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.

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.

Insight into cyanobacterial circadian timing from structural details of the KaiB-KaiC interaction.

Circadian timing in cyanobacteria is determined by the Kai system consisting of KaiA, KaiB, and KaiC. Interactions between Kai proteins change the phosphorylation status of KaiC, defining the phase of circadian timing. The KaiC-KaiB interaction is crucial for the circadian rhythm to enter the dephosphorylation phase but it is not well understood. Using mass spectrometry to characterize Kai complexes, we found that KaiB forms monomers, dimers, and tetramers. The monomer is the unit that interacts with KaiC, with six KaiB monomers binding to one KaiC hexamer. Hydrogen-deuterium exchange MS reveals structural changes in KaiC upon binding of KaiB in both the CI and CII domains, showing allosteric coupling upon KaiB binding. Based on this information we propose a model of the KaiB-KaiC complex and hypothesize that the allosteric changes observed upon complex formation relate to coupling KaiC ATPase activity with KaiB binding and to sequestration of KaiA dimers into KaiCBA complexes.

Prediction of homoprotein and heteroprotein complexes by protein docking and template-based modeling: A CASP-CAPRI experiment.

We present the results for CAPRI Round 30, the first joint CASP-CAPRI experiment, which brought together experts from the protein structure prediction and protein-protein docking communities. The Round comprised 25 targets from amongst those submitted for the CASP11 prediction experiment of 2014. The targets included mostly homodimers, a few homotetramers, and two heterodimers, and comprised protein chains that could readily be modeled using templates from the Protein Data Bank. On average 24 CAPRI groups and 7 CASP groups submitted docking predictions for each target, and 12 CAPRI groups per target participated in the CAPRI scoring experiment. In total more than 9500 models were assessed against the 3D structures of the corresponding target complexes. Results show that the prediction of homodimer assemblies by homology modeling techniques and docking calculations is quite successful for targets featuring large enough subunit interfaces to represent stable associations. Targets with ambiguous or inaccurate oligomeric state assignments, often featuring crystal contact-sized interfaces, represented a confounding factor. For those, a much poorer prediction performance was achieved, while nonetheless often providing helpful clues on the correct oligomeric state of the protein. The prediction performance was very poor for genuine tetrameric targets, where the inaccuracy of the homology-built subunit models and the smaller pair-wise interfaces severely limited the ability to derive the correct assembly mode. Our analysis also shows that docking procedures tend to perform better than standard homology modeling techniques and that highly accurate models of the protein components are not always required to identify their association modes with acceptable accuracy. Proteins 2016; 84(Suppl 1):323-348. © 2016 Wiley Periodicals, Inc.

Blind prediction of interfacial water positions in CAPRI.

We report the first assessment of blind predictions of water positions at protein-protein interfaces, performed as part of the critical assessment of predicted interactions (CAPRI) community-wide experiment. Groups submitting docking predictions for the complex of the DNase domain of colicin E2 and Im2 immunity protein (CAPRI Target 47), were invited to predict the positions of interfacial water molecules using the method of their choice. The predictions-20 groups submitted a total of 195 models-were assessed by measuring the recall fraction of water-mediated protein contacts. Of the 176 high- or medium-quality docking models-a very good docking performance per se-only 44% had a recall fraction above 0.3, and a mere 6% above 0.5. The actual water positions were in general predicted to an accuracy level no better than 1.5 Å, and even in good models about half of the contacts represented false positives. This notwithstanding, three hotspot interface water positions were quite well predicted, and so was one of the water positions that is believed to stabilize the loop that confers specificity in these complexes. Overall the best interface water predictions was achieved by groups that also produced high-quality docking models, indicating that accurate modelling of the protein portion is a determinant factor. The use of established molecular mechanics force fields, coupled to sampling and optimization procedures also seemed to confer an advantage. Insights gained from this analysis should help improve the prediction of protein-water interactions and their role in stabilizing protein complexes.

The HADDOCK2.4 web server for integrative modeling of biomolecular complexes.

