Accurate de novo design of membrane-traversing macrocycles.

We use computational design coupled with experimental characterization to systematically investigate the design principles for macrocycle membrane permeability and oral bioavailability. We designed 184 6-12 residue macrocycles with a wide range of predicted structures containing noncanonical backbone modifications and experimentally determined structures of 35; 29 are very close to the computational models. With such control, we show that membrane permeability can be systematically achieved by ensuring all amide (NH) groups are engaged in internal hydrogen bonding interactions. 84 designs over the 6-12 residue size range cross membranes with an apparent permeability greater than 1 × 10-6 cm/s. Designs with exposed NH groups can be made membrane permeable through the design of an alternative isoenergetic fully hydrogen-bonded state favored in the lipid membrane. The ability to robustly design membrane-permeable and orally bioavailable peptides with high structural accuracy should contribute to the next generation of designed macrocycle therapeutics.

Computational design of transmembrane pores.

Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.

Insights into antiparallel microtubule crosslinking by PRC1, a conserved nonmotor microtubule binding protein.

Formation of microtubule architectures, required for cell shape maintenance in yeast, directional cell expansion in plants and cytokinesis in eukaryotes, depends on antiparallel microtubule crosslinking by the conserved MAP65 protein family. Here, we combine structural and single molecule fluorescence methods to examine how PRC1, the human MAP65, crosslinks antiparallel microtubules. We find that PRC1's microtubule binding is mediated by a structured domain with a spectrin-fold and an unstructured Lys/Arg-rich domain. These two domains, at each end of a homodimer, are connected by a linkage that is flexible on single microtubules, but forms well-defined crossbridges between antiparallel filaments. Further, we show that PRC1 crosslinks are compliant and do not substantially resist filament sliding by motor proteins in vitro. Together, our data show how MAP65s, by combining structural flexibility and rigidity, tune microtubule associations to establish crosslinks that selectively "mark" antiparallel overlap in dynamic cytoskeletal networks.

Programmable design of orthogonal protein heterodimers.

Specificity of interactions between two DNA strands, or between protein and DNA, is often achieved by varying bases or side chains coming off the DNA or protein backbone-for example, the bases participating in Watson-Crick pairing in the double helix, or the side chains contacting DNA in TALEN-DNA complexes. By contrast, specificity of protein-protein interactions usually involves backbone shape complementarity1, which is less modular and hence harder to generalize. Coiled-coil heterodimers are an exception, but the restricted geometry of interactions across the heterodimer interface (primarily at the heptad a and d positions2) limits the number of orthogonal pairs that can be created simply by varying side-chain interactions3,4. Here we show that protein-protein interaction specificity can be achieved using extensive and modular side-chain hydrogen-bond networks. We used the Crick generating equations5 to produce millions of four-helix backbones with varying degrees of supercoiling around a central axis, identified those accommodating extensive hydrogen-bond networks, and used Rosetta to connect pairs of helices with short loops and to optimize the remainder of the sequence. Of 97 such designs expressed in Escherichia coli, 65 formed constitutive heterodimers, and the crystal structures of four designs were in close agreement with the computational models and confirmed the designed hydrogen-bond networks. In cells, six heterodimers were fully orthogonal, and in vitro-following mixing of 32 chains from 16 heterodimer designs, denaturation in 5 M guanidine hydrochloride and reannealing-almost all of the interactions observed by native mass spectrometry were between the designed cognate pairs. The ability to design orthogonal protein heterodimers should enable sophisticated protein-based control logic for synthetic biology, and illustrates that nature has not fully explored the possibilities for programmable biomolecular interaction modalities.

De novo protein design by citizen scientists.

