Biology subjects

Castells-Graells, R.

Publications and source records attributed to Castells-Graells, R..

10 recordsLinked to original sources

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↗

bioRxiv2025

Scalable prediction of symmetric protein complex structures

All life relies on proteins to function, yet accurately modeling protein structures that exceed {approx} 10, 000 amino acids or have higher-order geometries remains difficult. Existing solutions are limited to specific scenarios, require considerable computational resources, or are otherwise unscalable. Consequently, many large, disease-relevant protein complexes in the human proteome, as well as nearly all viruses and numerous other classes, are impractical to model with high fidelity for drug development. To modulate these protein complexes and viruses, structural information is eminently valuable, and often essential. In the last two years, machine learning based-tools that can generate binders to a given target structure with high hit rates have emerged. Combined with high-throughput screening, these technologies can far outpace traditional drug discovery. However, they cannot function well without accurate models of their target structures. Thus, to unlock the full power of AI-driven drug discovery, a scalable method must be developed to predict large protein complex structures. To overcome this bottleneck, we introduce Plica-1, a physics-based method to rapidly and accurately predict the structure of arbitrarily large, symmetric protein complexes. Validated across 4 major symmetry classes (icosahedral, tetrahedral, octahedral, and cyclic), the method consistently achieves near-experimental levels of accuracy, i.e., RMSD < 5[A]. In test cases, the method runs in < 5 minutes on consumer hardware, 103-105 times faster than the closest comparable software. The largest structure currently built, at {approx}40,000 amino acids, is > 8 times the limit of existing machine learning methods. The results demonstrate that protein complexes can be modeled at significantly improved speeds and scales, making Plica-1 a promising tool for protein engineering and drug development.

bioengineering↗

bioRxiv2025

Highly Conserved Core Residues Define Old-World Alphaviruses and Trace Early Evolutionary Divergence

Alphaviruses are positive-sense, single-stranded RNA viruses that assemble into striking double-icosahedral particles. During budding, their nucleocapsid core forms in the cytoplasm and adopts a T=4 icosahedral symmetry, a hallmark of Alphaviruses. Here, we combine structural and phylogenetic analyses to identify the amino acids most likely to govern capsomer formation (pentamers and hexamers) and core organization. We find that these residues are highly conserved in present-day Old-World alphaviruses but are divergent in New-World lineages. This suggests that the common ancestor of both groups likely assembled cores using the same molecular interactions seen in present-day Old-World viruses. We propose that early divergence in these interactions weakened core assembly efficiency, potentially contributing to the attenuation observed in encephalitic New-World alphaviruses. This attenuation may reflect an adaptive trade-off, in which reduced assembly efficiency lowers viral replication and virulence, supporting long-term persistence in enzootic cycles. Revealing how specific residues control capsid architecture and tracing their evolutionary history, this study provides key insights into alphavirus assembly mechanisms, opening new avenues for antiviral strategies and rational vaccine design.

evolutionary biology↗

bioRxiv2024

Unravelling the Maturation Pathway of a Eukaryotic Virus through Cryo-EM

The importance of virus maturation has been appreciated for nearly 70 years 1 as it provides models for large-scale protein reorganization resulting in functional activation as well as being a target for antiviral therapies 2. However, a detailed description of the pathway from the initial assembly product (procapsid) to the mature, infectious particle (virion) has been elusive. This is due to the "in cell" nature of the natural process, the 2- state behavior of maturation (no detectable intermediates) in some viruses in vitro 3 and heterogeneous populations of particle intermediates that are only partially matured in other systems 4. The non-enveloped, T=4, ssRNA-containing, Nudaurelia capensis omega virus (N{omega}V), is a highly accessible model system that exemplifies the maturation process of a eukaryotic virus. During maturation the particle shrinks in outer diameter from 482 [A] (pH 7.5) to 428 [A] (pH 5.0). It is possible to mimic the maturation process in vitro by lowering the pH of a population of procapsids produced in heterologous systems5. Indeed, by controlling the pH in vitro it is possible to produce homogenous populations of intermediate N{omega}V virus-like particles (VLPs) that occur too fleetingly to be observed in vivo 6. Here we report structural models, based on cryo-electron microscopy (cryo-EM), of five intermediates in the N{omega}V maturation process. The structures of the intermediate particles reveal unique, quaternary position-dependent trajectories and refolding of subunit N and C-terminal regions, including the formation of the autocatalytic cleavage site at N570. The detailed structures reported here, coupled with previously determined structures of the procapsids and mature particles, allows the maturation pathway to be described in detail for the first time for a eukaryotic virus.

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↗

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↗

bioRxiv2020

Host-interactor screens of Phytophthora infestans RXLR proteins reveal vesicle trafficking as a major effector-targeted process

Pathogens modulate plant cell structure and function by secreting effectors into host tissues. Effectors typically function by associating with host molecules and modulating their activities. This study aimed to identify the host processes targeted by the RXLR class of host-translocated effectors of the potato blight pathogen Phytophthora infestans. To this end, we performed an in planta protein-protein interaction screen by transiently expressing P. infestans RXLR effectors in Nicotiana benthamiana leaves followed by co-immunoprecipitation (co-IP) and liquid chromatography tandem mass spectrometry (LC-MS/MS). This screen generated an effector-host protein interactome matrix of 59 P. infestans RXLR effectors x 586 N. benthamiana proteins. Classification of the host interactors into putative functional categories revealed over 35 biological processes possibly targeted by P. infestans. We further characterized the PexRD12/31 family of RXLR-WY effectors, which associate and co-localize with components of the vesicle trafficking machinery. One member of this family, PexRD31, increased the number of FYVE positive vesicles in N. benthamiana cells. FYVE positive vesicles also accumulated in leaf cells near P. infestans hyphae, indicating that the pathogen may enhance endosomal trafficking during infection. We anticipate that the interactome dataset we generated will serve as a useful community resource for functional studies of P. infestans effectors and of effector-targeted host processes.

plant biology↗

bioRxiv2019

A CRISPR/Cas9 mediated 53 kb deletion of the NRC4 gene cluster of tomato does not affect bacterial flagellin-triggered immunity

Plants utilise cell surface pattern recognition receptors (PRRs) and intracellular nucleotide-binding domain leucine-rich repeat containing receptors (NLRs) to fend off invading pathogens. Although PRR- and NLR-triggered immunity are generally thought to activate distinct pathways, they can induce similar outputs. However, whether these two pathways converge at some point to potentiate and strengthen the immune response remains unclear. For instance, the extent to which the tomato NLR helper NRC4 is implicated in response to the bacterial flagellin peptide flg22 needs to be elucidated. One challenge is that the tomato NRC4 gene cluster consists of three paralogues and the related NRC5 gene. Here, we took advantage of the CRISPR/Cas9 system to generate a tomato mutant with a 53 kb deletion that encompasses the four NRC genes. Although this mutant failed to respond to the NRC4-dependent NLR Rpi-blb2, it remained unaltered in flg22-induced responses. We conclude that the NRC4 genes are not essential for flg22-induced responses in tomato.

plant biology↗