Interactions between macromolecules, such as proteins and nucleic acids, are essential for cellular functions. Experimental methods can fail to provide all the information required to fully model biomolecular complexes at atomic resolution, particularly for large and heterogeneous assemblies. Integrative computational approaches have, therefore, gained popularity, complementing traditional experimental methods in structural biology. Here, we introduce HADDOCK2.4, an integrative modeling platform, and its updated web interface ( https://wenmr.science.uu.nl/haddock2.4 ). The platform seamlessly integrates diverse experimental and theoretical data to generate high-quality models of macromolecular complexes. The user-friendly web server offers automated parameter settings, access to distributed computing resources, and pre- and post-processing steps that enhance the user experience. To present the web server's various interfaces and features, we demonstrate two different applications: (i) we predict the structure of an antibody-antigen complex by using NMR data for the antigen and knowledge of the hypervariable loops for the antibody, and (ii) we perform coarse-grained modeling of PRC1 with a nucleosome particle guided by mutagenesis and functional data. The described protocols require some basic familiarity with molecular modeling and the Linux command shell. This new version of our widely used HADDOCK web server allows structural biologists and non-experts to explore intricate macromolecular assemblies encompassing various molecule types.

Dynamic control of selectivity in the ubiquitination pathway revealed by an ASP to GLU substitution in an intra-molecular salt-bridge network.

Ubiquitination relies on a subtle balance between selectivity and promiscuity achieved through specific interactions between ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). Here, we report how a single aspartic to glutamic acid substitution acts as a dynamic switch to tip the selectivity balance of human E2s for interaction toward E3 RING-finger domains. By combining molecular dynamic simulations, experimental yeast-two-hybrid screen of E2-E3 (RING) interactions and mutagenesis, we reveal how the dynamics of an internal salt-bridge network at the rim of the E2-E3 interaction surface controls the balance between an "open", binding competent, and a "closed", binding incompetent state. The molecular dynamic simulations shed light on the fine mechanism of this molecular switch and allowed us to identify its components, namely an aspartate/glutamate pair, a lysine acting as the central switch and a remote aspartate. Perturbations of single residues in this network, both inside and outside the interaction surface, are sufficient to switch the global E2 interaction selectivity as demonstrated experimentally. Taken together, our results indicate a new mechanism to control E2-E3 interaction selectivity at an atomic level, highlighting how minimal changes in amino acid side-chain affecting the dynamics of intramolecular salt-bridges can be crucial for protein-protein interactions. These findings indicate that the widely accepted sequence-structure-function paradigm should be extended to sequence-structure-dynamics-function relationship and open new possibilities for control and fine-tuning of protein interaction selectivity.

Supramolecular structure of membrane-associated polypeptides by combining solid-state NMR and molecular dynamics simulations.

Elemental biological functions such as molecular signal transduction are determined by the dynamic interplay between polypeptides and the membrane environment. Determining such supramolecular arrangements poses a significant challenge for classical structural biology methods. We introduce an iterative approach that combines magic-angle spinning solid-state NMR spectroscopy and atomistic molecular dynamics simulations for the determination of the structure and topology of membrane-bound systems with a resolution and level of accuracy difficult to obtain by either method alone. Our study focuses on the Shaker B ball peptide that is representative for rapid N-type inactivating domains of voltage-gated K(+) channels, associated with negatively charged lipid bilayers.

Clustering biomolecular complexes by residue contacts similarity.

Inaccuracies in computational molecular modeling methods are often counterweighed by brute-force generation of a plethora of putative solutions. These are then typically sieved via structural clustering based on similarity measures such as the root mean square deviation (RMSD) of atomic positions. Albeit widely used, these measures suffer from several theoretical and technical limitations (e.g., choice of regions for fitting) that impair their application in multicomponent systems (N > 2), large-scale studies (e.g., interactomes), and other time-critical scenarios. We present here a simple similarity measure for structural clustering based on atomic contacts--the fraction of common contacts--and compare it with the most used similarity measure of the protein docking community--interface backbone RMSD. We show that this method produces very compact clusters in remarkably short time when applied to a collection of binary and multicomponent protein-protein and protein-DNA complexes. Furthermore, it allows easy clustering of similar conformations of multicomponent symmetrical assemblies in which chain permutations can occur. Simple contact-based metrics should be applicable to other structural biology clustering problems, in particular for time-critical or large-scale endeavors.

