Flexible structural arrangement and DNA-binding properties of protein p6 from Bacillus subtillis phage φ29.

The genome-organizing protein p6 of Bacillus subtilis bacteriophage φ29 plays an essential role in viral development by activating the initiation of DNA replication and participating in the early-to-late transcriptional switch. These activities require the formation of a nucleoprotein complex in which the DNA adopts a right-handed superhelix wrapping around a multimeric p6 scaffold, restraining positive supercoiling and compacting the viral genome. Due to the absence of homologous structures, prior attempts to unveil p6's structural architecture failed. Here, we employed AlphaFold2 to engineer rational p6 constructs yielding crystals for three-dimensional structure determination. Our findings reveal a novel fold adopted by p6 that sheds light on its self-association mechanism and its interaction with DNA. By means of protein-DNA docking and molecular dynamic simulations, we have generated a comprehensive structural model for the nucleoprotein complex that consistently aligns with its established biochemical and thermodynamic parameters. Besides, through analytical ultracentrifugation, we have confirmed the hydrodynamic properties of the nucleocomplex, further validating in solution our proposed model. Importantly, the disclosed structure not only provides a highly accurate explanation for previously experimental data accumulated over decades, but also enhances our holistic understanding of the structural and functional attributes of protein p6 during φ29 infection.

Predicting protein stability changes upon mutation using a simple orientational potential.

MOTIVATION: Structure-based stability prediction upon mutation is crucial for protein engineering and design, and for understanding genetic diseases or drug resistance events. For this task, we adopted a simple residue-based orientational potential that considers only three backbone atoms, previously applied in protein modeling. Its application to stability prediction only requires parametrizing 12 amino acid-dependent weights using cross-validation strategies on a curated dataset in which we tried to reduce the mutations that belong to protein-protein or protein-ligand interfaces, extreme conditions and the alanine over-representation. RESULTS: Our method, called KORPM, accurately predicts mutational effects on an independent benchmark dataset, whether the wild-type or mutated structure is used as starting point. Compared with state-of-the-art methods on this balanced dataset, our approach obtained the lowest root mean square error (RMSE) and the highest correlation between predicted and experimental ΔΔG measures, as well as better receiver operating characteristics and precision-recall curves. Our method is almost anti-symmetric by construction, and it performs thus similarly for the direct and reverse mutations with the corresponding wild-type and mutated structures. Despite the strong limitations of the available experimental mutation data in terms of size, variability, and heterogeneity, we show competitive results with a simple sum of energy terms, which is more efficient and less prone to overfitting. AVAILABILITY AND IMPLEMENTATION: https://github.com/chaconlab/korpm. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

InterEvDock3: a combined template-based and free docking server with increased performance through explicit modeling of complex homologs and integration of covariation-based contact maps.

The InterEvDock3 protein docking server exploits the constraints of evolution by multiple means to generate structural models of protein assemblies. The server takes as input either several sequences or 3D structures of proteins known to interact. It returns a set of 10 consensus candidate complexes, together with interface predictions to guide further experimental validation interactively. Three key novelties were implemented in InterEvDock3 to help obtain more reliable models: users can (i) generate template-based structural models of assemblies using close and remote homologs of known 3D structure, detected through an automated search protocol, (ii) select the assembly models most consistent with contact maps from external methods that implement covariation-based contact prediction with or without deep learning and (iii) exploit a novel coevolution-based scoring scheme at atomic level, which leads to significantly higher free docking success rates. The performance of the server was validated on two large free docking benchmark databases, containing respectively 230 unbound targets (Weng dataset) and 812 models of unbound targets (PPI4DOCK dataset). Its effectiveness has also been proven on a number of challenging examples. The InterEvDock3 web interface is available at http://bioserv.rpbs.univ-paris-diderot.fr/services/InterEvDock3/.

KORP-PL: a coarse-grained knowledge-based scoring function for protein-ligand interactions.

