Available online at www.sciencedirect.com
ScienceDirect New designed protein assemblies Sabina Bozˇicˇ1, Tibor Doles1,2, Helena Gradisˇar1,2 and Roman Jerala1,2,3 Self-assembly is an essential concept of all organisms. Polypeptides self-assemble either within a single polypeptide chain or through assembly of protein domains. Recent advances in designed protein assemblies were achieved by genetic or chemical linkage of oligomerization domains and by engineering new interaction interfaces, which resulted in formation of lattices and cage-like protein assemblies. The absence of new experimentally determined protein folds in the last few years underlines the challenge of designing new folds. Recently a new strategy for designing self-assembly of a polypeptide fold, based on the topological arrangement of coiled-coil modules as the protein origami, has been proposed. The polypeptide tetrahedron was designed from a single chain concatenating of coiled-coil forming building modules interspersed with flexible hinges. In this strategy the order of coiled-coil segments defines the fold of the polypeptide nanostructure. Addresses 1 Department of Biotechnology, National Institute of Chemistry, Ljubljana, Slovenia 2 Excellent NMR – Future Innovation for Sustainable Technologies Centre of Excellence, Ljubljana, Slovenia 3 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia Corresponding author: Jerala, Roman ([email protected])
Current Opinion in Chemical Biology 2013, 17:940–945 This review comes from a themed issue Synthetic biomolecules Edited by Shang-Cheng Hung and Derek N Woolfson For a complete overview see the Issue and the Editorial Available online 31st October 2013 1367-5931/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbpa.2013.10.014
Introduction Proteins form the most versatile structures in nature, both in terms of structural definition at the nanoscale and their functionality. Tertiary structures of proteins underlie their versatile functionality, such as catalysis, molecular recognition, assembly of cellular scaffolds and many others. Considerable numbers of natural proteins have evolved to form supramolecular structures based on the oligomerization domains, such as oligomeric enzymes or viral protein capsids. Oligomerization subunits are most often symmetric since this requires the minimal number of different domains and interaction surfaces. Oligomerization saturates all of the available binding sites and can lead to closed structures of precisely defined stoichiometry [1]. A recent study proposed that Current Opinion in Chemical Biology 2013, 17:940–945
symmetric structures are common because symmetric interfaces are over-represented among the set of all energetically favourable interactions and represent the evolutionary accessible targets [2]. In comparison to other man-made nanostructures protein assemblies can form much more detailed as well as asymmetric nanoscale structures and are as such of considerable interest for many technological applications. Self-assembly of protein domains can lead to the formation of lattices or assemblies of discrete size, such as various cages, comprising from few to tens and hundreds of subunits. In the last two years excellent reviews covered the topic of designed protein assemblies [3,4]. Engineering of natural proteins has been used to modify their functionality but design of new protein folds, not based on the natural template still represents a challenge. Native protein domains are typically composed of packed secondary structure elements stabilized by a large number of weak interactions between the non-consecutive amino acid residues that define the fold, with notable contribution of hydrophobic interactions defining the protein core. Currently the folds of only few small domains can be accurately predicted [5–8], while design of completely new folds is even more challenging [9]. There is a consensus that natural proteins self-assemble into a limited number of folds, estimated to comprise at most few thousand members [10–12]. Currently we can count approximately 1200 protein domain folds among the experimentally determined protein structures. However the absence of any new protein folds, deposited in the PDB since 2008, despite the determination of almost 9000 new structures each year, indicates that we have probably already sampled the large majority of the existing natural protein folds. Therefore de novo formation of new protein folds represents an additional challenge.
Design of symmetric intermolecular protein assemblies Assemblies based on linked protein oligomerization domains
Designed protein oligomerization based on the symmetry was aimed to create a tetrahedral symmetric cage, composed of 12 subunits. Each subunit was composed of one dimerization and one trimerization domain (Figure 1a). These two domains were connected by a continuous semi rigid a-helix, selected to define the relative orientation of the neighbouring domains in the resulting assembly. The designed protein formed cage-like assemblies whose sizes were too heterogeneous to confirm the anticipated structure [13]. Recent introduction of a small number of amino acid modifications into the original protein www.sciencedirect.com
New designed protein assemblies Bozˇicˇ et al. 941
Figure 1
(a) Fusion via alpha-helical linker
(b)
Natural trimeric protein
Engineered interaction surface Current Opinion in Chemical Biology
Design of symmetric intermolecular protein assemblies. (a) Different oligomerization domains are linked together to obtain two or more interacting interfaces within a single polypeptide building block. Fusion of a dimerization domain (red) to a trimerization domain (green), where the predetermined geometry of their symmetry axes is held in place by a semi rigid a-helical linker (blue), resulted in the formation of a tetrahedral cage-like structure (4d9j) (right), composed of 12 subunits [13]. (b) Engineering of new protein-protein interfaces into the naturally oligomerizing building blocks leads to the formation of desired symmetric assemblies [22]. By computational docking and interface design new interaction surfaces (marked red) have been introduced into a naturally oligomeric protein, leading to the formation of assemblies with tetrahedral and octahedral symmetries (4ddf) (right).