Building macromolecular assemblies by information-driven docking: introducing the HADDOCK multibody docking server.

Over the last years, large scale proteomics studies have generated a wealth of information of biomolecular complexes. Adding the structural dimension to the resulting interactomes represents a major challenge that classical structural experimental methods alone will have difficulties to confront. To meet this challenge, complementary modeling techniques such as docking are thus needed. Among the current docking methods, HADDOCK (High Ambiguity-Driven DOCKing) distinguishes itself from others by the use of experimental and/or bioinformatics data to drive the modeling process and has shown a strong performance in the critical assessment of prediction of interactions (CAPRI), a blind experiment for the prediction of interactions. Although most docking programs are limited to binary complexes, HADDOCK can deal with multiple molecules (up to six), a capability that will be required to build large macromolecular assemblies. We present here a novel web interface of HADDOCK that allows the user to dock up to six biomolecules simultaneously. This interface allows the inclusion of a large variety of both experimental and/or bioinformatics data and supports several types of cyclic and dihedral symmetries in the docking of multibody assemblies. The server was tested on a benchmark of six cases, containing five symmetric homo-oligomeric protein complexes and one symmetric protein-DNA complex. Our results reveal that, in the presence of either bioinformatics and/or experimental data, HADDOCK shows an excellent performance: in all cases, HADDOCK was able to generate good to high quality solutions and ranked them at the top, demonstrating its ability to model symmetric multicomponent assemblies. Docking methods can thus play an important role in adding the structural dimension to interactomes. However, although the current docking methodologies were successful for a vast range of cases, considering the variety and complexity of macromolecular assemblies, inclusion of some kind of experimental information (e.g. from mass spectrometry, nuclear magnetic resonance, cryoelectron microscopy, etc.) will remain highly desirable to obtain reliable results.

Robust detection of translocations in lymphoma FFPE samples using targeted locus capture-based sequencing.

In routine diagnostic pathology, cancer biopsies are preserved by formalin-fixed, paraffin-embedding (FFPE) procedures for examination of (intra-) cellular morphology. Such procedures inadvertently induce DNA fragmentation, which compromises sequencing-based analyses of chromosomal rearrangements. Yet, rearrangements drive many types of hematolymphoid malignancies and solid tumors, and their manifestation is instructive for diagnosis, prognosis, and treatment. Here, we present FFPE-targeted locus capture (FFPE-TLC) for targeted sequencing of proximity-ligation products formed in FFPE tissue blocks, and PLIER, a computational framework that allows automated identification and characterization of rearrangements involving selected, clinically relevant, loci. FFPE-TLC, blindly applied to 149 lymphoma and control FFPE samples, identifies the known and previously uncharacterized rearrangement partners. It outperforms fluorescence in situ hybridization (FISH) in sensitivity and specificity, and shows clear advantages over standard capture-NGS methods, finding rearrangements involving repetitive sequences which they typically miss. FFPE-TLC is therefore a powerful clinical diagnostics tool for accurate targeted rearrangement detection in FFPE specimens.

Strengths and weaknesses of data-driven docking in critical assessment of prediction of interactions.

The recent CAPRI rounds have introduced new docking challenges in the form of protein-RNA complexes, multiple alternative interfaces, and an unprecedented number of targets for which homology modeling was required. We present here the performance of HADDOCK and its web server in the CAPRI experiment and discuss the strengths and weaknesses of data-driven docking. HADDOCK was successful for 6 out of 9 complexes (6 out of 11 targets) and accurately predicted the individual interfaces for two more complexes. The HADDOCK server, which is the first allowing the simultaneous docking of generic multi-body complexes, was successful in 4 out of 7 complexes for which it participated. In the scoring experiment, we predicted the highest number of targets of any group. The main weakness of data-driven docking revealed from these last CAPRI results is its vulnerability for incorrect experimental data related to the interface or the stoichiometry of the complex. At the same time, the use of experimental and/or predicted information is also the strength of our approach as evidenced for those targets for which accurate experimental information was available (e.g., the 10 three-stars predictions for T40!). Even when the models show a wrong orientation, the individual interfaces are generally well predicted with an average coverage of 60% ± 26% over all targets. This makes data-driven docking particularly valuable in a biological context to guide experimental studies like, for example, targeted mutagenesis.

