De novo design of modular and tunable protein biosensors.

Naturally occurring protein switches have been repurposed for the development of biosensors and reporters for cellular and clinical applications1. However, the number of such switches is limited, and reengineering them is challenging. Here we show that a general class of protein-based biosensors can be created by inverting the flow of information through de novo designed protein switches in which the binding of a peptide key triggers biological outputs of interest2. The designed sensors are modular molecular devices with a closed dark state and an open luminescent state; analyte binding drives the switch from the closed to the open state. Because the sensor is based on the thermodynamic coupling of analyte binding to sensor activation, only one target binding domain is required, which simplifies sensor design and allows direct readout in solution. We create biosensors that can sensitively detect the anti-apoptosis protein BCL-2, the IgG1 Fc domain, the HER2 receptor, and Botulinum neurotoxin B, as well as biosensors for cardiac troponin I and an anti-hepatitis B virus antibody with the high sensitivity required to detect these molecules clinically. Given the need for diagnostic tools to track the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)3, we used the approach to design sensors for the SARS-CoV-2 spike protein and antibodies against the membrane and nucleocapsid proteins. The former, which incorporates a de novo designed spike receptor binding domain (RBD) binder4, has a limit of detection of 15 pM and a luminescence signal 50-fold higher than the background level. The modularity and sensitivity of the platform should enable the rapid construction of sensors for a wide range of analytes, and highlights the power of de novo protein design to create multi-state protein systems with new and useful functions.

Publisher Correction: Modular and tunable biological feedback control using a de novo protein switch.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

Modular and tunable biological feedback control using a de novo protein switch.

De novo-designed proteins1-3 hold great promise as building blocks for synthetic circuits, and can complement the use of engineered variants of natural proteins4-7. One such designer protein-degronLOCKR, which is based on 'latching orthogonal cage-key proteins' (LOCKR) technology8-is a switch that degrades a protein of interest in vivo upon induction by a genetically encoded small peptide. Here we leverage the plug-and-play nature of degronLOCKR to implement feedback control of endogenous signalling pathways and synthetic gene circuits. We first generate synthetic negative and positive feedback in the yeast mating pathway by fusing degronLOCKR to endogenous signalling molecules, illustrating the ease with which this strategy can be used to rewire complex endogenous pathways. We next evaluate feedback control mediated by degronLOCKR on a synthetic gene circuit9, to quantify the feedback capabilities and operational range of the feedback control circuit. The designed nature of degronLOCKR proteins enables simple and rational modifications to tune feedback behaviour in both the synthetic circuit and the mating pathway. The ability to engineer feedback control into living cells represents an important milestone in achieving the full potential of synthetic biology10,11,12. More broadly, this work demonstrates the large and untapped potential of de novo design of proteins for generating tools that implement complex synthetic functionalities in cells for biotechnological and therapeutic applications.

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 design of bioactive protein switches.

Allosteric regulation of protein function is widespread in biology, but is challenging for de novo protein design as it requires the explicit design of multiple states with comparable free energies. Here we explore the possibility of designing switchable protein systems de novo, through the modulation of competing inter- and intramolecular interactions. We design a static, five-helix 'cage' with a single interface that can interact either intramolecularly with a terminal 'latch' helix or intermolecularly with a peptide 'key'. Encoded on the latch are functional motifs for binding, degradation or nuclear export that function only when the key displaces the latch from the cage. We describe orthogonal cage-key systems that function in vitro, in yeast and in mammalian cells with up to 40-fold activation of function by key. The ability to design switchable protein functions that are controlled by induced conformational change is a milestone for de novo protein design, and opens up new avenues for synthetic biology and cell engineering.

Macromolecular modeling and design in Rosetta: recent methods and frameworks.

The Rosetta software for macromolecular modeling, docking and design is extensively used in laboratories worldwide. During two decades of development by a community of laboratories at more than 60 institutions, Rosetta has been continuously refactored and extended. Its advantages are its performance and interoperability between broad modeling capabilities. Here we review tools developed in the last 5 years, including over 80 methods. We discuss improvements to the score function, user interfaces and usability. Rosetta is available at http://www.rosettacommons.org.