Online citizen science projects such as GalaxyZoo1, Eyewire2 and Phylo3 have proven very successful for data collection, annotation and processing, but for the most part have harnessed human pattern-recognition skills rather than human creativity. An exception is the game EteRNA4, in which game players learn to build new RNA structures by exploring the discrete two-dimensional space of Watson-Crick base pairing possibilities. Building new proteins, however, is a more challenging task to present in a game, as both the representation and evaluation of a protein structure are intrinsically three-dimensional. We posed the challenge of de novo protein design in the online protein-folding game Foldit5. Players were presented with a fully extended peptide chain and challenged to craft a folded protein structure and an amino acid sequence encoding that structure. After many iterations of player design, analysis of the top-scoring solutions and subsequent game improvement, Foldit players can now-starting from an extended polypeptide chain-generate a diversity of protein structures and sequences that encode them in silico. One hundred forty-six Foldit player designs with sequences unrelated to naturally occurring proteins were encoded in synthetic genes; 56 were found to be expressed and soluble in Escherichia coli, and to adopt stable monomeric folded structures in solution. The diversity of these structures is unprecedented in de novo protein design, representing 20 different folds-including a new fold not observed in natural proteins. High-resolution structures were determined for four of the designs, and are nearly identical to the player models. This work makes explicit the considerable implicit knowledge that contributes to success in de novo protein design, and shows that citizen scientists can discover creative new solutions to outstanding scientific challenges such as the protein design problem.

Accurate computational design of three-dimensional protein crystals.

Protein crystallization plays a central role in structural biology. Despite this, the process of crystallization remains poorly understood and highly empirical, with crystal contacts, lattice packing arrangements and space group preferences being largely unpredictable. Programming protein crystallization through precisely engineered side-chain-side-chain interactions across protein-protein interfaces is an outstanding challenge. Here we develop a general computational approach for designing three-dimensional protein crystals with prespecified lattice architectures at atomic accuracy that hierarchically constrains the overall number of degrees of freedom of the system. We design three pairs of oligomers that can be individually purified, and upon mixing, spontaneously self-assemble into >100 µm three-dimensional crystals. The structures of these crystals are nearly identical to the computational design models, closely corresponding in both overall architecture and the specific protein-protein interactions. The dimensions of the crystal unit cell can be systematically redesigned while retaining the space group symmetry and overall architecture, and the crystals are extremely porous and highly stable. Our approach enables the computational design of protein crystals with high accuracy, and the designed protein crystals, which have both structural and assembly information encoded in their primary sequences, provide a powerful platform for biological materials engineering.

De novo design of a fluorescence-activating ß-barrel.

The regular arrangements of ß-strands around a central axis in ß-barrels and of α-helices in coiled coils contrast with the irregular tertiary structures of most globular proteins, and have fascinated structural biologists since they were first discovered. Simple parametric models have been used to design a wide range of α-helical coiled-coil structures, but to date there has been no success with ß-barrels. Here we show that accurate de novo design of ß-barrels requires considerable symmetry-breaking to achieve continuous hydrogen-bond connectivity and eliminate backbone strain. We then build ensembles of ß-barrel backbone models with cavity shapes that match the fluorogenic compound DFHBI, and use a hierarchical grid-based search method to simultaneously optimize the rigid-body placement of DFHBI in these cavities and the identities of the surrounding amino acids to achieve high shape and chemical complementarity. The designs have high structural accuracy and bind and fluorescently activate DFHBI in vitro and in Escherichia coli, yeast and mammalian cells. This de novo design of small-molecule binding activity, using backbones custom-built to bind the ligand, should enable the design of increasingly sophisticated ligand-binding proteins, sensors and catalysts that are not limited by the backbone geometries available in known protein structures.

Computational design of a synthetic PD-1 agonist.

Programmed cell death protein-1 (PD-1) expressed on activated T cells inhibits T cell function and proliferation to prevent an excessive immune response, and disease can result if this delicate balance is shifted in either direction. Tumor cells often take advantage of this pathway by overexpressing the PD-1 ligand PD-L1 to evade destruction by the immune system. Alternatively, if there is a decrease in function of the PD-1 pathway, unchecked activation of the immune system and autoimmunity can result. Using a combination of computation and experiment, we designed a hyperstable 40-residue miniprotein, PD-MP1, that specifically binds murine and human PD-1 at the PD-L1 interface with a Kd of ∼100 nM. The apo crystal structure shows that the binder folds as designed with a backbone RMSD of 1.3 Što the design model. Trimerization of PD-MP1 resulted in a PD-1 agonist that strongly inhibits murine T cell activation. This small, hyperstable PD-1 binding protein was computationally designed with an all-beta interface, and the trimeric agonist could contribute to treatments for autoimmune and inflammatory diseases.