MOTIVATION: Despite the progress made in studying protein-ligand interactions and the widespread application of docking and affinity prediction tools, improving their precision and efficiency still remains a challenge. Computational approaches based on the scoring of docking conformations with statistical potentials constitute a popular alternative to more accurate but costly physics-based thermodynamic sampling methods. In this context, a minimalist and fast sidechain-free knowledge-based potential with a high docking and screening power can be very useful when screening a big number of putative docking conformations. RESULTS: Here, we present a novel coarse-grained potential defined by a 3D joint probability distribution function that only depends on the pairwise orientation and position between protein backbone and ligand atoms. Despite its extreme simplicity, our approach yields very competitive results with the state-of-the-art scoring functions, especially in docking and screening tasks. For example, we observed a twofold improvement in the median 5% enrichment factor on the DUD-E benchmark compared to Autodock Vina results. Moreover, our results prove that a coarse sidechain-free potential is sufficient for a very successful docking pose prediction. AVAILABILITYAND IMPLEMENTATION: The standalone version of KORP-PL with the corresponding tests and benchmarks are available at https://team.inria.fr/nano-d/korp-pl/ and https://chaconlab.org/modeling/korp-pl. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Atomic-level evolutionary information improves protein-protein interface scoring.

MOTIVATION: The crucial role of protein interactions and the difficulty in characterizing them experimentally strongly motivates the development of computational approaches for structural prediction. Even when protein-protein docking samples correct models, current scoring functions struggle to discriminate them from incorrect decoys. The previous incorporation of conservation and coevolution information has shown promise for improving protein-protein scoring. Here, we present a novel strategy to integrate atomic-level evolutionary information into different types of scoring functions to improve their docking discrimination. RESULTS: We applied this general strategy to our residue-level statistical potential from InterEvScore and to two atomic-level scores, SOAP-PP and Rosetta interface score (ISC). Including evolutionary information from as few as 10 homologous sequences improves the top 10 success rates of individual atomic-level scores SOAP-PP and Rosetta ISC by 6 and 13.5 percentage points, respectively, on a large benchmark of 752 docking cases. The best individual homology-enriched score reaches a top 10 success rate of 34.4%. A consensus approach based on the complementarity between different homology-enriched scores further increases the top 10 success rate to 40%. AVAILABILITY AND IMPLEMENTATION: All data used for benchmarking and scoring results, as well as a Singularity container of the pipeline, are available at http://biodev.cea.fr/interevol/interevdata/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Local Normal Mode Analysis for Fast Loop Conformational Sampling.

We propose and validate a novel method to efficiently explore local protein loop conformations based on a new formalism for constrained normal mode analysis (NMA) in internal coordinates. The manifold of possible loop configurations imposed by the position and orientation of the fixed loop ends is reduced to an orthogonal set of motions (or modes) encoding concerted rotations of all the backbone dihedral angles. We validate the sampling power on a set of protein loops with highly variable experimental structures and demonstrate that our approach can efficiently explore the conformational space of closed loops. We also show an acceptable resemblance of the ensembles around equilibrium conformations generated by long molecular simulations and constrained NMA on a set of exposed and diverse loops. In comparison with other methods, the main advantage is the lack of restrictions on the number of dihedrals that can be altered simultaneously. Furthermore, the method is computationally efficient since it only requires the diagonalization of a tiny matrix, and the modes of motions are energetically contextualized by the elastic network model, which includes both the loop and the neighboring residues.

Structure of promoter-bound TFIID and model of human pre-initiation complex assembly.

The general transcription factor IID (TFIID) plays a central role in the initiation of RNA polymerase II (Pol II)-dependent transcription by nucleating pre-initiation complex (PIC) assembly at the core promoter. TFIID comprises the TATA-binding protein (TBP) and 13 TBP-associated factors (TAF1-13), which specifically interact with a variety of core promoter DNA sequences. Here we present the structure of human TFIID in complex with TFIIA and core promoter DNA, determined by single-particle cryo-electron microscopy at sub-nanometre resolution. All core promoter elements are contacted by subunits of TFIID, with TAF1 and TAF2 mediating major interactions with the downstream promoter. TFIIA bridges the TBP-TATA complex with lobe B of TFIID. We also present the cryo-electron microscopy reconstruction of a fully assembled human TAF-less PIC. Superposition of common elements between the two structures provides novel insights into the general role of TFIID in promoter recognition, PIC assembly, and transcription initiation.

