De novo design of high-affinity binders of bioactive helical peptides.

Many peptide hormones form an α-helix on binding their receptors1-4, and sensitive methods for their detection could contribute to better clinical management of disease5. De novo protein design can now generate binders with high affinity and specificity to structured proteins6,7. However, the design of interactions between proteins and short peptides with helical propensity is an unmet challenge. Here we describe parametric generation and deep learning-based methods for designing proteins to address this challenge. We show that by extending RFdiffusion8 to enable binder design to flexible targets, and to refining input structure models by successive noising and denoising (partial diffusion), picomolar-affinity binders can be generated to helical peptide targets by either refining designs generated with other methods, or completely de novo starting from random noise distributions without any subsequent experimental optimization. The RFdiffusion designs enable the enrichment and subsequent detection of parathyroid hormone and glucagon by mass spectrometry, and the construction of bioluminescence-based protein biosensors. The ability to design binders to conformationally variable targets, and to optimize by partial diffusion both natural and designed proteins, should be broadly useful.

De novo design of protein structure and function with RFdiffusion.

There has been considerable recent progress in designing new proteins using deep-learning methods1-9. Despite this progress, a general deep-learning framework for protein design that enables solution of a wide range of design challenges, including de novo binder design and design of higher-order symmetric architectures, has yet to be described. Diffusion models10,11 have had considerable success in image and language generative modelling but limited success when applied to protein modelling, probably due to the complexity of protein backbone geometry and sequence-structure relationships. Here we show that by fine-tuning the RoseTTAFold structure prediction network on protein structure denoising tasks, we obtain a generative model of protein backbones that achieves outstanding performance on unconditional and topology-constrained protein monomer design, protein binder design, symmetric oligomer design, enzyme active site scaffolding and symmetric motif scaffolding for therapeutic and metal-binding protein design. We demonstrate the power and generality of the method, called RoseTTAFold diffusion (RFdiffusion), by experimentally characterizing the structures and functions of hundreds of designed symmetric assemblies, metal-binding proteins and protein binders. The accuracy of RFdiffusion is confirmed by the cryogenic electron microscopy structure of a designed binder in complex with influenza haemagglutinin that is nearly identical to the design model. In a manner analogous to networks that produce images from user-specified inputs, RFdiffusion enables the design of diverse functional proteins from simple molecular specifications.

Design of protein-binding proteins from the target structure alone.

The design of proteins that bind to a specific site on the surface of a target protein using no information other than the three-dimensional structure of the target remains a challenge1-5. Here we describe a general solution to this problem that starts with a broad exploration of the vast space of possible binding modes to a selected region of a protein surface, and then intensifies the search in the vicinity of the most promising binding modes. We demonstrate the broad applicability of this approach through the de novo design of binding proteins to 12 diverse protein targets with different shapes and surface properties. Biophysical characterization shows that the binders, which are all smaller than 65 amino acids, are hyperstable and, following experimental optimization, bind their targets with nanomolar to picomolar affinities. We succeeded in solving crystal structures of five of the binder-target complexes, and all five closely match the corresponding computational design models. Experimental data on nearly half a million computational designs and hundreds of thousands of point mutants provide detailed feedback on the strengths and limitations of the method and of our current understanding of protein-protein interactions, and should guide improvements of both. Our approach enables the targeted design of binders to sites of interest on a wide variety of proteins for therapeutic and diagnostic applications.

Geometry and expression enhance enrichment of functional yeast-displayed ligands via cell panning.

Yeast surface display has proven to be an effective tool in the discovery and evolution of ligands with new or improved binding activity. Selections for binding activity are generally carried out using immobilized or fluorescently labeled soluble domains of target molecules such as recombinant ectodomain fragments. While this method typically provides ligands with high affinity and specificity for the soluble molecular target, translation to binding true membrane-bound cellular target is commonly problematic. Direct selections against mammalian cell surfaces can be carried out either exclusively or in combination with soluble target-based selections to further direct towards ligands for genuine cellular target. Using a series of fibronectin domain, affibody, and Gp2 ligands and human cell lines expressing a range of their targets, epidermal growth factor receptor and carcinoembryonic antigen, this study quantitatively identifies the elements that dictate ligand enrichment and yield. Most notably, extended flexible linkers between ligand and yeast enhance enrichment ratios from 1.4 ± 0.8 to 62 ± 57 for a low-affinity (>600 nM) binder on cells with high target expression and from 14 ± 13 to 74 ± 25 for a high-affinity binder (2 nM) on cells with medium valency. Inversion of the yeast display fusion from C-terminal display to N-terminal display still enables enrichment albeit with 40-97% reduced efficacy. Collectively, this study further enlightens the conditions-while highlighting new approaches-that yield successful enrichment of yeast-displayed binding ligands via panning on mammalian cells. Biotechnol. Bioeng. 2016;113: 2328-2341. © 2016 Wiley Periodicals, Inc.

Single-shot quantitative x-ray imaging using a primary modulator and dual-layer detector.

