Biology subjects

Yeates, T. O.

Publications and source records attributed to Yeates, T. O..

10 recordsLinked to original sources

bioRxiv2026

Design of full-length agonist and antagonist antibodies for hard targets

Therapeutic antibodies have transformed modern medicine, yet conventional discovery relies on lengthy experimental workflows, offers limited control over how antibodies engage their targets, and struggles to deliver candidates across many important target classes. Despite recent advances in computational protein design, generating full-length antibodies with desired functional activity and favorable developability profiles remains challenging. Here we present an end-to-end computational design pipeline built around Pythia-1, an all-atom generative model that reasons jointly over amino acid sequence and three-dimensional atomic structure under explicit design constraints. We generated candidates in full-length human immunoglobulin G (IgG) format across 22 therapeutic targets spanning receptor ectodomains, cytokines, growth factors, and G protein-coupled receptors (GPCRs), a particularly challenging target class for antibody discovery. Following a single computational design round, experimental screening of at most 48 candidates per target identified antibodies with single-digit nanomolar affinities for soluble proteins and single-digit nanomolar cell-binding EC50 values for GPCRs. The pipeline's ability to target specified epitopes and conformational states enabled the design of antibodies with agonist and antagonist activity. In vitro characterization demonstrated target-specific engagement and potent functional modulation, with single-digit nanomolar inhibitory potency in functional assays for a range of targets, including multiple GPCRs. To our knowledge, this study provides the first demonstration of both GPCR agonism and antagonism by de novo designed full-length IgG antibodies. We incorporated key developability and manufacturability requirements into the initial designs by conditioning generation on human germline frameworks and applying sequence constraints to limit known liabilities. The resulting antibody designs exhibited biophysical profiles comparable to or better than those of clinical-stage therapeutic controls. We provide the complete sequences of all lead candidates to support independent experimental validation and further development. Together, these findings demonstrate that our technology can deliver antibodies with programmable function and key properties expected of lead candidates, opening opportunities for first-in-class therapeutics against hard-to-drug targets.

molecular biology↗

bioRxiv2026

Design and structure of protein cages based on helical fusion and machine learning

Self-assembling protein cages are versatile nanoscale architectures with broad applications in drug delivery, vaccine development, and structural biology. Historically, two main strategies have been used to construct such cages: genetic fusion of oligomeric domains connected by helical linkers, and computational interface design using either physics-based or machine learning-based methods. Here, we extend the original fusion approach using modern AI algorithms and more sophisticated treatments of helix bending to create protein cages with novel architectures composed exclusively of trimeric building blocks arranged in tetrahedral symmetry. Of fifteen designs tested experimentally, multiple sequence variants of two of these designs assembled predominantly into soluble, monodisperse particles of the expected size, with native molecular masses of 633 kDa (T33-Fus-1A, B) and 638 kDa (T33-Fus-2). Cryo-electron microscopy (cryo-EM) structures of three distinct sequence variants spanning from 3.0-3.9 [A] in resolution confirmed the intended structures in atomic detail, with C-alpha RSMD values over the entire assemblies as low as 2 [A]. The predicted modes of helix bending were similarly validated. The results highlight the impact of methodological improvements for achieving a level of regularity and design precision that has largely evaded prior applications of the fusion approach. These findings expand the prospects and accessible design space for self-assembling protein nanomaterials.

biochemistry↗

bioRxiv2024

Designed fluorescent protein cages as fiducial markers for targeted cell imaging

Understanding how proteins function within their cellular environments is essential for cellular biology and biomedical research. However, current imaging techniques exhibit limitations, particularly in the study of small complexes and individual proteins within cells. Previously, protein cages have been employed as imaging scaffolds to study purified small proteins using cryo-electron microscopy (cryo-EM). Here we demonstrate an approach to deliver designed protein cages - endowed with fluorescence and targeted binding properties - into cells, thereby serving as fiducial markers for cellular imaging. We used protein cages with anti-GFP DARPin domains to target a mitochondrial protein (MFN1) expressed in mammalian cells, which was genetically fused to GFP. We demonstrate that the protein cages can penetrate cells, are directed to specific subcellular locations, and are detectable with confocal microscopy. This innovation represents a milestone in developing tools for in-depth cellular exploration, especially in conjunction with methods such as cryo-correlative light and electron microscopy (cryo-CLEM).