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.

The coming of age of de novo protein design.

There are 20(200) possible amino-acid sequences for a 200-residue protein, of which the natural evolutionary process has sampled only an infinitesimal subset. De novo protein design explores the full sequence space, guided by the physical principles that underlie protein folding. Computational methodology has advanced to the point that a wide range of structures can be designed from scratch with atomic-level accuracy. Almost all protein engineering so far has involved the modification of naturally occurring proteins; it should now be possible to design new functional proteins from the ground up to tackle current challenges in biomedicine and nanotechnology.

The SH3 domains of the protein kinases ITK and LCK compete for adjacent sites on T cell-specific adapter protein.

T-cell activation requires stimulation of specific intracellular signaling pathways in which protein-tyrosine kinases, phosphatases, and adapter proteins interact to transmit signals from the T-cell receptor to the nucleus. Interactions of LCK proto-oncogene, SRC family tyrosine kinase (LCK), and the IL-2-inducible T cell kinase (ITK) with the T cell-specific adapter protein (TSAD) promotes LCK-mediated phosphorylation and thereby ITK activation. Both ITK and LCK interact with TSAD's proline-rich region (PRR) through their Src homology 3 (SH3) domains. Whereas LCK may also interact with TSAD through its SH2 domain, ITK interacts with TSAD only through its SH3 domain. To begin to understand on a molecular level how the LCK SH3 and ITK SH3 domains interact with TSAD in human HEK293T cells, here we combined biochemical analyses with NMR spectroscopy. We found that the ITK and LCK SH3 domains potentially have adjacent and overlapping binding sites within the TSAD PRR amino acids (aa) 239-274. Pulldown experiments and NMR spectroscopy revealed that both domains may bind to TSAD aa 239-256 and aa 257-274. Co-immunoprecipitation experiments further revealed that both domains may also bind simultaneously to TSAD aa 242-268. Accordingly, NMR spectroscopy indicated that the SH3 domains may compete for these two adjacent binding sites. We propose that once the associations of ITK and LCK with TSAD promote the ITK and LCK interaction, the interactions among TSAD, ITK, and LCK are dynamically altered by ITK phosphorylation status.

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.

An Autoinhibitory Role for the Pleckstrin Homology Domain of Interleukin-2-Inducible Tyrosine Kinase and Its Interplay with Canonical Phospholipid Recognition.

Pleckstrin homology (PH) domains are well-known as phospholipid binding modules, yet evidence that PH domain function extends beyond lipid recognition is mounting. In this work, we characterize a protein binding function for the PH domain of interleukin-2-inducible tyrosine kinase (ITK), an immune cell specific signaling protein that belongs to the TEC family of nonreceptor tyrosine kinases. Its N-terminal PH domain is a well-characterized lipid binding module that localizes ITK to the membrane via phosphatidylinositol 3,4,5-trisphosphate (PIP3) binding. Using a combination of nuclear magnetic resonance spectroscopy and mutagenesis, we have mapped an autoregulatory protein interaction site on the ITK PH domain that makes direct contact with the catalytic kinase domain of ITK, inhibiting the phospho-transfer reaction. Moreover, we have elucidated an important interplay between lipid binding by the ITK PH domain and the stability of the autoinhibitory complex formed by full length ITK. The ITK activation loop in the kinase domain becomes accessible to phosphorylation to the exogenous kinase LCK upon binding of the ITK PH domain to PIP3. By clarifying the allosteric role of the ITK PH domain in controlling ITK function, we have expanded the functional repertoire of the PH domain generally and opened the door to alternative strategies to target this specific kinase in the context of immune cell signaling.

Modeling oblong proteins and water-mediated interfaces with RosettaDock in CAPRI rounds 28-35.