Modular repeat protein sculpting using rigid helical junctions.

The ability to precisely design large proteins with diverse shapes would enable applications ranging from the design of protein binders that wrap around their target to the positioning of multiple functional sites in specified orientations. We describe a protein backbone design method for generating a wide range of rigid fusions between helix-containing proteins and use it to design 75,000 structurally unique junctions between monomeric and homo-oligomeric de novo designed and ankyrin repeat proteins (RPs). Of the junction designs that were experimentally characterized, 82% have circular dichroism and solution small-angle X-ray scattering profiles consistent with the design models and are stable at 95 °C. Crystal structures of four designed junctions were in close agreement with the design models with rmsds ranging from 0.9 to 1.6 Å. Electron microscopic images of extended tetrameric structures and ∼10-nm-diameter "L" and "V" shapes generated using the junctions are close to the design models, demonstrating the control the rigid junctions provide for protein shape sculpting over multiple nanometer length scales.

An enumerative algorithm for de novo design of proteins with diverse pocket structures.

To create new enzymes and biosensors from scratch, precise control over the structure of small-molecule binding sites is of paramount importance, but systematically designing arbitrary protein pocket shapes and sizes remains an outstanding challenge. Using the NTF2-like structural superfamily as a model system, we developed an enumerative algorithm for creating a virtually unlimited number of de novo proteins supporting diverse pocket structures. The enumerative algorithm was tested and refined through feedback from two rounds of large-scale experimental testing, involving in total the assembly of synthetic genes encoding 7,896 designs and assessment of their stability on yeast cell surface, detailed biophysical characterization of 64 designs, and crystal structures of 5 designs. The refined algorithm generates proteins that remain folded at high temperatures and exhibit more pocket diversity than naturally occurring NTF2-like proteins. We expect this approach to transform the design of small-molecule sensors and enzymes by enabling the creation of binding and active site geometries much more optimal for specific design challenges than is accessible by repurposing the limited number of naturally occurring NTF2-like proteins.

Tight and specific lanthanide binding in a de novo TIM barrel with a large internal cavity designed by symmetric domain fusion.

De novo protein design has succeeded in generating a large variety of globular proteins, but the construction of protein scaffolds with cavities that could accommodate large signaling molecules, cofactors, and substrates remains an outstanding challenge. The long, often flexible loops that form such cavities in many natural proteins are difficult to precisely program and thus challenging for computational protein design. Here we describe an alternative approach to this problem. We fused two stable proteins with C2 symmetry-a de novo designed dimeric ferredoxin fold and a de novo designed TIM barrel-such that their symmetry axes are aligned to create scaffolds with large cavities that can serve as binding pockets or enzymatic reaction chambers. The crystal structures of two such designs confirm the presence of a 420 cubic Ångström chamber defined by the top of the designed TIM barrel and the bottom of the ferredoxin dimer. We functionalized the scaffold by installing a metal-binding site consisting of four glutamate residues close to the symmetry axis. The protein binds lanthanide ions with very high affinity as demonstrated by tryptophan-enhanced terbium luminescence. This approach can be extended to other metals and cofactors, making this scaffold a modular platform for the design of binding proteins and biocatalysts.

Computational design of closely related proteins that adopt two well-defined but structurally divergent folds.