biochemistry↗

bioRxiv2024

Structural determinants of co-translational protein complex assembly

The assembly of proteins into functional complexes is critical to lifes processes. While textbooks depict complex assembly as occurring between fully synthesized proteins, we know today that thousands of proteins in the human proteome assemble co-translationally during their synthesis. Why this process takes place, however, remains unknown. We show that co-translational assembly is governed by biophysical and structural characteristics of the protein complex, and involves mutually stabilized, intertwined subunits. Consequently, these subunits are also co-regulated across the central dogma, from transcription to protein degradation. Leveraging structural signatures with AlphaFold2-based predictions enables us to accurately predict co-translational assembly on a proteome-wide scale, which we validated by ribosome profiling, genetic perturbations, and smFISH experiments. Notably, the latter showed that co-translationally assembling subunits exhibit co-localized mRNAs. This work unveils a fundamental connection between protein structure and the translation process, highlighting the overarching impact of three-dimensional structure on gene expression, mRNA localization, and proteostasis. One Sentence SummaryProtein complexes with topologically intertwined subunits require co-translational assembly and synchronized proteostasis of subunits, with implications in protein stability, mRNA localization, and evolution. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=195 SRC="FIGDIR/small/576408v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@712a2org.highwire.dtl.DTLVardef@58894borg.highwire.dtl.DTLVardef@9db5c6org.highwire.dtl.DTLVardef@8eae4_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2023

Design of a symmetry-broken tetrahedral protein cage by a method of internal steric occlusion

Methods in protein design have made it possible to create large and complex, self-assembling protein cages with diverse applications. These have largely been based on highly symmetric forms exemplified by the Platonic solids. Prospective applications of protein cages would be expanded by strategies for breaking the designed symmetry, e.g., so that only one or a few (instead of many) copies of an exterior domain or motif might be displayed on their surfaces. Here we demonstrate a straightforward design approach for creating symmetry-broken protein cages able to display singular copies of outward-facing domains. We modify the subunit of an otherwise symmetric protein cage through fusion to a small inward-facing domain, only one copy of which can be accommodated in the cage interior. Using biochemical methods and native mass spectrometry, we show that co-expression of the original subunit and the modified subunit, which is further fused to an outward-facing anti-GFP DARPin domain, leads to self-assembly of a protein cage presenting just one copy of the DARPin protein on its exterior. This strategy of designed occlusion provides a facile route for creating new types of protein cages with unique properties.

biochemistry↗

bioRxiv2023

A Suite of Designed Protein Cages Using Machine Learning Algorithms and Protein Fragment-Based Protocols

Designed protein cages and related materials provide unique opportunities for applications in biotechnology and medicine, while methods for their creation remain challenging and unpredictable. In the present study, we apply new computational approaches to design a suite of new tetrahedrally symmetric, self-assembling protein cages. For the generation of docked poses, we emphasize a protein fragment-based approach, while for de novo interface design, a comparison of computational protocols highlights the power and increased experimental success achieved using the machine learning program ProteinMPNN. In relating information from docking and design, we observe that agreement between fragment-based sequence preferences and ProteinMPNN sequence inference correlates with experimental success. Additional insights for designing polar interactions are highlighted by experimentally testing larger and more polar interfaces. In all, using X-ray crystallography and cryo-EM, we report five structures for seven protein cages, with atomic resolution in the best case reaching 2.0 [A]. We also report structures of two incompletely assembled protein cages, providing unique insights into one type of assembly failure. The new set of designed cages and their structures add substantially to the body of available protein nanoparticles, and to methodologies for their creation.

biochemistry↗

bioRxiv2022

Rigidified Scaffolds for 3-Angstrom Resolution Cryo-EM of Small Therapeutic Protein Targets