sequence made it possible to remove the potential steric conflicts and deviations on the designed orientation of the symmetry axes. After those modifications the X-ray crystal structure of the homogenous 12-subunit assembly has been determined. The structure confirmed the tetrahedral geometry of the assembly measuring approximately 16 nm in diameter and deviation of 8 A˚ from the perfect symmetry [14,15]. Several other strategies utilizing linkage of protein oligomerization domains have been explored: utilizing protein chemical modifications, small molecule interactions or genetic fusions. C4-symmetric tetrameric aldolase and D2 tetrameric streptavidin were used to produce a quadrangular lattice, linked together with a pair of tethered biotin molecules. Arrays of limited size were obtained, probably owing to the flexibility of the biotin linker and imperfect control over the relative orientation of the component oligomers [16]. In another study the protein nanorings were prepared by chemically induced self-assembly of dihydrofolate reductase (DHFR) and histidine triad nucleotide binding 1 (Hint1) fusion proteins. The dimensions of nanorings could be modulated by the length and composition of the peptide linking fusion proteins, in the range from 10 to 70 nm [17]. One study prepared gyraseB-based regulated assembly/disassembly of a fusion polypeptide between gyrase www.sciencedirect.com
and a trimerization protein domain. The addition of a pseudo-dimeric gyrase B ligand coumermycin induced formation of gyrase B dimers and led to the formation of hexagonal assemblies and its dissociation by a monomeric ligand novobiocin [18]. The main challenge of the oligomerization domain-based assembly is connecting two oligomerization domains in a fixed relative orientation. The key advance was introduction of an extended fusion strategy. Fusion protein, composed of the two oligomerization domains, can generate two or more connections between the adjacent oligomers if the two domains are joined along an axis of symmetry that they both share. The symmetry-matching fusion protein strategy successfully generated linear filaments, 2D arrays extending up to 5 mm and large solid aggregates having crystal-like morphology; however this strategy was only used to construct periodic arrays, but not finite structures like molecular cages [19]. Engineering new protein interaction surfaces
Although definition of the relative orientation of fusion oligomerizing protein domain partners has been addressed in the studies described above, it requires careful selection of the natural protein domains with specific geometries and arrangements of the symmetry axes, thereby limiting its use as a general platform for the Current Opinion in Chemical Biology 2013, 17:940–945
942 Synthetic biomolecules
construction of new protein assemblies. De novo design of interaction surfaces allows greater potential versatility and functionality. Grueninger et al. introduced only few nonpolar side chains into the surface of natural monomeric or dimeric proteins, in order to generate homo oligomers with different symmetries [20]. Even though the established oligomer structures were not in the best agreement with the intended assemblies, this study demonstrated that a given protein can be engineered to form new contact interfaces. On the basis of development of powerful algorithms for modeling proteinprotein interactions, such as Rosetta [21], it is now possible to generate de novo interacting interfaces [7], which can drive the self-assembly of protein building blocks into symmetric complexes. This strategy has been successfully implemented in a recent study [22], describing a computational method for designing proteins that self-assemble into nanostructures with tetrahedral and octahedral point group symmetries (Figure 1b). This approach consists of two steps: first, docking of protein building blocks into the target symmetrical structure and second, design of low-energy protein–protein interaction interfaces between the building blocks, which are the driving force of the self-assembly. After selection of few candidates this strategy resulted in monodisperse protein nanostructures with atomic level accuracy, demonstrating its applicability to a broad range of new symmetric assemblies. New interacting surfaces were prepared by a metaltemplated interface redesign [23]. A monomeric protein is converted into a self-assembling form by introducing metal-chelating groups, followed by the introduction of stabilizing complementary interactions on the interfaces. The strategy can also be performed the other way around — a native self-assembling protein was redesigned into a form that requires metal for oligomerization [24]. Similarly, introduction of chelating histidines into the cytochrome accompanying the interface design resulted in metal-induced oligomerization [25]. Engineering protein surfaces recently led to the first report of a de novo 3D crystal from designed three-helix coiled coils [26]. Engineering polypeptide self-assembly from secondary structure modules
a-Helices and b-strands play an important role in protein self-assembly as they were recognized as an attractive protein folding motifs [27–29]. b-Strands interactions are the driving force of amyloid formation, and have been used to design many artificial amyloidlike structures, assembling in the form of fibrils and gels with different potential applications [30–34]. Rules governing the b-strand mediated protein domain assembly are being established [35] which could accelerate the future use of this structural element for designed selfassembly beyond amyloid fibrils. However it remains to Current Opinion in Chemical Biology 2013, 17:940–945
be seen what degree of orthogonality b-strands can offer as the building elements for de novo designed protein assemblies [36]. On the other hand, helical periodicity allows precise design of the polypeptide surface for interactions with other helices as well as for the interaction with other molecules and surfaces. Helical peptide design has been used to combine interactions between peptide building blocks to optimize the interaction with single-walled carbon nanotubes [37]. Coiled-coil dimers and oligomers represent one of the ubiquitous facilitators of inter-molecular and intramolecular protein–protein interactions. The rules governing coiled-coil formation, their oligomerization state and interaction partner specificity have been elucidated over the last decades [38,39], supporting development of sets of orthogonal designed coiled coils as the toolkit for the designed protein self-assembly [40,41,42,43]. Responsive, self-assembling a-helical peptide hydrogels were rationally designed and prepared from two components [44]. Peptide chains that self-assembled into homogeneous aggregates of dodecahedral symmetry were composed of two coiled-coil domains, one encoding a trimeric and another a dimeric oligomerization state, joined by a short linker segment [45]. Discrete circular assemblies of defined stoichiometry as well as flexible fibers have been prepared from coiled-coil forming segments [46], while the short dipeptide linker had limited flexibility and formed only linear fibers. Longer peptide linkers between coiled-coil forming segments comprising 6–10 residues induced formation of discrete self-assemblies corresponding to trimers and tetramers. Disulfide tethering a heterodimeric coiled-coil forming peptide with a trimerizing coiled-coil peptide led to the self-assembly of homogeneous cages measuring approximately 100 nm in diameter [47], presumably due to the intrinsic small curvature of each building block unit. This design allowed modulation of the size of assemblies by varying the affinity of heterodimeric peptides.