The 28th-35th rounds of the Critical Assessment of PRotein Interactions (CAPRI) served as a practical benchmark for our RosettaDock protein-protein docking protocols, highlighting strengths and weaknesses of the approach. We achieved acceptable or better quality models in three out of 11 targets. For the two α-repeat protein-green fluorescent protein (αrep-GFP) complexes, we used a novel ellipsoidal partial-global docking method (Ellipsoidal Dock) to generate models with 2.2 Å/1.5 Å interface RMSD, capturing 49%/42% of the native contacts, for the 7-/5-repeat αrep complexes. For the DNase-immunity protein complex, we used a new predictor of hydrogen-bonding networks, HBNet with Bridging Waters, to place individual water models at the complex interface; models were generated with 1.8 Å interface RMSD and 12% native water contacts recovered. The targets for which RosettaDock failed to create an acceptable model were typically difficult in general, as six had no acceptable models submitted by any CAPRI predictor. The UCH-L5-RPN13 and UCH-L5-INO80G de-ubiquitinating enzyme-inhibitor complexes comprised inhibitors undergoing significant structural changes upon binding, with the partners being highly interwoven in the docked complexes. Our failure to predict the nucleosome-enzyme complex in Target 95 was largely due to tight constraints we placed on our model based on sparse biochemical data suggesting two specific cross-interface interactions, preventing the correct structure from being sampled. While RosettaDock's three successes show that it is a state-of-the-art docking method, the difficulties with highly flexible and multi-domain complexes highlight the need for better flexible docking and domain-assembly methods. Proteins 2017; 85:479-486. © 2016 Wiley Periodicals, Inc.

Dynamic Allostery Mediated by a Conserved Tryptophan in the Tec Family Kinases.

Bruton's tyrosine kinase (Btk) is a Tec family non-receptor tyrosine kinase that plays a critical role in immune signaling and is associated with the immunological disorder X-linked agammaglobulinemia (XLA). Our previous findings showed that the Tec kinases are allosterically activated by the adjacent N-terminal linker. A single tryptophan residue in the N-terminal 17-residue linker mediates allosteric activation, and its mutation to alanine leads to the complete loss of activity. Guided by hydrogen/deuterium exchange mass spectrometry results, we have employed Molecular Dynamics simulations, Principal Component Analysis, Community Analysis and measures of node centrality to understand the details of how a single tryptophan mediates allostery in Btk. A specific tryptophan side chain rotamer promotes the functional dynamic allostery by inducing coordinated motions that spread across the kinase domain. Either a shift in the rotamer population, or a loss of the tryptophan side chain by mutation, drastically changes the coordinated motions and dynamically isolates catalytically important regions of the kinase domain. This work also identifies a new set of residues in the Btk kinase domain with high node centrality values indicating their importance in transmission of dynamics essential for kinase activation. Structurally, these node residues appear in both lobes of the kinase domain. In the N-lobe, high centrality residues wrap around the ATP binding pocket connecting previously described Catalytic-spine residues. In the C-lobe, two high centrality node residues connect the base of the R- and C-spines on the αF-helix. We suggest that the bridging residues that connect the catalytic and regulatory architecture within the kinase domain may be a crucial element in transmitting information about regulatory spine assembly to the catalytic machinery of the catalytic spine and active site.

Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport.

Gram-negative bacteria, such as Escherichia coli, frequently use tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel various toxic compounds from the cell. The efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions. No previous structural information was available for the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here we describe the crystal structures of the inner-membrane transporter CusA in the absence and presence of bound Cu(I) or Ag(I). These CusA structures provide new structural information about the HME subfamily of RND efflux pumps. The structures suggest that the metal-binding sites, formed by a three-methionine cluster, are located within the cleft region of the periplasmic domain. This cleft is closed in the apo-CusA form but open in the CusA-Cu(I) and CusA-Ag(I) structures, which directly suggests a plausible pathway for ion export. Binding of Cu(I) and Ag(I) triggers significant conformational changes in both the periplasmic and transmembrane domains. The crystal structure indicates that CusA has, in addition to the three-methionine metal-binding site, four methionine pairs-three located in the transmembrane region and one in the periplasmic domain. Genetic analysis and transport assays suggest that CusA is capable of actively picking up metal ions from the cytosol, using these methionine pairs or clusters to bind and export metal ions. These structures suggest a stepwise shuttle mechanism for transport between these sites.

Rates of molecular evolution vary in vertebrates for insulin-like growth factor-1 (IGF-1), a pleiotropic locus that regulates life history traits.