KORP: knowledge-based 6D potential for fast protein and loop modeling.

MOTIVATION: Knowledge-based statistical potentials constitute a simpler and easier alternative to physics-based potentials in many applications, including folding, docking and protein modeling. Here, to improve the effectiveness of the current approximations, we attempt to capture the six-dimensional nature of residue-residue interactions from known protein structures using a simple backbone-based representation. RESULTS: We have developed KORP, a knowledge-based pairwise potential for proteins that depends on the relative position and orientation between residues. Using a minimalist representation of only three backbone atoms per residue, KORP utilizes a six-dimensional joint probability distribution to outperform state-of-the-art statistical potentials for native structure recognition and best model selection in recent critical assessment of protein structure prediction and loop-modeling benchmarks. Compared with the existing methods, our side-chain independent potential has a lower complexity and better efficiency. The superior accuracy and robustness of KORP represent a promising advance for protein modeling and refinement applications that require a fast but highly discriminative energy function. AVAILABILITY AND IMPLEMENTATION: http://chaconlab.org/modeling/korp. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Near-atomic cryo-EM structure of PRC1 bound to the microtubule.

Proteins that associate with microtubules (MTs) are crucial to generate MT arrays and establish different cellular architectures. One example is PRC1 (protein regulator of cytokinesis 1), which cross-links antiparallel MTs and is essential for the completion of mitosis and cytokinesis. Here we describe a 4-Å-resolution cryo-EM structure of monomeric PRC1 bound to MTs. Residues in the spectrin domain of PRC1 contacting the MT are highly conserved and interact with the same pocket recognized by kinesin. We additionally found that PRC1 promotes MT assembly even in the presence of the MT stabilizer taxol. Interestingly, the angle of the spectrin domain on the MT surface corresponds to the previously observed cross-bridge angle between MTs cross-linked by full-length, dimeric PRC1. This finding, together with molecular dynamic simulations describing the intrinsic flexibility of PRC1, suggests that the MT-spectrin domain interface determines the geometry of the MT arrays cross-linked by PRC1.

RCD+: Fast loop modeling server.

Modeling loops is a critical and challenging step in protein modeling and prediction. We have developed a quick online service (http://rcd.chaconlab.org) for ab initio loop modeling combining a coarse-grained conformational search with a full-atom refinement. Our original Random Coordinate Descent (RCD) loop closure algorithm has been greatly improved to enrich the sampling distribution towards near-native conformations. These improvements include a new workflow optimization, MPI-parallelization and fast backbone angle sampling based on neighbor-dependent Ramachandran probability distributions. The server starts by efficiently searching the vast conformational space from only the loop sequence information and the environment atomic coordinates. The generated closed loop models are subsequently ranked using a fast distance-orientation dependent energy filter. Top ranked loops are refined with the Rosetta energy function to obtain accurate all-atom predictions that can be interactively inspected in an user-friendly web interface. Using standard benchmarks, the average root mean squared deviation (RMSD) is 0.8 and 1.4 Šfor 8 and 12 residues loops, respectively, in the challenging modeling scenario in where the side chains of the loop environment are fully remodeled. These results are not only very competitive compared to those obtained with public state of the art methods, but also they are obtained ∼10-fold faster.

FRODOCK 2.0: fast protein-protein docking server.

UNLABELLED: The prediction of protein-protein complexes from the structures of unbound components is a challenging and powerful strategy to decipher the mechanism of many essential biological processes. We present a user-friendly protein-protein docking server based on an improved version of FRODOCK that includes a complementary knowledge-based potential. The web interface provides a very effective tool to explore and select protein-protein models and interactively screen them against experimental distance constraints. The competitive success rates and efficiency achieved allow the retrieval of reliable potential protein-protein binding conformations that can be further refined with more computationally demanding strategies. AVAILABILITY AND IMPLEMENTATION: The server is free and open to all users with no login requirement at http://frodock.chaconlab.org CONTACT: pablo@chaconlab.org SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

iMODS: internal coordinates normal mode analysis server.