The plasticity of naturally occurring protein structures, which can change shape considerably in response to changes in environmental conditions, is critical to biological function. While computational methods have been used for de novo design of proteins that fold to a single state with a deep free-energy minimum [P.-S. Huang, S. E. Boyken, D. Baker, Nature 537, 320-327 (2016)], and to reengineer natural proteins to alter their dynamics [J. A. Davey, A. M. Damry, N. K. Goto, R. A. Chica, Nat. Chem. Biol. 13, 1280-1285 (2017)] or fold [P. A. Alexander, Y. He, Y. Chen, J. Orban, P. N. Bryan, Proc. Natl. Acad. Sci. U.S.A. 106, 21149-21154 (2009)], the de novo design of closely related sequences which adopt well-defined but structurally divergent structures remains an outstanding challenge. We designed closely related sequences (over 94% identity) that can adopt two very different homotrimeric helical bundle conformations-one short (∼66 Šheight) and the other long (∼100 Šheight)-reminiscent of the conformational transition of viral fusion proteins. Crystallographic and NMR spectroscopic characterization shows that both the short- and long-state sequences fold as designed. We sought to design bistable sequences for which both states are accessible, and obtained a single designed protein sequence that populates either the short state or the long state depending on the measurement conditions. The design of sequences which are poised to adopt two very different conformations sets the stage for creating large-scale conformational switches between structurally divergent forms.

Self-Assembling 2D Arrays with de Novo Protein Building Blocks.

Modular self-assembly of biomolecules in two dimensions (2D) is straightforward with DNA but has been difficult to realize with proteins, due to the lack of modular specificity similar to Watson-Crick base pairing. Here we describe a general approach to design 2D arrays using de novo designed pseudosymmetric protein building blocks. A homodimeric helical bundle was reconnected into a monomeric building block, and the surface was redesigned in Rosetta to enable self-assembly into a 2D array in the C12 layer symmetry group. Two out of ten designed arrays assembled to micrometer scale under negative stain electron microscopy, and displayed the designed lattice geometry with assembly size up to 100 nm under atomic force microscopy. The design of 2D arrays with pseudosymmetric building blocks is an important step toward the design of programmable protein self-assembly via pseudosymmetric patterning of orthogonal binding interfaces.

Structure of the branched intermediate in protein splicing.

Inteins are autoprocessing domains that cut themselves out of host proteins in a traceless manner. This process, known as protein splicing, involves multiple chemical steps that must be coordinated to ensure fidelity in the process. The committed step in splicing involves attack of a conserved Asn side-chain amide on the adjacent backbone amide, leading to an intein-succinimide product and scission of that peptide bond. This cleavage reaction is stimulated by formation of a branched intermediate in the splicing process. The mechanism by which the Asn side-chain becomes activated as a nucleophile is not understood. Here we solve the crystal structure of an intein trapped in the branched intermediate step in protein splicing. Guided by this structure, we use protein-engineering approaches to show that intein-succinimide formation is critically dependent on a backbone-to-side-chain hydrogen-bond. We propose that this interaction serves to both position the side-chain amide for attack and to activate its nitrogen as a nucleophile. Collectively, these data provide an unprecedented view of an intein poised to carry out the rate-limiting step in protein splicing, shedding light on how a nominally nonnucleophilic group, a primary amide, can become activated in a protein active site.

Precisely patterned nanofibres made from extendable protein multiplexes.

Molecular systems with coincident cyclic and superhelical symmetry axes have considerable advantages for materials design as they can be readily lengthened or shortened by changing the length of the constituent monomers. Among proteins, alpha-helical coiled coils have such symmetric, extendable architectures, but are limited by the relatively fixed geometry and flexibility of the helical protomers. Here we describe a systematic approach to generating modular and rigid repeat protein oligomers with coincident C2 to C8 and superhelical symmetry axes that can be readily extended by repeat propagation. From these building blocks, we demonstrate that a wide range of unbounded fibres can be systematically designed by introducing hydrophilic surface patches that force staggering of the monomers; the geometry of such fibres can be precisely tuned by varying the number of repeat units in the monomer and the placement of the hydrophilic patches.

Computational design of constitutively active cGAS.

Cyclic GMP-AMP synthase (cGAS) is a pattern recognition receptor critical for the innate immune response to intracellular pathogens, DNA damage, tumorigenesis and senescence. Binding to double-stranded DNA (dsDNA) induces conformational changes in cGAS that activate the enzyme to produce 2'-3' cyclic GMP-AMP (cGAMP), a second messenger that initiates a potent interferon (IFN) response through its receptor, STING. Here, we combined two-state computational design with informatics-guided design to create constitutively active, dsDNA ligand-independent cGAS (CA-cGAS). We identified CA-cGAS mutants with IFN-stimulating activity approaching that of dsDNA-stimulated wild-type cGAS. DNA-independent adoption of the active conformation was directly confirmed by X-ray crystallography. In vivo expression of CA-cGAS in tumor cells resulted in STING-dependent tumor regression, demonstrating that the designed proteins have therapeutically relevant biological activity. Our work provides a general framework for stabilizing active conformations of enzymes and provides CA-cGAS variants that could be useful as genetically encoded adjuvants and tools for understanding inflammatory diseases.