Molecular dynamics characterization of the conformational landscape of small peptides: A series of hands-on collaborative practical sessions for undergraduate students.

Molecular modelling and simulations are nowadays an integral part of research in areas ranging from physics to chemistry to structural biology, as well as pharmaceutical drug design. This popularity is due to the development of high-performance hardware and of accurate and efficient molecular mechanics algorithms by the scientific community. These improvements are also benefitting scientific education. Molecular simulations, their underlying theory, and their applications are particularly difficult to grasp for undergraduate students. Having hands-on experience with the methods contributes to a better understanding and solidification of the concepts taught during the lectures. To this end, we have created a computer practical class, which has been running for the past five years, composed of several sessions where students characterize the conformational landscape of small peptides using molecular dynamics simulations in order to gain insights on their binding to protein receptors. In this report, we detail the ingredients and recipe necessary to establish and carry out this practical, as well as some of the questions posed to the students and their expected results. Further, we cite some examples of the students' written reports, provide statistics, and share their feedbacks on the structure and execution of the sessions. These sessions were implemented alongside a theoretical molecular modelling course but have also been used successfully as a standalone tutorial during specialized workshops. The availability of the material on our web page also facilitates this integration and dissemination and lends strength to the thesis of open-source science and education.

dMM-PBSA: A New HADDOCK Scoring Function for Protein-Peptide Docking.

Molecular-docking programs coupled with suitable scoring functions are now established and very useful tools enabling computational chemists to rapidly screen large chemical databases and thereby to identify promising candidate compounds for further experimental processing. In a broader scenario, predicting binding affinity is one of the most critical and challenging components of computer-aided structure-based drug design. The development of a molecular docking scoring function which in principle could combine both features, namely ranking putative poses and predicting complex affinity, would be of paramount importance. Here, we systematically investigated the performance of the MM-PBSA approach, using two different Poisson-Boltzmann solvers (APBS and DelPhi), in the currently rising field of protein-peptide interactions (PPIs), identifying the correct binding conformations of 19 different protein-peptide complexes and predicting their binding free energies. First, we scored the decoy structures from HADDOCK calculation via the MM-PBSA approach in order to assess the capability of retrieving near-native poses in the best-scoring clusters and of evaluating the corresponding free energies of binding. MM-PBSA behaves well in finding the poses corresponding to the lowest binding free energy, however the built-in HADDOCK score shows a better performance. In order to improve the MM-PBSA-based scoring function, we dampened the MM-PBSA solvation and coulombic terms by 0.2, as proposed in the HADDOCK score and LIE approaches. The new dampened MM-PBSA (dMM-PBSA) outperforms the original MM-PBSA and ranks the decoys structures as the HADDOCK score does. Second, we found a good correlation between the dMM-PBSA and HADDOCK scores for the near-native clusters of each system and the experimental binding energies, respectively. Therefore, we propose a new scoring function, dMM-PBSA, to be used together with the built-in HADDOCK score in the context of protein-peptide docking simulations.

Enhancers reside in a unique epigenetic environment during early zebrafish development.

BACKGROUND: Enhancers, not promoters, are the most dynamic in their DNA methylation status throughout development and differentiation. Generally speaking, enhancers that are primed to or actually drive gene expression are characterized by relatively low levels of DNA methylation (hypo-methylation), while inactive enhancers display hyper-methylation of the underlying DNA. The direct functional significance of the DNA methylation state of enhancers is, however, unclear for most loci. RESULTS: In contrast to conventional epigenetic interactions at enhancers, we find that DNA methylation status and enhancer activity during early zebrafish development display very unusual correlation characteristics: hypo-methylation is a unique feature of primed enhancers whereas active enhancers are generally hyper-methylated. The hypo-methylated enhancers that we identify (hypo-enhancers) are enriched close to important transcription factors that act later in development. Interestingly, hypo-enhancers are de-methylated shortly before the midblastula transition and reside in a unique epigenetic environment. Finally, we demonstrate that hypo-enhancers do become active at later developmental stages and that they are physically associated with the transcriptional start site of target genes, irrespective of target gene activity. CONCLUSIONS: We demonstrate that early development in zebrafish embodies a time window characterized by non-canonical DNA methylation-enhancer relationships, including global DNA hypo-methylation of inactive enhancers and DNA hyper-methylation of active enhancers.