Modular topological polypeptide selfassembly Recent approach to the polypeptide fold self-assembly from a single chain is based on the topological arrangement of pairs of interacting coiled-coil dimerizing modules [48]. In this approach long range interactions occur between coiled-coil forming segments, which dimerize independently (Figure 2a). Modular self-assembly of coiled-coil segments resembles in many aspects the principles of DNA nanostructures [49–51], where polyhedra had been constructed based on the complementary DNA segments. www.sciencedirect.com
New designed protein assemblies Bozˇicˇ et al. 943
Figure 2
(a)
(b) Toolbox of coiled-coil forming modules
Deconstruction of a polyhedron into rigid building blocks
P3 : P4
GCNSH : GCNSH
P5 : P6
APH : APH
P7 : P8
BCR : BCR
Sequential order of concatenated coiledcoil forming modules
Self-assembled tetrahedron Current Opinion in Chemical Biology
Modular topological design of a protein fold from a single polypeptide chain. (a) The desired shape of a polyhedron is decomposed into the edges, which are composed of rigid coiled coil dimers. (b) Building blocks for coiled-coil dimeric edges of a polyhedron are selected from a toolbox of orthogonal coiled-coil dimers. The polypeptide path is threaded through the edges of a tetrahedron traversing each edge exactly twice, so that the path interlocks the structure into a stable shape stabilized by the six coiled-coil dimers, where some of them have to be parallel and some antiparallel. Coiled-coil dimer forming segments are concatenated in a defined order into a single polypeptide chain with flexible peptide linker hinges, which selfassembles into the stable structure [48].
Orthogonality of used coiled-coil building modules ensures that each segment binds preferably to its designated partner segment within the same polypeptide chain. Shape of the polyhedron is deconstructed into the edges composed of orthogonal coiled-coil dimers followed by threading the polypeptide chain through all of the coiled-coil forming segments in a single path, interlocking the chain into a stable structure (Figure 2b). The final topology is therefore defined by the sequential order of coiled-coil segments. Path traversing the polyhedron (demonstrated on a tetrahedron with approx. 5 nm edges) can be constructed in several mathematically allowed paths that can be circularly permutated as the beginning and end of the chain have to meet at the same vertex. There are three distinct solutions for the tetrahedral paths that have to combine parallel as well as antiparallel coiled-coil dimers. These types of protein folds had to be slowly annealed into the final structure and the order of segments defines the fold, similar as the order of amino acids in the native proteins. The modular topological design of the polypeptide fold is based on the interactions between secondary structure elements, whose folding is designed independently. In contrast to native protein structures, which are stabilized by the hydrophobic core, the topological fold comprises large cavity bounded by coiled-coil dimers.
Future directions Engineering of designed protein assemblies has recently advanced based on the developments of computational www.sciencedirect.com
molecular design and application of inventive design concepts. Even computer modeling-based design is still a challenging task and currently several solutions have to be tested experimentally. Nevertheless the design of protein assemblies has matured beyond the proof of principle and is ready to face more complex challenges. Design of new topological polypeptide folds based on modules introduced a principle that has not been found in nature. The impact of this strategy and the achievable limits of complexity remain to be established in the future. The main determinants will probably represent the size of the set of orthogonal peptide module set and kinetic limitations connected to the self-assembly of more complex polyhedra. Translation of the developments of new designed protein assemblies into different applications, such as drug delivery, vaccine design, introduction of binding and catalytic sites can be expected to follow in the near future.
Acknowledgements This work was financed by the program and projects from the Slovenian Research Agency and in part by funds of the European Union to the Centre of excellence EN-FIST.
