The structural landscape of the immunoglobulin fold by large-scale de novo design.

De novo designing immunoglobulin-like frameworks that allow for functional loop diversification shows great potential for crafting antibody-like scaffolds with fully customizable structures and functions. In this work, we combined de novo parametric design with deep-learning methods for protein structure prediction and design to explore the structural landscape of 7-stranded immunoglobulin domains. After screening folding of nearly 4 million designs, we have assembled a structurally diverse library of ~50,000 immunoglobulin domains with high-confidence AlphaFold2 predictions and structures diverging from naturally occurring ones. The designed dataset enabled us to identify structural requirements for the correct folding of immunoglobulin domains, shed light on ß-sheet-ß-sheet rotational preferences and how these are linked to functional properties. Our approach eliminates the need for preset loop conformations and opens the route to large-scale de novo design of immunoglobulin-like frameworks.

Impact of AlphaFold on structure prediction of protein complexes: The CASP15-CAPRI experiment.

We present the results for CAPRI Round 54, the 5th joint CASP-CAPRI protein assembly prediction challenge. The Round offered 37 targets, including 14 homodimers, 3 homo-trimers, 13 heterodimers including 3 antibody-antigen complexes, and 7 large assemblies. On average ~70 CASP and CAPRI predictor groups, including more than 20 automatics servers, submitted models for each target. A total of 21 941 models submitted by these groups and by 15 CAPRI scorer groups were evaluated using the CAPRI model quality measures and the DockQ score consolidating these measures. The prediction performance was quantified by a weighted score based on the number of models of acceptable quality or higher submitted by each group among their five best models. Results show substantial progress achieved across a significant fraction of the 60+ participating groups. High-quality models were produced for about 40% of the targets compared to 8% two years earlier. This remarkable improvement is due to the wide use of the AlphaFold2 and AlphaFold2-Multimer software and the confidence metrics they provide. Notably, expanded sampling of candidate solutions by manipulating these deep learning inference engines, enriching multiple sequence alignments, or integration of advanced modeling tools, enabled top performing groups to exceed the performance of a standard AlphaFold2-Multimer version used as a yard stick. This notwithstanding, performance remained poor for complexes with antibodies and nanobodies, where evolutionary relationships between the binding partners are lacking, and for complexes featuring conformational flexibility, clearly indicating that the prediction of protein complexes remains a challenging problem.

Single-chain dimers from de novo immunoglobulins as robust scaffolds for multiple binding loops.

Antibody derivatives have sought to recapitulate the antigen binding properties of antibodies, but with improved biophysical attributes convenient for therapeutic, diagnostic and research applications. However, their success has been limited by the naturally occurring structure of the immunoglobulin dimer displaying hypervariable binding loops, which is hard to modify by traditional engineering approaches. Here, we devise geometrical principles for de novo designing single-chain immunoglobulin dimers, as a tunable two-domain architecture that optimizes biophysical properties through more favorable dimer interfaces. Guided by these principles, we computationally designed protein scaffolds that were hyperstable, structurally accurate and robust for accommodating multiple functional loops, both individually and in combination, as confirmed through biochemical assays and X-ray crystallography. We showcase the modularity of this architecture by deep-learning-based diversification, opening up the possibility for tailoring the number, positioning, and relative orientation of ligand-binding loops targeting one or two distal epitopes. Our results provide a route to custom-design robust protein scaffolds for harboring multiple functional loops.

The LightDock Server: Artificial Intelligence-powered modeling of macromolecular interactions.

Computational docking is an instrumental method of the structural biology toolbox. Specifically, integrative modeling software, such as LightDock, arise as complementary and synergetic methods to experimental structural biology techniques. Ubiquitousness and accessibility are fundamental features to promote ease of use and to improve user experience. With this goal in mind, we have developed the LightDock Server, a web server for the integrative modeling of macromolecular interactions, along with several dedicated usage modes. The server builds upon the LightDock macromolecular docking framework, which has proved useful for modeling medium-to-high flexible complexes, antibody-antigen interactions, or membrane-associated protein assemblies. We believe that this free-to-use resource will be a valuable addition to the structural biology community and can be accessed online at: https://server.lightdock.org/.

De novo design of immunoglobulin-like domains.