Normal mode analysis (NMA) in internal (dihedral) coordinates naturally reproduces the collective functional motions of biological macromolecules. iMODS facilitates the exploration of such modes and generates feasible transition pathways between two homologous structures, even with large macromolecules. The distinctive internal coordinate formulation improves the efficiency of NMA and extends its applicability while implicitly maintaining stereochemistry. Vibrational analysis, motion animations and morphing trajectories can be easily carried out at different resolution scales almost interactively. The server is versatile; non-specialists can rapidly characterize potential conformational changes, whereas advanced users can customize the model resolution with multiple coarse-grained atomic representations and elastic network potentials. iMODS supports advanced visualization capabilities for illustrating collective motions, including an improved affine-model-based arrow representation of domain dynamics. The generated all-heavy-atoms conformations can be used to introduce flexibility for more advanced modeling or sampling strategies. The server is free and open to all users with no login requirement at http://imods.chaconlab.org.

Understanding nucleotide-regulated FtsZ filament dynamics and the monomer assembly switch with large-scale atomistic simulations.

Bacterial cytoskeletal protein FtsZ assembles in a head-to-tail manner, forming dynamic filaments that are essential for cell division. Here, we study their dynamics using unbiased atomistic molecular simulations from representative filament crystal structures. In agreement with experimental data, we find different filament curvatures that are supported by a nucleotide-regulated hinge motion between consecutive FtsZ monomers. Whereas GTP-FtsZ filaments bend and twist in a preferred orientation, thereby burying the nucleotide, the differently curved GDP-FtsZ filaments exhibit a heterogeneous distribution of open and closed interfaces between monomers. We identify a coordinated Mg(2+) ion as the key structural element in closing the nucleotide site and stabilizing GTP filaments, whereas the loss of the contacts with loop T7 from the next monomer in GDP filaments leads to open interfaces that are more prone to depolymerization. We monitored the FtsZ monomer assembly switch, which involves opening/closing of the cleft between the C-terminal domain and the H7 helix, and observed the relaxation of isolated and filament minus-end monomers into the closed-cleft inactive conformation. This result validates the proposed switch between the low-affinity monomeric closed-cleft conformation and the active open-cleft FtsZ conformation within filaments. Finally, we observed how the antibiotic PC190723 suppresses the disassembly switch and allosterically induces closure of the intermonomer interfaces, thus stabilizing the filament. Our studies provide detailed structural and dynamic insights into modulation of both the intrinsic curvature of the FtsZ filaments and the molecular switch coupled to the high-affinity end-wise association of FtsZ monomers.

Influence of sex on the timing of coronary angiography and the prescription of antiplatelet therapy in patients with nonST-segment elevation myocardial infarction. / Impacto del sexo en los tiempos de cateterismo y en el uso de pretratamiento antiagregante plaquetario en pacientes con síndrome coronario agudo sin elevación del segmento ST (SCASEST).

OBJECTIVES: To assess differences in the clinical management of nonST-segment elevation myocardial infarction (NSTEMI), including in-hospital events, according to biological sex. MATERIAL AND METHODS: Prospective observational multicenter study of patients diagnosed with NSTEMI and atherosclerosis who underwent coronary angiography. RESULTS: We enrolled 1020 patients in April and May 2022; 240 (23.5%) were women. Women were older than men on average (72.6 vs 66.5 years, P .001), and more women were frail (17.1% vs 5.6%, P .001). No difference was observed in pretreatment with any P2Y12 inhibitor (prescribed in 68.8% of women vs 70.2% of men, P = .67); however, more women than men were prescribed clopidogrel (56% vs 44%, P = .009). Women prescribed clopidogrel were more often under the age of 75 years and not frail. Coronary angiography was performed within 24 hours less corooften in women (29.8% vs 36.9%, P = .03) even when high risk was recognized. Frailty was independently associated with deferring coronary angiography in the adjusted analysis; biological sex by itself was not related. The frequency and type of revascularization were the same in both sexes, and there were no differences in in-hospital cardiovascular events. CONCLUSION: Women were more often prescribed less potent antithrombotic therapy than men. Frailty, but not sex, correlated independently with deferral of coronary angiography. However, we detected no differences in the frequency of coronary revascularization or in-hospital events according to sex.