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10. Fernandez-Fuentes N, Dybas JM, Fiser A: Structural characteristics of novel protein folds. PLoS Comput Biol 2010, 6:e1000750. 11. Zhang CO, DeLisi C: Estimating the number of protein folds. J Mol Biol 1998, 284:1301-1305. 12. Liu XS, Fan K, Wang W: The number of protein folds and their distribution over families in nature. Proteins – Struct Funct Genet 2004, 54:491-499. 13. Padilla JE, Colovos C, Yeates TO: Nanohedra: using symmetry to design self assembling protein cages, layers, crystals, and filaments. Proc Natl Acad Sci U S A 2001, 98:2217-2221. 14. Lai YT, Cascio D, Yeates TO: Structure of a 16-nm cage designed by using protein oligomers. Science 2012, 336:1129. The crystal structure of a nanoscale tetrahedron obtained by fusing oligomeric domains was determined. 15. Lai YT, Tsai KL, Sawaya MR, Asturias FJ, Yeates TO: Structure and flexibility of nanoscale protein cages designed by symmetric self-assembly. J Am Chem Soc 2013, 135:7738-7743. 16. Ringler P, Schulz GE: Self-assembly of proteins into designed networks. Science 2003, 302:106-109. 17. Chou TF, So C, White BR, Carlson JC, Sarikaya M, Wagner CR: Enzyme nanorings. ACS Nano 2008, 2:2519-2525. 18. Doles T, Bozic S, Gradisar H, Jerala R: Functional selfassembling polypeptide bionanomaterials. Biochem Soc Trans 2012, 40:629-634. 19. Sinclair JC, Davies KM, Venien-Bryan C, Noble ME: Generation of protein lattices by fusing proteins with matching rotational symmetry. Nat Nanotechnol 2011, 6:558-562. By genetic fusion of oligomerizing protein subunits with matching rotational symmetry, the authors demonstrated the self-association of resulting building blocks into well-ordered one-dimensional and two-dimensional arrays. 20. Grueninger D, Treiber N, Ziegler MO, Koetter JW, Schulze MS, Schulz GE: Designed protein–protein association. Science 2008, 319:206-209. 21. Leaver-Fay A, Tyka M, Lewis SM, Lange OF, Thompson J, Jacak R, Kaufman K, Renfrew PD, Smith CA, Sheffler W et al.: ROSETTA3: an object-oriented software suite for the simulation and design of macromolecules. Methods Enzymol 2011, 487:545-574. 22. King NP, Sheffler W, Sawaya MR, Vollmar BS, Sumida JP, Andre I, Gonen T, Yeates TO, Baker D: Computational design of selfassembling protein nanomaterials with atomic level accuracy. Science 2012, 336:1171-1174. Current Opinion in Chemical Biology 2013, 17:940–945
This paper shows how the introduction of a large novel self-associating surface into naturally oligomeric subunits can result in the formation of atomic level accuracy cage-like structures with octahedral and tetrahedral symmetries. 23. Salgado EN, Ambroggio XI, Brodin JD, Lewis RA, Kuhlman B, Tezcan FA: Metal templated design of protein interfaces. Proc Natl Acad Sci U S A 2010, 107:1827-1832. 24. Huard DJ, Kane KM, Tezcan FA: Re-engineering protein interfaces yields copper-inducible ferritin cage assembly. Nat Chem Biol 2013, 9:169-176. 25. Brodin JD, Ambroggio XI, Tang C, Parent KN, Baker TS, Tezcan FA: Metal-directed, chemically tunable assembly of one-, two- and three-dimensional crystalline protein arrays. Nat Chem 2012, 4:375-382. 26. Lanci CJ, MacDermaid CM, Kang SG, Acharya R, North B, Yang X, Qiu XJ, DeGrado WF, Saven JG: Computational design of a protein crystal. Proc Natl Acad Sci U S A 2012, 109:7304-7309. 27. Apostolovic B, Danial M, Klok HA: Coiled coils: attractive protein folding motifs for the fabrication of self-assembled, responsive and bioactive materials. Chem Soc Rev 2010, 39:3541-3575. 28. Bromley EH, Sessions RB, Thomson AR, Woolfson DN: Designed alpha-helical tectons for constructing multicomponent synthetic biological systems. J Am Chem Soc 2009, 131:928-930. 29. Woolfson DN, Bartlett GJ, Bruning M, Thomson AR: New currency for old rope: from coiled-coil assemblies to alphahelical barrels. Curr Opin Struct Biol 2012, 22:432-441. 30. Haines LA, Rajagopal K, Ozbas B, Salick DA, Pochan DJ, Schneider JP: Light-activated hydrogel formation via the triggered folding and self-assembly of a designed peptide. J Am Chem Soc 2005, 127:17025-17029. 31. Lamm MS, Rajagopal K, Schneider JP, Pochan DJ: Laminated morphology of nontwisting beta-sheet fibrils constructed via peptide self-assembly. J Am Chem Soc 2005, 127:16692-16700. 32. Marini DM, Hwang W, Lauffenburger DA, Zhang SG, Kamm RD: Left-handed helical ribbon intermediates in the self-assembly of a beta-sheet peptide. Nano Lett 2002, 2:295-299. 