Design of multi-scale protein complexes by hierarchical building block fusion.

A systematic and robust approach to generating complex protein nanomaterials would have broad utility. We develop a hierarchical approach to designing multi-component protein assemblies from two classes of modular building blocks: designed helical repeat proteins (DHRs) and helical bundle oligomers (HBs). We first rigidly fuse DHRs to HBs to generate a large library of oligomeric building blocks. We then generate assemblies with cyclic, dihedral, and point group symmetries from these building blocks using architecture guided rigid helical fusion with new software named WORMS. X-ray crystallography and cryo-electron microscopy characterization show that the hierarchical design approach can accurately generate a wide range of assemblies, including a 43 nm diameter icosahedral nanocage. The computational methods and building block sets described here provide a very general route to de novo designed protein nanomaterials.

Crystal structures of the glycopeptide sulfotransferase Teg12 in a complex with the teicoplanin aglycone.

The TEG gene cluster, a glycopeptide biosynthetic gene cluster that is predicted to encode the biosynthesis of a polysulfated glycopeptide congener, was recently cloned from DNA extracted directly from desert soil. This predicted glycopeptide gene cluster contains three closely related sulfotransferases (Teg12, -13, and -14) that sulfate teicoplanin-like glycopeptides at three unique sites. Here we report a series of structures: an apo structure of Teg12, Teg12 bound to the desulfated cosubstrate 3'-phosphoadenosine 5'-phosphate, and Teg12 bound to the teicoplanin aglycone. Teg12 appears to undergo a series of significant conformational rearrangements during glycopeptide recruitment, binding, and catalysis. Loop regions that exhibit the most conformational flexibility show the least sequence conservation between TEG sulfotransferases. Site-directed mutagenesis guided by our structural studies confirmed the importance of key catalytic residues as well as the importance of residues found throughout the conformationally flexible loop regions.

The 2.7†Šresolution structure of the glycopeptide sulfotransferase Teg14.

The TEG gene cluster was recently isolated from an environmental DNA library and is predicted to encode the biosynthesis of a polysulfated glycopeptide congener. Three closely related sulfotransferases found in the TEG gene cluster (Teg12, Teg13 and Teg14) have been shown to sulfate the teicoplanin aglycone at three unique sites. Crystal structures of the first sulfotransferase from the TEG cluster, Teg12, in complex with the teicoplanin aglycone and its desulfated cosubstrate PAP have recently been reported [Bick et al. (2010), Biochemistry, 49, 4159-4168]. Here, the 2.7 Šresolution crystal structure of the apo form of Teg14 is reported. Teg14 sulfates the hydroxyphenylglycine at position 4 in the teicoplanin aglycone. The Teg14 structure is discussed and is compared with those of other bacterial 3'-phosphoadenosine 5'-phosphosulfate-dependent sulfotransferases.

Designed protein logic to target cells with precise combinations of surface antigens.

Precise cell targeting is challenging because most mammalian cell types lack a single surface marker that distinguishes them from other cells. A solution would be to target cells using specific combinations of proteins present on their surfaces. In this study, we design colocalization-dependent protein switches (Co-LOCKR) that perform AND, OR, and NOT Boolean logic operations. These switches activate through a conformational change only when all conditions are met, generating rapid, transcription-independent responses at single-cell resolution within complex cell populations. We implement AND gates to redirect T cell specificity against tumor cells expressing two surface antigens while avoiding off-target recognition of single-antigen cells, and three-input switches that add NOT or OR logic to avoid or include cells expressing a third antigen. Thus, de novo designed proteins can perform computations on the surface of cells, integrating multiple distinct binding interactions into a single output.