Despite decades of work by structural biologists, there are still ~5200 protein families with unknown structure outside the range of comparative modeling. We show that Rosetta structure prediction guided by residue-residue contacts inferred from evolutionary information can accurately model proteins that belong to large families and that metagenome sequence data more than triple the number of protein families with sufficient sequences for accurate modeling. We then integrate metagenome data, contact-based structure matching, and Rosetta structure calculations to generate models for 614 protein families with currently unknown structures; 206 are membrane proteins and 137 have folds not represented in the Protein Data Bank. This approach provides the representative models for large protein families originally envisioned as the goal of the Protein Structure Initiative at a fraction of the cost.

Interleukin-6 (IL-6) family cytokines signal through multimeric receptor complexes, providing unique opportunities to create novel ligand-based therapeutics. The cardiotrophin-like cytokine factor 1 (CLCF1) ligand has been shown to play a role in cancer, osteoporosis, and atherosclerosis. Once bound to ciliary neurotrophic factor receptor (CNTFR), CLCF1 mediates interactions to coreceptors glycoprotein 130 (gp130) and leukemia inhibitory factor receptor (LIFR). By increasing CNTFR-mediated binding to these coreceptors we generated a receptor superagonist which surpassed the potency of natural CNTFR ligands in neuronal signaling. Through additional mutations, we generated a receptor antagonist with increased binding to CNTFR but lack of binding to the coreceptors that inhibited tumor progression in murine xenograft models of nonsmall cell lung cancer. These studies further validate the CLCF1-CNTFR signaling axis as a therapeutic target and highlight an approach of engineering cytokine activity through a small number of mutations.

A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. Here we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix-loop-helix-loop structural motif. Eighty-three designs with sequences unrelated to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.

Synthetic protein switches controlled with user-defined inputs are powerful tools for studying and controlling dynamic cellular processes. To date, these approaches have relied primarily on intermolecular regulation. Here we report a computationally guided framework for engineering intramolecular regulation of protein function. We utilize this framework to develop chemically inducible activator of RAS (CIAR), a single-component RAS rheostat that directly activates endogenous RAS in response to a small molecule. Using CIAR, we show that direct RAS activation elicits markedly different RAS-ERK signaling dynamics from growth factor stimulation, and that these dynamics differ among cell types. We also found that the clinically approved RAF inhibitor vemurafenib potently primes cells to respond to direct wild-type RAS activation. These results demonstrate the utility of CIAR for quantitatively interrogating RAS signaling. Finally, we demonstrate the general utility of our approach in design of intramolecularly regulated protein tools by applying it to the Rho family of guanine nucleotide exchange factors.

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 Å height) and the other long (∼100 Å height)-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 dodecahedron [corrected] is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The dodecahedron [corrected] is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent ‘standard candles’ for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.

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 Å height) and the other long (∼100 Å height)-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 regular arrangements of β-strands around a central axis in β-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 β-barrels. Here we show that accurate de novo design of β-barrels requires considerable symmetry-breaking to achieve continuous hydrogen-bond connectivity and eliminate backbone strain. We then build ensembles of β-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.

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.