33. de la Paz ML, Goldie K, Zurdo J, Lacroix E, Dobson CM, Hoenger A, Serrano L: De novo designed peptide-based amyloid fibrils. Proc Natl Acad Sci U S A 2002, 99:16052-16057. 34. Waterhouse SH, Gerrard JA: Amyloid fibrils in bionanotechnology. Aust J Chem 2004, 57:519-523. 35. Feverati G, Achoch M, Zrimi J, Vuillon L, Lesieur C: Beta-strand interfaces of non-dimeric protein oligomers are characterized by scattered charged residue patterns. PLoS ONE 2012, 7:e32558. 36. Wang W, Hecht MH: Rationally designed mutations convert de novo amyloid-like fibrils into monomeric beta-sheet proteins. Proc Natl Acad Sci U S A 2002, 99:2760-2765. 37. Grigoryan G, Kim YH, Acharya R, Axelrod K, Jain RM, Willis L, Drndic M, Kikkawa JM, DeGrado WF: Computational design of virus-like protein assemblies on carbon nanotube surfaces. Science 2011, 332:1071-1076. A good example of how computationally designed peptides can be further modified in order to bind a specific surface. 38. Woolfson DN: The design of coiled-coil structures and assemblies. Adv Protein Chem 2005, 70:79-112. 39. Mason JM, Muller KM, Arndt KM: Considerations in the design and optimization of coiled coil structures. Methods Mol Biol 2007, 352:35-70. 40. Gradisar H, Jerala R: De novo design of orthogonal peptide pairs forming parallel coiled-coil heterodimers. J Pept Sci 2011, 17:100-106. Strategy for design of artificial set of coiled-coil-forming peptide pairs and experimental verification of their characteristics. 41. Fletcher JM, Boyle AL, Bruning M, Bartlett GJ, Vincent TL, Zaccai NR, Armstrong CT, Bromley EH, Booth PJ, Brady RL et al.: A basis set of de novo coiled-coil Peptide oligomers for www.sciencedirect.com
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rational protein design and synthetic biology. ACS Synth Biol 2012, 1:240-250. Report of de novo designed a-helical coiled-coil peptides that adopt defined and well-characterized parallel dimeric, trimeric, and tetrameric states. 42. Thompson KE, Bashor CJ, Lim WA, Keating AE: SYNZIP protein interaction toolbox: in vitro and in vivo specifications of heterospecific coiled-coil interaction domains. ACS Synth Biol 2012, 1:118-129. 43. Zaccai NR, Chi B, Thomson AR, Boyle AL, Bartlett GJ, Bruning M, Linden N, Sessions RB, Booth PJ, Brady RL et al.: A de novo peptide hexamer with a mutable channel. Nat Chem Biol 2011, 7:935-941. 44. Banwell EF, Abelardo ES, Adams DJ, Birchall MA, Corrigan A, Donald AM, Kirkland M, Serpell LC, Butler MF, Woolfson DN: Rational design and application of responsive alpha-helical peptide hydrogels. Nat Mater 2009, 8:596-600. 45. Raman S, Machaidze G, Lustig A, Aebi U, Burkhard P: Structurebased design of peptides that self-assemble into regular polyhedral nanoparticles. Nanomedicine 2006, 2:95-102. 46. Boyle AL, Bromley EH, Bartlett GJ, Sessions RB, Sharp TH, Williams CL, Curmi PM, Forde NR, Linke H, Woolfson DN: Squaring the circle in peptide assembly: from fibers to discrete nanostructures by de novo design. J Am Chem Soc 2012, 134:15457-15467.
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47. Fletcher JM, Harniman RL, Barnes FR, Boyle AL, Collins A, Mantell J, Sharp TH, Antognozzi M, Booth PJ, Linden N et al.: Selfassembling cages from coiled-coil peptide modules. Science 2013, 340:595-599. Using short heterodimeric and homotrimeric coiled-coils, the authors were able to obtain unilamellar spheres of approximately 100 nm in diameter. 48. Gradisar H, Bozic S, Doles T, Vengust D, Hafner-Bratkovic I, Mertelj A, Webb B, Sali A, Klavzar S, Jerala R: Design of a singlechain polypeptide tetrahedron assembled from coiled-coil segments. Nat Chem Biol 2013, 9:362-366. A new approach in the design of self-assembling nanostructures from a single polypeptide chain is shown. Orthogonal coiled-coils are used as building modules — their sequential order and specific dimerization are the driving force for the self-assembly of a polypeptide chain into a tetrahedron. 49. Rothemund PW: Folding DNA to create nanoscale shapes and patterns. Nature 2006, 440:297-302. 50. Goodman RP, Schaap IA, Tardin CF, Erben CM, Berry RM, Schmidt CF, Turberfield AJ: Rapid chiral assembly of rigid DNA building blocks for molecular nanofabrication. Science 2005, 310:1661-1665. 51. He Y, Ye T, Su M, Zhang C, Ribbe AE, Jiang W, Mao CD: Hierarchical self-assembly of DNA into symmetric supramolecular polyhedra. Nature 2008, 452:U141-U198.
