes CCPO design an attractive tool for targeted drug delivery and for molecular cages.
Control over proteins' three dimensional rearrangement can readily be achieved by grafting flexible conformational hinges within rigid structural elements. In the context of CCPO structures, CC formation involves a conformational transition from unfolded monomers to structurally rigid dimeric units, an example of folding-upon-binding behavior, resembling some intrinsically disordered proteins.119 In the context of CCPO, the reversibility of the folding transition offers the possibility to further engineer CCPO folds into dynamic assemblies able to assume different conformations upon interaction.
Modular CC-based designs exhibit several properties that make them appealing for therapeutic purposes. Such properties are also gaining importance in biotechnology, in particular in controlled catalysis, where the precise stoichiometric and spatial clustering of catalytic elements is important.121,122 In this regard, CCPO nanostructures may offer high customization. Furthermore, the biocompatibility of these novel folds, already demonstrated by in vivo studies,21 provides solid foundations for further development. In particular, understanding these novel folds and repurposing them for geometrical rearrangement of grafted moieties and molecular shielding represent interesting perspectives towards biotechnological applications, and while these challenges may require substantial efforts, recent advances in designing CC-based nanostructures offer all the reasons to be optimistic.
RJ is the author of the patent application on the design of self-assembling polypeptide polyhedra.
The authors acknowledge the support of Slovenian Research Agency (program no. P4-0176, project N4-0037), MSC-RTN 642157 TOLLerant H2020 (R. J. and F. L.) and the ERANET SynBio project Bioorigami (ERASYNBIO1-006) and COST action CM1306. Dr Prof. B. Bromley, Dr L. Small and Dr Prof. A. Keating kindly provided the models used in Fig. 2a and b.
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