OBJETIVO: Evaluar las diferencias en el manejo clínico y eventos intrahospitalarios en una cohorte de pacientes con síndrome coronario agudo sin elevación del segmento ST (SCASEST) en función del sexo. METODO: Estudio observacional, prospectivo y multicéntrico que incluyó pacientes consecutivos con diagnóstico de SCASEST sometidos a coronariografía con enfermedad ateroesclerótica responsable. RESULTADOS: Entre abril y mayo de 2022 se incluyeron 1.020 pacientes; de ellos, 240 eran mujeres (23,5%). En comparación con los hombres, las mujeres fueron mayores (72,6 años vs 66,5 años; p 0,001) y más frágiles (17,1% vs 5,6%; p 0,001). No hubo diferencias en el pretratamiento con un inhibidor del receptor P2Y12 (68,8% vs 70,2%, p = 0,67), aunque las mujeres recibieron más pretratamiento con clopidogrel (56% vs 44%, p = 0,009), principalmente aquellas de edad 75 años y sin fragilidad. En las mujeres se realizaron menos coronariografías precoces (# 24 h) (29,8% vs 36,9%; p = 0,03) a pesar de presentar la misma indicación (criterios de alto riesgo). En el análisis ajustado, la fragilidad, pero no el sexo, se asoció de forma independiente con la realización de una coronariografía diferida. La tasa y el tipo de revascularización fue igual en ambos sexos, y no hubo diferencias en los eventos cardiovasculares intrahospitalarios. CONCLUSIONES: Las mujeres recibieron con mayor frecuencia un tratamiento antitrombótico menos potente. La fragilidad y no el sexo se asoció con la realización de coronariografía diferida. Sin embargo, no hubo diferencias en la tasa de revascularización coronaria ni en los eventos intrahospitalarios en función del sexo.

Timing of coronary angiography and use of antiplatelet pretreatment in patients with NSTEACS in Spain.

INTRODUCTION AND OBJECTIVES: The optimal timing of coronary angiography in patients admitted with non-ST-segment elevation acute coronary syndrome (NSTEACS) as well as the need for pretreatment are controversial. The main objective of the IMPACT-TIMING-GO registry was to assess the proportion of patients undergoing an early invasive strategy (0-24hours) without dual antiplatelet therapy (no pretreatment strategy) in Spain. METHODS: This observational, prospective, and multicenter study included consecutive patients with NSTEACS who underwent coronary angiography that identified a culprit lesion. RESULTS: Between April and May 2022, we included 1021 patients diagnosed with NSTEACS, with a mean age of 67±12 years (23.6% women). A total of 87% of the patients were deemed at high risk (elevated troponin; electrocardiogram changes; GRACE score>140) but only 37.8% underwent an early invasive strategy, and 30.3% did not receive pretreatment. Overall, 13.6% of the patients underwent an early invasive strategy without pretreatment, while the most frequent strategy was a deferred angiography under antiplatelet pretreatment (46%). During admission, 9 patients (0.9%) died, while major bleeding occurred in 34 (3.3%). CONCLUSIONS: In Spain, only 13.6% of patients with NSTEACS undergoing coronary angiography received an early invasive strategy without pretreatment. The incidence of cardiovascular and severe bleeding events during admission was low.

iMODFIT: efficient and robust flexible fitting based on vibrational analysis in internal coordinates.

Here, we employed the collective motions extracted from Normal Mode Analysis (NMA) in internal coordinates (torsional space) for the flexible fitting of atomic-resolution structures into electron microscopy (EM) density maps. The proposed methodology was validated using a benchmark of simulated cases, highlighting its robustness over the full range of EM resolutions and even over coarse-grained representations. A systematic comparison with other methods further showcased the advantages of this proposed methodology, especially at medium to lower resolutions. Using this method, computational costs and potential overfitting problems are naturally reduced by constraining the search in low-frequency NMA space, where covalent geometry is implicitly maintained. This method also effectively captures the macromolecular changes of a representative set of experimental test cases. We believe that this novel approach will extend the currently available EM hybrid methods to the atomic-level interpretation of large conformational changes and their functional implications.