Current Opinion in Chemical Biology 2013, 17:940–945
ScienceDirect New designed protein assemblies Sabina Bozˇicˇ1, Tibor Doles1,2, Helena Gradisˇar1,2 and Roman Jerala1,2,3 Self-assembly is an essential concept of all organisms. Polypeptides self-assemble either within a single polypeptide chain or through assembly of protein domains. Recent advances in designed protein assemblies were achieved by genetic or chemical linkage of oligomerization domains and by engineering new interaction interfaces, which resulted in formation of lattices and cage-like protein assemblies. The absence of new experimentally determined protein folds in the last few years underlines the challenge of designing new folds. Recently a new strategy for designing self-assembly of a polypeptide fold, based on the topological arrangement of coiled-coil modules as the protein origami, has been proposed. The polypeptide tetrahedron was designed from a single chain concatenating of coiled-coil forming building modules interspersed with flexible hinges. In this strategy the order of coiled-coil segments defines the fold of the polypeptide nanostructure. Addresses 1 Department of Biotechnology, National Institute of Chemistry, Ljubljana, Slovenia 2 Excellent NMR – Future Innovation for Sustainable Technologies Centre of Excellence, Ljubljana, Slovenia 3 Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia Corresponding author: Jerala, Roman ([email protected])
Current Opinion in Chemical Biology 2013, 17:940–945 This review comes from a themed issue Synthetic biomolecules Edited by Shang-Cheng Hung and Derek N Woolfson For a complete overview see the Issue and the Editorial Available online 31st October 2013 1367-5931/$ – see front matter, # 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbpa.2013.10.014
Introduction Proteins form the most versatile structures in nature, both in terms of structural definition at the nanoscale and their functionality. Tertiary structures of proteins underlie their versatile functionality, such as catalysis, molecular recognition, assembly of cellular scaffolds and many others. Considerable numbers of natural proteins have evolved to form supramolecular structures based on the oligomerization domains, such as oligomeric enzymes or viral protein capsids. Oligomerization subunits are most often symmetric since this requires the minimal number of different domains and interaction surfaces. Oligomerization saturates all of the available binding sites and can lead to closed structures of precisely defined stoichiometry [1]. A recent study proposed that Current Opinion in Chemical Biology 2013, 17:940–945
symmetric structures are common because symmetric interfaces are over-represented among the set of all energetically favourable interactions and represent the evolutionary accessible targets [2]. In comparison to other man-made nanostructures protein assemblies can form much more detailed as well as asymmetric nanoscale structures and are as such of considerable interest for many technological applications. Self-assembly of protein domains can lead to the formation of lattices or assemblies of discrete size, such as various cages, comprising from few to tens and hundreds of subunits. In the last two years excellent reviews covered the topic of designed protein assemblies [3,4]. Engineering of natural proteins has been used to modify their functionality but design of new protein folds, not based on the natural template still represents a challenge. Native protein domains are typically composed of packed secondary structure elements stabilized by a large number of weak interactions between the non-consecutive amino acid residues that define the fold, with notable contribution of hydrophobic interactions defining the protein core. Currently the folds of only few small domains can be accurately predicted [5–8], while design of completely new folds is even more challenging [9]. There is a consensus that natural proteins self-assemble into a limited number of folds, estimated to comprise at most few thousand members [10–12]. Currently we can count approximately 1200 protein domain folds among the experimentally determined protein structures. However the absence of any new protein folds, deposited in the PDB since 2008, despite the determination of almost 9000 new structures each year, indicates that we have probably already sampled the large majority of the existing natural protein folds. Therefore de novo formation of new protein folds represents an additional challenge.
Design of symmetric intermolecular protein assemblies Assemblies based on linked protein oligomerization domains
Designed protein oligomerization based on the symmetry was aimed to create a tetrahedral symmetric cage, composed of 12 subunits. Each subunit was composed of one dimerization and one trimerization domain (Figure 1a). These two domains were connected by a continuous semi rigid a-helix, selected to define the relative orientation of the neighbouring domains in the resulting assembly. The designed protein formed cage-like assemblies whose sizes were too heterogeneous to confirm the anticipated structure [13]. Recent introduction of a small number of amino acid modifications into the original protein www.sciencedirect.com
New designed protein assemblies Bozˇicˇ et al. 941
Figure 1
(a) Fusion via alpha-helical linker
(b)
Natural trimeric protein
Engineered interaction surface Current Opinion in Chemical Biology
Design of symmetric intermolecular protein assemblies. (a) Different oligomerization domains are linked together to obtain two or more interacting interfaces within a single polypeptide building block. Fusion of a dimerization domain (red) to a trimerization domain (green), where the predetermined geometry of their symmetry axes is held in place by a semi rigid a-helical linker (blue), resulted in the formation of a tetrahedral cage-like structure (4d9j) (right), composed of 12 subunits [13]. (b) Engineering of new protein-protein interfaces into the naturally oligomerizing building blocks leads to the formation of desired symmetric assemblies [22]. By computational docking and interface design new interaction surfaces (marked red) have been introduced into a naturally oligomeric protein, leading to the formation of assemblies with tetrahedral and octahedral symmetries (4ddf) (right).