Antibodies, and antibody derivatives such as nanobodies, contain immunoglobulin-like (Ig) ß-sandwich scaffolds which anchor the hypervariable antigen-binding loops and constitute the largest growing class of drugs. Current engineering strategies for this class of compounds rely on naturally existing Ig frameworks, which can be hard to modify and have limitations in manufacturability, designability and range of action. Here, we develop design rules for the central feature of the Ig fold architecture-the non-local cross-ß structure connecting the two ß-sheets-and use these to design highly stable Ig domains de novo, confirm their structures through X-ray crystallography, and show they can correctly scaffold functional loops. Our approach opens the door to the design of antibody-like scaffolds with tailored structures and superior biophysical properties.

Prediction of protein assemblies, the next frontier: The CASP14-CAPRI experiment.

We present the results for CAPRI Round 50, the fourth joint CASP-CAPRI protein assembly prediction challenge. The Round comprised a total of twelve targets, including six dimers, three trimers, and three higher-order oligomers. Four of these were easy targets, for which good structural templates were available either for the full assembly, or for the main interfaces (of the higher-order oligomers). Eight were difficult targets for which only distantly related templates were found for the individual subunits. Twenty-five CAPRI groups including eight automatic servers submitted ~1250 models per target. Twenty groups including six servers participated in the CAPRI scoring challenge submitted ~190 models per target. The accuracy of the predicted models was evaluated using the classical CAPRI criteria. The prediction performance was measured by a weighted scoring scheme that takes into account the number of models of acceptable quality or higher submitted by each group as part of their five top-ranking models. Compared to the previous CASP-CAPRI challenge, top performing groups submitted such models for a larger fraction (70-75%) of the targets in this Round, but fewer of these models were of high accuracy. Scorer groups achieved stronger performance with more groups submitting correct models for 70-80% of the targets or achieving high accuracy predictions. Servers performed less well in general, except for the MDOCKPP and LZERD servers, who performed on par with human groups. In addition to these results, major advances in methodology are discussed, providing an informative overview of where the prediction of protein assemblies currently stands.

The gutSMASH web server: automated identification of primary metabolic gene clusters from the gut microbiota.

Anaerobic bacteria from the human microbiome produce a wide array of molecules at high concentrations that can directly or indirectly affect the host. The production of these molecules, mostly derived from their primary metabolism, is frequently encoded in metabolic gene clusters (MGCs). However, despite the importance of microbiome-derived primary metabolites, no tool existed to predict the gene clusters responsible for their production. For this reason, we recently introduced gutSMASH. gutSMASH can predict 41 different known pathways, including MGCs involved in bioenergetics, but also putative ones that are candidates for novel pathway discovery. To make the tool more user-friendly and accessible, we here present the gutSMASH web server, hosted at https://gutsmash.bioinformatics.nl/. The user can either input the GenBank assembly accession or upload a genome file in FASTA or GenBank format. Optionally, the user can enable additional analyses to obtain further insights into the predicted MGCs. An interactive HTML output (viewable online or downloadable for offline use) provides a user-friendly way to browse functional gene annotations and sequence comparisons with reference gene clusters as well as gene clusters predicted in other genomes. Thus, this web server provides the community with a streamlined and user-friendly interface to analyze the metabolic potential of gut microbiomes.

Integrative modeling of membrane-associated protein assemblies.

Membrane proteins are among the most challenging systems to study with experimental structural biology techniques. The increased number of deposited structures of membrane proteins has opened the route to modeling their complexes by methods such as docking. Here, we present an integrative computational protocol for the modeling of membrane-associated protein assemblies. The information encoded by the membrane is represented by artificial beads, which allow targeting of the docking toward the binding-competent regions. It combines efficient, artificial intelligence-based rigid-body docking by LightDock with a flexible final refinement with HADDOCK to remove potential clashes at the interface. We demonstrate the performance of this protocol on eighteen membrane-associated complexes, whose interface lies between the membrane and either the cytosolic or periplasmic regions. In addition, we provide a comparison to another state-of-the-art docking software, ZDOCK. This protocol should shed light on the still dark fraction of the interactome consisting of membrane proteins.

Coarse-grained (hybrid) integrative modeling of biomolecular interactions.