Plasmid replication initiator RepB forms a hexamer reminiscent of ring helicases and has mobile nuclease domains.

RepB initiates plasmid rolling-circle replication by binding to a triple 11-bp direct repeat (bind locus) and cleaving the DNA at a specific distant site located in a hairpin loop within the nic locus of the origin. The structure of native full-length RepB reveals a hexameric ring molecule, where each protomer has two domains. The origin-binding and catalytic domains show a three-layer alpha-beta-alpha sandwich fold. The active site is positioned at one of the faces of the beta-sheet and coordinates a Mn2+ ion at short distance from the essential nucleophilic Y99. The oligomerization domains (ODs), each consisting of four alpha-helices, together define a compact ring with a central channel, a feature found in ring helicases. The toroidal arrangement of RepB suggests that, similar to ring helicases, it encircles one of the DNA strands during replication to confer processivity to the replisome complex. The catalytic domains appear to be highly mobile with respect to ODs. This mobility may account for the adaptation of the protein to two distinct DNA recognition sites.

A physiologically enhanced muscle spindle model: using a Hill-type model for extrafusal fibers as template for intrafusal fibers.

The muscle spindle is an essential proprioceptor, significantly involved in sensing limb position and movement. Although biological spindle models exist for years, the gold-standard for motor control in biomechanics are still sensors built of homogenized spindle output models due to their simpler combination with neuro-musculoskeletal models. Aiming to improve biomechanical simulations, this work establishes a more physiological model of the muscle spindle, aligned to the advantage of easy integration into large-scale musculoskeletal models. We implemented four variations of a spindle model in Matlab/Simulink®: the Mileusnic et al. (2006) model, Mileusnic model without mass, our enhanced Hill-type model, and our enhanced Hill-type model with parallel damping element (PDE). Different stretches in the intrafusal fibers were simulated in all model variations following the spindle afferent recorded in previous experiments in feline soleus muscle. Additionally, the enhanced Hill-type models had their parameters extensively optimized to match the experimental conditions, and the resulting model was validated against data from rats' triceps surae muscle. As result, the Mileusnic models present a better overall performance generating the afferent firings compared to the common data evaluated. However, the enhanced Hill-type model with PDE exhibits a more stable performance than the original Mileusnic model, at the same time that presents a well-tuned Hill-type model as muscle spindle fibers, and also accounts for real sarcomere force-length and force-velocity aspects. Finally, our activation dynamics is similar to the one applied to Hill-type model for extrafusal fibers, making our proposed model more easily integrated in multi-body simulations.

Non-Sterile to Sterile Compounding: An Unconventional Response During the COVID-19 Pandemic.

The drug supply chain has suffered many interruptions over the past decade. The COVID-19 pandemic exacerbated an already fragile infrastructure for supplying critical medications to hospitals and health-systems. The purpose of this paper is to provide insight to the history, thought-processes, and response to critical medication shortages during the COVID-19 pandemic, with a focus on hydromorphone infusions and the action steps taken to engage in non-sterile to sterile (NSTS) compounding. Over a period of 6 weeks, we compounded 1,613 NSTS hydromorphone infusion bags. All lots were cleared for sterility, particulate, potency, and endotoxin testing by an outside FDA registered laboratory. We did not have any safety reports filed specific to the NSTS compounded hydromorphone infusion bags. Over a period of 15 weeks, 715 infusions were consumed. The drug supply chain suffers frequent interruptions and critical shortages, particularly in times of a natural disaster or a global pandemic. Non-sterile to sterile compounding is often associated with risks of inaccuracies, impurities, and contamination. There are instances in which non-sterile to sterile compounding is appropriate and should be considered in times of drug shortages to support the care of hospitalized patients.