BACKGROUND: Conventional x-ray imaging and fluoroscopy have limitations in quantitation due to several challenges, including scatter, beam hardening, and overlapping tissues. Dual-energy (DE) imaging, with its capability to quantify area density of specific materials, is well-suited to address such limitations, but only if the dual-energy projections are acquired with perfect spatial and temporal alignment and corrected for scatter. PURPOSE: In this work, we propose single-shot quantitative imaging (SSQI) by combining the use of a primary modulator (PM) and dual-layer (DL) detector, which enables motion-free DE imaging with scatter correction in a single exposure. METHODS: The key components of our SSQI setup include a PM and DL detector, where the former enables scatter correction for the latter while the latter enables beam hardening correction for the former. The SSQI algorithm allows simultaneous recovery of two material-specific images and two scatter images using four sub-measurements from the PM encoding. The concept was first demonstrated using simulation of chest x-ray imaging for a COVID patient. For validation, we set up SSQI geometry on our tabletop system and imaged acrylic and copper slabs with known thicknesses (acrylic: 0-22.5 cm; copper: 0-0.9 mm), estimated scatter with our SSQI algorithm, and compared the material decomposition (MD) for different combinations of the two materials with ground truth. Second, we imaged an anthropomorphic chest phantom containing contrast in the coronary arteries and compared the MD with and without SSQI. Lastly, to evaluate SSQI in dynamic applications, we constructed a flow phantom that enabled dynamic imaging of iodine contrast. RESULTS: Our simulation study demonstrated that SSQI led to accurate scatter correction and MD, particularly for smaller focal blur and finer PM pitch. In the validation study, we found that the root mean squared error (RMSE) of SSQI estimation was 0.13 cm for acrylic and 0.04 mm for copper. For the anthropomorphic phantom, direct MD resulted in incorrect interpretation of contrast and soft tissue, while SSQI successfully distinguished them quantitatively, reducing RMSE in material-specific images by 38%-92%. For the flow phantom, SSQI was able to perform accurate dynamic quantitative imaging, separating contrast from the background. CONCLUSIONS: We demonstrated the potential of SSQI for robust quantitative x-ray imaging. The integration of SSQI is straightforward with the addition of a PM and upgrade to a DL detector, which may enable its widespread adoption, including in techniques such as radiography and dynamic imaging (i.e., real-time image guidance and cone-beam CT).

Atomically accurate de novo design of single-domain antibodies.

Despite the central role that antibodies play in modern medicine, there is currently no way to rationally design novel antibodies to bind a specific epitope on a target. Instead, antibody discovery currently involves time-consuming immunization of an animal or library screening approaches. Here we demonstrate that a fine-tuned RFdiffusion network is capable of designing de novo antibody variable heavy chains (VHH's) that bind user-specified epitopes. We experimentally confirm binders to four disease-relevant epitopes, and the cryo-EM structure of a designed VHH bound to influenza hemagglutinin is nearly identical to the design model both in the configuration of the CDR loops and the overall binding pose.

Improving de novo protein binder design with deep learning.

Recently it has become possible to de novo design high affinity protein binding proteins from target structural information alone. There is, however, considerable room for improvement as the overall design success rate is low. Here, we explore the augmentation of energy-based protein binder design using deep learning. We find that using AlphaFold2 or RoseTTAFold to assess the probability that a designed sequence adopts the designed monomer structure, and the probability that this structure binds the target as designed, increases design success rates nearly 10-fold. We find further that sequence design using ProteinMPNN rather than Rosetta considerably increases computational efficiency.

Designed Endocytosis-Triggering Proteins mediate Targeted Degradation.

Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by interaction with endogenous ligands. Therapeutic approaches such as LYTAC1,2 and KineTAC3, have taken advantage of this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. While powerful, these approaches can be limited by possible competition with the endogenous ligand(s), the requirement in some cases for chemical modification that limits genetic encodability and can complicate manufacturing, and more generally, there may not be natural ligands which stimulate endocytosis through a given receptor. Here we describe general protein design approaches for designing endocytosis triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for the IGF-2R, ASGPR, Sortillin, and Transferrin receptors, and show that fusing these tags to proteins which bind to soluble or transmembrane protein leads to lysosomal trafficking and target degradation; as these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. The modularity and genetic encodability of EndoTags enables AND gate control for higher specificity targeted degradation, and the localized secretion of degraders from engineered cells. The tunability and modularity of our genetically encodable EndoTags should contribute to deciphering the relationship between receptor engagement and cellular trafficking, and they have considerable therapeutic potential as targeted degradation inducers, signaling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody drug and RNA conjugates.

Scaffolding protein functional sites using deep learning.

The binding and catalytic functions of proteins are generally mediated by a small number of functional residues held in place by the overall protein structure. Here, we describe deep learning approaches for scaffolding such functional sites without needing to prespecify the fold or secondary structure of the scaffold. The first approach, "constrained hallucination," optimizes sequences such that their predicted structures contain the desired functional site. The second approach, "inpainting," starts from the functional site and fills in additional sequence and structure to create a viable protein scaffold in a single forward pass through a specifically trained RoseTTAFold network. We use these two methods to design candidate immunogens, receptor traps, metalloproteins, enzymes, and protein-binding proteins and validate the designs using a combination of in silico and experimental tests.

High-CO2 Requirement as a Mechanism for the Containment of Genetically Modified Cyanobacteria.

As researchers engineer cyanobacteria for biotechnological applications, we must consider potential environmental release of these organisms. Previous theoretical work has considered cyanobacterial containment through elimination of the CO2-concentrating mechanism (CCM) to impose a high-CO2 requirement (HCR), which could be provided in the cultivation environment but not in the surroundings. In this work, we experimentally implemented an HCR containment mechanism in Synechococcus sp. strain PCC7002 (PCC7002) through deletion of carboxysome shell proteins and showed that this mechanism contained cyanobacteria in a 5% CO2 environment. We considered escape through horizontal gene transfer (HGT) and reduced the risk of HGT escape by deleting competence genes. We showed that the HCR containment mechanism did not negatively impact the performance of a strain of PCC7002 engineered for L-lactate production. We showed through coculture experiments of HCR strains with ccm-containing strains that this HCR mechanism reduced the frequency of escape below the NIH recommended limit for recombinant organisms of one escape event in 108 CFU.