Insulin-like growth factor-1 (IGF-1) is a member of the vertebrate insulin/insulin-like growth factor/relaxin gene family necessary for growth, reproduction, and survival at both the cellular and organismal level. Its sequence, protein structure, and function have been characterized in mammals, birds, and fish; however, a notable gap in our current knowledge of the function of IGF-1 and its molecular evolution is information in ectothermic reptiles. To address this disparity, we sequenced the coding region of IGF-1 in 11 reptile species-one crocodilian, three turtles, three lizards, and four snakes. Complete sequencing of the full mRNA transcript of a snake revealed the Ea-isoform, the predominant isoform of IGF-1 also reported in other vertebrate groups. A gene tree of the IGF-1 protein-coding region that incorporated sequences from diverse vertebrate groups showed similarity to the species phylogeny, with the exception of the placement of Testudines as sister group to Aves, due to their high nucleotide sequence similarity. In contrast, long-branch lengths indicate more rapid divergence in IGF-1 among lizards and snakes. Additionally, lepidosaurs (i.e., lizards and snakes) had higher rates of non-synonymous:synonymous substitutions (dN/dS) relative to archosaurs (i.e., birds and crocodilians) and turtles. Tests for positive selection on specific codons within branches and evaluation of the changes in the amino acid properties, suggested positive selection in lepidosaurs on the C domain of IGF-1, which is involved in binding affinity to the IGF-1 receptor. Predicted structural changes suggest that major alterations in protein structure and function may have occurred in reptiles. These data propose new insights into the molecular co-evolution of IGF-1 and its receptors, and ultimately the evolution of IGF-1's role in regulating life-history traits across vertebrates.

De novo design of obligate ABC-type heterotrimeric proteins.

The de novo design of three protein chains that associate to form a heterotrimer (but not any of the possible two-chain heterodimers) and that can drive the assembly of higher-order branching structures is an important challenge for protein design. We designed helical heterotrimers with specificity conferred by buried hydrogen bond networks and large aromatic residues to enhance shape complementary packing. We obtained ten designs for which all three chains cooperatively assembled into heterotrimers with few or no other species present. Crystal structures of a helical bundle heterotrimer and extended versions, with helical repeat proteins fused to individual subunits, showed all three chains assembling in the designed orientation. We used these heterotrimers as building blocks to construct larger cyclic oligomers, which were structurally validated by electron microscopy. Our three-way junction designs provide new routes to complex protein nanostructures and enable the scaffolding of three distinct ligands for modulation of cell signaling.

Multivalent designed proteins neutralize SARS-CoV-2 variants of concern and confer protection against infection in mice.

New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.

Multivalent designed proteins protect against SARS-CoV-2 variants of concern.

Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC 50 values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness. ONE-SENTENCE SUMMARY: We designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.

Design and structure of two new protein cages illustrate successes and ongoing challenges in protein engineering.

In recent years, new protein engineering methods have produced more than a dozen symmetric, self-assembling protein cages whose structures have been validated to match their design models with near-atomic accuracy. However, many protein cage designs that are tested in the lab do not form the desired assembly, and improving the success rate of design has been a point of recent emphasis. Here we present two protein structures solved by X-ray crystallography of designed protein oligomers that form two-component cages with tetrahedral symmetry. To improve on the past tendency toward poorly soluble protein, we used a computational protocol that favors the formation of hydrogen-bonding networks over exclusively hydrophobic interactions to stabilize the designed protein-protein interfaces. Preliminary characterization showed highly soluble expression, and solution studies indicated successful cage formation by both designed proteins. For one of the designs, a crystal structure confirmed at high resolution that the intended tetrahedral cage was formed, though several flipped amino acid side chain rotamers resulted in an interface that deviates from the precise hydrogen-bonding pattern that was intended. A structure of the other designed cage showed that, under the conditions where crystals were obtained, a noncage structure was formed wherein a porous 3D protein network in space group I21 3 is generated by an off-target twofold homomeric interface. These results illustrate some of the ongoing challenges of developing computational methods for polar interface design, and add two potentially valuable new entries to the growing list of engineered protein materials for downstream applications.