Numerous technical advances have made cryo-EM an attractive method for atomic structure determination. Cryo-EM is ideally suited for large macromolecular structures, while problems of low signal-to-noise prevent routine structure determination of proteins smaller than about 50 kDa. This size limitation excludes large numbers of important cellular proteins from structural characterization by this powerful technique, including many cell-signaling proteins of high therapeutic interest. In the present work, we use molecular engineering techniques to rigidify an imaging scaffold, based on a designed protein cage, to the point where 3 [A] resolution can be achieved, even for very small proteins. After optimizing the design of the rigidified scaffold on test proteins, we apply this imaging system to the key oncogenic signaling protein KRAS, which represents an outstanding challenge in the area of structure-based drug design. Despite its 19 kDa size, we show that the structure of KRAS, in multiple mutant forms, and bound to its GDP ligand, can be readily interpreted at a resolution slightly better than 3.0 [A]. This advance further expands the capability of cryo-EM to become an essentially universal method for protein structure determination, including for applications to small therapeutic protein targets.

biochemistry↗

bioRxiv2021

A fragment-based protein interface design algorithm for symmetric assemblies

Theoretical and experimental advances in protein engineering have led to the creation of precisely defined, novel protein assemblies of great size and complexity, with diverse applications. One powerful approach involves designing a new attachment or binding interface between two simpler symmetric oligomeric protein components. The required methods of design, which present both similarities and key differences compared to problems in protein docking, remain challenging, and are not yet routine. With the aim of more fully enabling this emerging area of protein material engineering, we developed a computer program, Nanohedra, to introduce two key advances. First, we encoded in the program the construction rules (i.e. the search space parameters) that underlie all possible symmetric material constructions. Second, we developed algorithms for rapidly identifying favorable docking/interface arrangements based on tabulations of empirical patterns of known protein fragment-pair associations. As a result, the candidate poses that Nanohedra generates for subsequent amino acid interface design appear highly native-like (at the protein backbone level), while simultaneously conforming to the exacting requirements for symmetry-based assembly. A retrospective computational analysis of successful vs failed experimental studies supports the expectation that this should improve the success rate for this challenging area of protein engineering.

synthetic biology↗

bioRxiv2021

MCPdb: The Bacterial Microcompartment Database

Bacterial microcompartments are organelle-like structures composed entirely of proteins. They have evolved to carry out several distinct and specialized metabolic functions in a wide variety of bacteria. Their outer shell is constructed from thousands of tessellating protein subunits, encapsulating enzymes that carry out the internal metabolic reactions. The shell proteins are varied, with single, tandem and permuted versions of the PF00936 protein family domain comprising the primary structural component of their polyhedral architecture, which is reminiscent of a viral capsid. While considerable amounts of structural and biophysical data have been generated in the last 15 years, current resources present challenges for understanding the functional and structural properties of microcompartments (MCPs) and their diversity. In order to make the remarkable structural features of bacterial microcompartments accessible to a broad community of scientists and non-specialists, we developed MCPdb: The Bacterial Microcompartment Database (https://mcpdb.mbi.ucla.edu/). MCPdb is a comprehensive resource that categorizes and organizes known microcompartment protein structures and their larger assemblies. To emphazise the critical roles symmetric assembly and architecture play in microcompartment function, each structure in the MCPdb is validated and annotated with respect to: (1) its predicted natural assembly state (2) tertiary structure and topology and (3) the metabolic compartment type from which it derives. The current database includes 134 structures and is available to the public with the anticipation that it will serve as a growing resource for scientists interested in understanding protein-based metabolic organelles in bacteria.

bioinformatics↗

bioRxiv2018

A 3.8 A Resolution Cryo-EM Structure of a Small Protein Bound to a Modular Imaging Scaffold

Proteins smaller than about 50 kDa are currently too small to be imaged by cryo-electron microscopy (cryo-EM), leaving most protein molecules in the cell beyond the reach of this powerful structural technique. Here we use a designed protein scaffold to bind and symmetrically display 12 copies of a small 26 kDa protein. We show that the bound cargo protein is held rigidly enough to visualize it at a resolution of 3.8 [A] by cryo-EM, where basic structural features of the protein are visible. The designed scaffold is modular and can be modified through modest changes in its amino acid sequence to bind and display diverse proteins for imaging, thus providing a general method to break through the lower size limitation in cryo-EM.

biochemistry↗