Information-driven modeling of protein-peptide complexes.

Despite their biological importance in many regulatory processes, protein-peptide recognition mechanisms are difficult to study experimentally at the structural level because of the inherent flexibility of peptides and the often transient interactions on which they rely. Complementary methods like biomolecular docking are therefore required. The prediction of the three-dimensional structure of protein-peptide complexes raises unique challenges for computational algorithms, as exemplified by the recent introduction of protein-peptide targets in the blind international experiment CAPRI (Critical Assessment of PRedicted Interactions). Conventional protein-protein docking approaches are often struggling with the high flexibility of peptides whose short sizes impede protocols and scoring functions developed for larger interfaces. On the other side, protein-small ligand docking methods are unable to cope with the larger number of degrees of freedom in peptides compared to small molecules and the typically reduced available information to define the binding site. In this chapter, we describe a protocol to model protein-peptide complexes using the HADDOCK web server, working through a test case to illustrate every steps. The flexibility challenge that peptides represent is dealt with by combining elements of conformational selection and induced fit molecular recognition theories.

Integrative Modeling of Biomolecular Complexes: HADDOCKing with Cryo-Electron Microscopy Data.

Protein-protein interactions play a central role in all cellular processes. Insight into their atomic architecture is therefore of paramount importance. Cryo-electron microscopy (cryo-EM) is capable of directly imaging large macromolecular complexes. Unfortunately, the resolution is usually not sufficient for a direct atomic interpretation. To overcome this, cryo-EM data are often combined with high-resolution atomic structures. However, current computational approaches typically do not include information from other experimental sources nor a proper physico-chemical description of the interfaces. Here we describe the integration of cryo-EM data into our data-driven docking program HADDOCK and its performance on a benchmark of 17 complexes. The approach is demonstrated on five systems using experimental cryo-EM data in the range of 8.5-21 Å resolution. For several cases, cryo-EM data are integrated with additional interface information, e.g. mutagenesis and hydroxyl radical footprinting data. The resulting models have high-quality interfaces, revealing novel details of the interactions.

A unified conformational selection and induced fit approach to protein-peptide docking.

Protein-peptide interactions are vital for the cell. They mediate, inhibit or serve as structural components in nearly 40% of all macromolecular interactions, and are often associated with diseases, making them interesting leads for protein drug design. In recent years, large-scale technologies have enabled exhaustive studies on the peptide recognition preferences for a number of peptide-binding domain families. Yet, the paucity of data regarding their molecular binding mechanisms together with their inherent flexibility makes the structural prediction of protein-peptide interactions very challenging. This leaves flexible docking as one of the few amenable computational techniques to model these complexes. We present here an ensemble, flexible protein-peptide docking protocol that combines conformational selection and induced fit mechanisms. Starting from an ensemble of three peptide conformations (extended, a-helix, polyproline-II), flexible docking with HADDOCK generates 79.4% of high quality models for bound/unbound and 69.4% for unbound/unbound docking when tested against the largest protein-peptide complexes benchmark dataset available to date. Conformational selection at the rigid-body docking stage successfully recovers the most relevant conformation for a given protein-peptide complex and the subsequent flexible refinement further improves the interface by up to 4.5 Šinterface RMSD. Cluster-based scoring of the models results in a selection of near-native solutions in the top three for ∼75% of the successfully predicted cases. This unified conformational selection and induced fit approach to protein-peptide docking should open the route to the modeling of challenging systems such as disorder-order transitions taking place upon binding, significantly expanding the applicability limit of biomolecular interaction modeling by docking.