The computational modeling field has vastly evolved over the past decades. The early developments of simplified protein systems represented a stepping stone towards establishing more efficient approaches to sample intricated conformational landscapes. Downscaling the level of resolution of biomolecules to coarser representations allows for studying protein structure, dynamics and interactions that are not accessible by classical atomistic approaches. The combination of different resolutions, namely hybrid modeling, has also been proved as an alternative when mixed levels of details are required. In this review, we provide an overview of coarse-grained/hybrid models focusing on their applicability in the modeling of biomolecular interactions. We give a detailed list of ready-to-use modeling software for studying biomolecular interactions allowing various levels of coarse-graining and provide examples of complexes determined by integrative coarse-grained/hybrid approaches in combination with experimental information.

LightDock goes information-driven.

MOTIVATION: The use of experimental information has been demonstrated to increase the success rate of computational macromolecular docking. Many methods use information to post-filter the simulation output while others drive the simulation based on experimental restraints, which can become problematic for more complex scenarios such as multiple binding interfaces. RESULTS: We present a novel method for including interface information into protein docking simulations within the LightDock framework. Prior to the simulation, irrelevant regions from the receptor are excluded for sampling (filter of initial swarms) and initial ligand poses are pre-oriented based on ligand input information. We demonstrate the applicability of this approach on the new 55 cases of the Protein-Protein Docking Benchmark 5, using different amounts of information. Even with incomplete or incorrect information, a significant improvement in performance is obtained compared to blind ab initio docking. AVAILABILITY AND IMPLEMENTATION: The software is supported and freely available from https://github.com/brianjimenez/lightdock and analysis data from https://github.com/brianjimenez/lightdock_bm5. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Modeling Antibody-Antigen Complexes by Information-Driven Docking.

Antibodies are Y-shaped proteins essential for immune response. Their capability to recognize antigens with high specificity makes them excellent therapeutic targets. Understanding the structural basis of antibody-antigen interactions is therefore crucial for improving our ability to design efficient biological drugs. Computational approaches such as molecular docking are providing a valuable and fast alternative to experimental structural characterization for these complexes. We investigate here how information about complementarity-determining regions and binding epitopes can be used to drive the modeling process, and present a comparative study of four different docking software suites (ClusPro, LightDock, ZDOCK, and HADDOCK) providing specific options for antibody-antigen modeling. Their performance on a dataset of 16 complexes is reported. HADDOCK, which includes information to drive the docking, is shown to perform best in terms of both success rate and quality of the generated models in both the presence and absence of information about the epitope on the antigen.

An overview of data-driven HADDOCK strategies in CAPRI rounds 38-45.

Our information-driven docking approach HADDOCK has demonstrated a sustained performance since the start of its participation to CAPRI. This is due, in part, to its ability to integrate data into the modeling process, and to the robustness of its scoring function. We participated in CAPRI both as server and manual predictors. In CAPRI rounds 38-45, we have used various strategies depending on the available information. These ranged from imposing restraints to a few residues identified from literature as being important for the interaction, to binding pockets identified from homologous complexes or template-based refinement/CA-CA restraint-guided docking from identified templates. When relevant, symmetry restraints were used to limit the conformational sampling. We also tested for a large decamer target a new implementation of the MARTINI coarse-grained force field in HADDOCK. Overall, we obtained acceptable or better predictions for 13 and 11 server and manual submissions, respectively, out of the 22 interfaces. Our server performance (acceptable or higher-quality models when considering the top 10) was better (59%) than the manual (50%) one, in which we typically experiment with various combinations of protocols and data sources. Again, our simple scoring function based on a linear combination of intermolecular van der Waals and electrostatic energies and an empirical desolvation term demonstrated a good performance in the scoring experiment with a 63% success rate across all 22 interfaces. An analysis of model quality indicates that, while we are consistently performing well in generating acceptable models, there is room for improvement for generating/identifying higher quality models.

MARTINI-Based Protein-DNA Coarse-Grained HADDOCKing.

Modeling biomolecular assemblies is an important field in computational structural biology. The inherent complexity of their energy landscape and the computational cost associated with modeling large and complex assemblies are major drawbacks for integrative modeling approaches. The so-called coarse-graining approaches, which reduce the degrees of freedom of the system by grouping several atoms into larger "pseudo-atoms," have been shown to alleviate some of those limitations, facilitating the identification of the global energy minima assumed to correspond to the native state of the complex, while making the calculations more efficient. Here, we describe and assess the implementation of the MARTINI force field for DNA into HADDOCK, our integrative modeling platform. We combine it with our previous implementation for protein-protein coarse-grained docking, enabling coarse-grained modeling of protein-nucleic acid complexes. The system is modeled using MARTINI topologies and interaction parameters during the rigid body docking and semi-flexible refinement stages of HADDOCK, and the resulting models are then converted back to atomistic resolution by an atom-to-bead distance restraints-guided protocol. We first demonstrate the performance of this protocol using 44 complexes from the protein-DNA docking benchmark, which shows an overall ~6-fold speed increase and maintains similar accuracy as compared to standard atomistic calculations. As a proof of concept, we then model the interaction between the PRC1 and the nucleosome (a former CAPRI target in round 31), using the same information available at the time the target was offered, and compare all-atom and coarse-grained models.