sequence made it possible to remove the potential steric conflicts and deviations on the designed orientation of the symmetry axes. After those modifications the X-ray crystal structure of the homogenous 12-subunit assembly has been determined. The structure confirmed the tetrahedral geometry of the assembly measuring approximately 16 nm in diameter and deviation of 8 A˚ from the perfect symmetry [14,15]. Several other strategies utilizing linkage of protein oligomerization domains have been explored: utilizing protein chemical modifications, small molecule interactions or genetic fusions. C4-symmetric tetrameric aldolase and D2 tetrameric streptavidin were used to produce a quadrangular lattice, linked together with a pair of tethered biotin molecules. Arrays of limited size were obtained, probably owing to the flexibility of the biotin linker and imperfect control over the relative orientation of the component oligomers [16]. In another study the protein nanorings were prepared by chemically induced self-assembly of dihydrofolate reductase (DHFR) and histidine triad nucleotide binding 1 (Hint1) fusion proteins. The dimensions of nanorings could be modulated by the length and composition of the peptide linking fusion proteins, in the range from 10 to 70 nm [17]. One study prepared gyraseB-based regulated assembly/disassembly of a fusion polypeptide between gyrase www.sciencedirect.com
and a trimerization protein domain. The addition of a pseudo-dimeric gyrase B ligand coumermycin induced formation of gyrase B dimers and led to the formation of hexagonal assemblies and its dissociation by a monomeric ligand novobiocin [18]. The main challenge of the oligomerization domain-based assembly is connecting two oligomerization domains in a fixed relative orientation. The key advance was introduction of an extended fusion strategy. Fusion protein, composed of the two oligomerization domains, can generate two or more connections between the adjacent oligomers if the two domains are joined along an axis of symmetry that they both share. The symmetry-matching fusion protein strategy successfully generated linear filaments, 2D arrays extending up to 5 mm and large solid aggregates having crystal-like morphology; however this strategy was only used to construct periodic arrays, but not finite structures like molecular cages [19]. Engineering new protein interaction surfaces
Although definition of the relative orientation of fusion oligomerizing protein domain partners has been addressed in the studies described above, it requires careful selection of the natural protein domains with specific geometries and arrangements of the symmetry axes, thereby limiting its use as a general platform for the Current Opinion in Chemical Biology 2013, 17:940–945
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construction of new protein assemblies. De novo design of interaction surfaces allows greater potential versatility and functionality. Grueninger et al. introduced only few nonpolar side chains into the surface of natural monomeric or dimeric proteins, in order to generate homo oligomers with different symmetries [20]. Even though the established oligomer structures were not in the best agreement with the intended assemblies, this study demonstrated that a given protein can be engineered to form new contact interfaces. On the basis of development of powerful algorithms for modeling proteinprotein interactions, such as Rosetta [21], it is now possible to generate de novo interacting interfaces [7], which can drive the self-assembly of protein building blocks into symmetric complexes. This strategy has been successfully implemented in a recent study [22], describing a computational method for designing proteins that self-assemble into nanostructures with tetrahedral and octahedral point group symmetries (Figure 1b). This approach consists of two steps: first, docking of protein building blocks into the target symmetrical structure and second, design of low-energy protein–protein interaction interfaces between the building blocks, which are the driving force of the self-assembly. After selection of few candidates this strategy resulted in monodisperse protein nanostructures with atomic level accuracy, demonstrating its applicability to a broad range of new symmetric assemblies. New interacting surfaces were prepared by a metaltemplated interface redesign [23]. A monomeric protein is converted into a self-assembling form by introducing metal-chelating groups, followed by the introduction of stabilizing complementary interactions on the interfaces. The strategy can also be performed the other way around — a native self-assembling protein was redesigned into a form that requires metal for oligomerization [24]. Similarly, introduction of chelating histidines into the cytochrome accompanying the interface design resulted in metal-induced oligomerization [25]. Engineering protein surfaces recently led to the first report of a de novo 3D crystal from designed three-helix coiled coils [26]. Engineering polypeptide self-assembly from secondary structure modules
a-Helices and b-strands play an important role in protein self-assembly as they were recognized as an attractive protein folding motifs [27–29]. b-Strands interactions are the driving force of amyloid formation, and have been used to design many artificial amyloidlike structures, assembling in the form of fibrils and gels with different potential applications [30–34]. Rules governing the b-strand mediated protein domain assembly are being established [35] which could accelerate the future use of this structural element for designed selfassembly beyond amyloid fibrils. However it remains to Current Opinion in Chemical Biology 2013, 17:940–945
be seen what degree of orthogonality b-strands can offer as the building elements for de novo designed protein assemblies [36]. On the other hand, helical periodicity allows precise design of the polypeptide surface for interactions with other helices as well as for the interaction with other molecules and surfaces. Helical peptide design has been used to combine interactions between peptide building blocks to optimize the interaction with single-walled carbon nanotubes [37]. Coiled-coil dimers and oligomers represent one of the ubiquitous facilitators of inter-molecular and intramolecular protein–protein interactions. The rules governing coiled-coil formation, their oligomerization state and interaction partner specificity have been elucidated over the last decades [38,39], supporting development of sets of orthogonal designed coiled coils as the toolkit for the designed protein self-assembly [40,41,42,43]. Responsive, self-assembling a-helical peptide hydrogels were rationally designed and prepared from two components [44]. Peptide chains that self-assembled into homogeneous aggregates of dodecahedral symmetry were composed of two coiled-coil domains, one encoding a trimeric and another a dimeric oligomerization state, joined by a short linker segment [45]. Discrete circular assemblies of defined stoichiometry as well as flexible fibers have been prepared from coiled-coil forming segments [46], while the short dipeptide linker had limited flexibility and formed only linear fibers. Longer peptide linkers between coiled-coil forming segments comprising 6–10 residues induced formation of discrete self-assemblies corresponding to trimers and tetramers. Disulfide tethering a heterodimeric coiled-coil forming peptide with a trimerizing coiled-coil peptide led to the self-assembly of homogeneous cages measuring approximately 100 nm in diameter [47], presumably due to the intrinsic small curvature of each building block unit. This design allowed modulation of the size of assemblies by varying the affinity of heterodimeric peptides.