Less Is More: Coarse-Grained Integrative Modeling of Large Biomolecular Assemblies with HADDOCK.

Predicting the 3D structure of protein interactions remains a challenge in the field of computational structural biology. This is in part due to difficulties in sampling the complex energy landscape of multiple interacting flexible polypeptide chains. Coarse-graining approaches, which reduce the number of degrees of freedom of the system, help address this limitation by smoothing the energy landscape, allowing an easier identification of the global energy minimum. They also accelerate the calculations, allowing for modeling larger assemblies. Here, we present the implementation of the MARTINI coarse-grained force field for proteins into HADDOCK, our integrative modeling platform. Docking and refinement are performed at the coarse-grained level, and the resulting models are then converted back to atomistic resolution through a distance restraints-guided morphing procedure. Our protocol, tested on the largest complexes of the protein docking benchmark 5, shows an overall ∼7-fold speed increase compared to standard all-atom calculations, while maintaining a similar accuracy and yielding substantially more near-native solutions. To showcase the potential of our method, we performed simultaneous 7 body docking to model the 1:6 KaiC-KaiB complex, integrating mutagenesis and hydrogen/deuterium exchange data from mass spectrometry with symmetry restraints, and validated the resulting models against a recently published cryo-EM structure.

Performance of HADDOCK and a simple contact-based protein-ligand binding affinity predictor in the D3R Grand Challenge 2.

We present the performance of HADDOCK, our information-driven docking software, in the second edition of the D3R Grand Challenge. In this blind experiment, participants were requested to predict the structures and binding affinities of complexes between the Farnesoid X nuclear receptor and 102 different ligands. The models obtained in Stage1 with HADDOCK and ligand-specific protocol show an average ligand RMSD of 5.1 Å from the crystal structure. Only 6/35 targets were within 2.5 Å RMSD from the reference, which prompted us to investigate the limiting factors and revise our protocol for Stage2. The choice of the receptor conformation appeared to have the strongest influence on the results. Our Stage2 models were of higher quality (13 out of 35 were within 2.5 Å), with an average RMSD of 4.1 Å. The docking protocol was applied to all 102 ligands to generate poses for binding affinity prediction. We developed a modified version of our contact-based binding affinity predictor PRODIGY, using the number of interatomic contacts classified by their type and the intermolecular electrostatic energy. This simple structure-based binding affinity predictor shows a Kendall's Tau correlation of 0.37 in ranking the ligands (7th best out of 77 methods, 5th/25 groups). Those results were obtained from the average prediction over the top10 poses, irrespective of their similarity/correctness, underscoring the robustness of our simple predictor. This results in an enrichment factor of 2.5 compared to a random predictor for ranking ligands within the top 25%, making it a promising approach to identify lead compounds in virtual screening.

LightDock: a new multi-scale approach to protein-protein docking.

Motivation: Computational prediction of protein-protein complex structure by docking can provide structural and mechanistic insights for protein interactions of biomedical interest. However, current methods struggle with difficult cases, such as those involving flexible proteins, low-affinity complexes or transient interactions. A major challenge is how to efficiently sample the structural and energetic landscape of the association at different resolution levels, given that each scoring function is often highly coupled to a specific type of search method. Thus, new methodologies capable of accommodating multi-scale conformational flexibility and scoring are strongly needed. Results: We describe here a new multi-scale protein-protein docking methodology, LightDock, capable of accommodating conformational flexibility and a variety of scoring functions at different resolution levels. Implicit use of normal modes during the search and atomic/coarse-grained combined scoring functions yielded improved predictive results with respect to state-of-the-art rigid-body docking, especially in flexible cases. Availability and implementation: The source code of the software and installation instructions are available for download at https://life.bsc.es/pid/lightdock/. Contact: juanf@bsc.es. Supplementary information: Supplementary data are available at Bioinformatics online.