Modular topological polypeptide selfassembly Recent approach to the polypeptide fold self-assembly from a single chain is based on the topological arrangement of pairs of interacting coiled-coil dimerizing modules [48]. In this approach long range interactions occur between coiled-coil forming segments, which dimerize independently (Figure 2a). Modular self-assembly of coiled-coil segments resembles in many aspects the principles of DNA nanostructures [49–51], where polyhedra had been constructed based on the complementary DNA segments. www.sciencedirect.com
New designed protein assemblies Bozˇicˇ et al. 943
Figure 2
(a)
(b) Toolbox of coiled-coil forming modules
Deconstruction of a polyhedron into rigid building blocks
P3 : P4
GCNSH : GCNSH
P5 : P6
APH : APH
P7 : P8
BCR : BCR
Sequential order of concatenated coiledcoil forming modules
Self-assembled tetrahedron Current Opinion in Chemical Biology
Modular topological design of a protein fold from a single polypeptide chain. (a) The desired shape of a polyhedron is decomposed into the edges, which are composed of rigid coiled coil dimers. (b) Building blocks for coiled-coil dimeric edges of a polyhedron are selected from a toolbox of orthogonal coiled-coil dimers. The polypeptide path is threaded through the edges of a tetrahedron traversing each edge exactly twice, so that the path interlocks the structure into a stable shape stabilized by the six coiled-coil dimers, where some of them have to be parallel and some antiparallel. Coiled-coil dimer forming segments are concatenated in a defined order into a single polypeptide chain with flexible peptide linker hinges, which selfassembles into the stable structure [48].
Orthogonality of used coiled-coil building modules ensures that each segment binds preferably to its designated partner segment within the same polypeptide chain. Shape of the polyhedron is deconstructed into the edges composed of orthogonal coiled-coil dimers followed by threading the polypeptide chain through all of the coiled-coil forming segments in a single path, interlocking the chain into a stable structure (Figure 2b). The final topology is therefore defined by the sequential order of coiled-coil segments. Path traversing the polyhedron (demonstrated on a tetrahedron with approx. 5 nm edges) can be constructed in several mathematically allowed paths that can be circularly permutated as the beginning and end of the chain have to meet at the same vertex. There are three distinct solutions for the tetrahedral paths that have to combine parallel as well as antiparallel coiled-coil dimers. These types of protein folds had to be slowly annealed into the final structure and the order of segments defines the fold, similar as the order of amino acids in the native proteins. The modular topological design of the polypeptide fold is based on the interactions between secondary structure elements, whose folding is designed independently. In contrast to native protein structures, which are stabilized by the hydrophobic core, the topological fold comprises large cavity bounded by coiled-coil dimers.
Future directions Engineering of designed protein assemblies has recently advanced based on the developments of computational www.sciencedirect.com
molecular design and application of inventive design concepts. Even computer modeling-based design is still a challenging task and currently several solutions have to be tested experimentally. Nevertheless the design of protein assemblies has matured beyond the proof of principle and is ready to face more complex challenges. Design of new topological polypeptide folds based on modules introduced a principle that has not been found in nature. The impact of this strategy and the achievable limits of complexity remain to be established in the future. The main determinants will probably represent the size of the set of orthogonal peptide module set and kinetic limitations connected to the self-assembly of more complex polyhedra. Translation of the developments of new designed protein assemblies into different applications, such as drug delivery, vaccine design, introduction of binding and catalytic sites can be expected to follow in the near future.
Acknowledgements This work was financed by the program and projects from the Slovenian Research Agency and in part by funds of the European Union to the Centre of excellence EN-FIST.
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