N4BP1 functions as a dimerization-dependent linear ubiquitin reader which regulates TNF signalling.

Signalling through TNFR1 modulates proinflammatory gene transcription and programmed cell death, and its impairment causes autoimmune diseases and cancer. NEDD4-binding protein 1 (N4BP1) is a critical suppressor of proinflammatory cytokine production that acts as a regulator of innate immune signalling and inflammation. However, our current understanding about the molecular properties that enable N4BP1 to exert its suppressive potential remain limited. Here, we show that N4BP1 is a novel linear ubiquitin reader that negatively regulates NFκB signalling by its unique dimerization-dependent ubiquitin-binding module that we named LUBIN. Dimeric N4BP1 strategically positions two non-selective ubiquitin-binding domains to ensure preferential recognition of linear ubiquitin. Under proinflammatory conditions, N4BP1 is recruited to the nascent TNFR1 signalling complex, where it regulates duration of proinflammatory signalling in LUBIN-dependent manner. N4BP1 deficiency accelerates TNFα-induced cell death by increasing complex II assembly. Under proapoptotic conditions, caspase-8 mediates proteolytic processing of N4BP1, resulting in rapid degradation of N4BP1 by the 26 S proteasome, and acceleration of apoptosis. In summary, our findings demonstrate that N4BP1 dimerization creates a novel type of ubiquitin reader that selectively recognises linear ubiquitin which enables the timely and coordinated regulation of TNFR1-mediated inflammation and cell death.

PyCoM: a python library for large-scale analysis of residue-residue coevolution data.

MOTIVATION: Computational methods to detect correlated amino acid positions in proteins have become a valuable tool to predict intra- and inter-residue protein contacts, protein structures, and effects of mutation on protein stability and function. While there are many tools and webservers to compute coevolution scoring matrices, there is no central repository of alignments and coevolution matrices for large-scale studies and pattern detection leveraging on biological and structural annotations already available in UniProt. RESULTS: We present a Python library, PyCoM, which enables users to query and analyze coevolution matrices and sequence alignments of 457 622 proteins, selected from UniProtKB/Swiss-Prot database (length ≤ 500 residues), from a precompiled coevolution matrix database (PyCoMdb). PyCoM facilitates the development of statistical analyses of residue coevolution patterns using filters on biological and structural annotations from UniProtKB/Swiss-Prot, with simple access to PyCoMdb for both novice and advanced users, supporting Jupyter Notebooks, Python scripts, and a web API access. The resource is open source and will help in generating data-driven computational models and methods to study and understand protein structures, stability, function, and design. AVAILABILITY AND IMPLEMENTATION: PyCoM code is freely available from https://github.com/scdantu/pycom and PyCoMdb and the Jupyter Notebook tutorials are freely available from https://pycom.brunel.ac.uk.

MDSubSampler: a posteriori sampling of important protein conformations from biomolecular simulations.

MOTIVATION: Molecular dynamics (MD) simulations have become routine tools for the study of protein dynamics and function. Thanks to faster GPU-based algorithms, atomistic and coarse-grained simulations are being used to explore biological functions over the microsecond timescale, yielding terabytes of data spanning multiple trajectories, thereby extracting relevant protein conformations without losing important information is often challenging. RESULTS: We present MDSubSampler, a Python library and toolkit for a posteriori subsampling of data from multiple trajectories. This toolkit provides access to uniform, random, stratified, weighted sampling, and bootstrapping sampling methods. Sampling can be performed under the constraint of preserving the original distribution of relevant geometrical properties. Possible applications include simulations post-processing, noise reduction, and structures selection for ensemble docking. AVAILABILITY AND IMPLEMENTATION: MDSubSampler is freely available at https://github.com/alepandini/MDSubSampler, along with guidance on installation and tutorials on how it can be used.

Recognizing the Binding Pattern and Dissociation Pathways of the p300 Taz2-p53 TAD2 Complex.

The dynamic association and dissociation between proteins are the basis of cellular signal transduction. This process becomes much more complicated if one or both interaction partners are intrinsically disordered because intrinsically disordered proteins can undergo disorder-to-order transitions upon binding to their partners. p53, a transcription factor with disordered regions, plays significant roles in many cellular signaling pathways. It is critical to understand the binding/unbinding mechanism involving these disordered regions of p53 at the residue level to reveal how p53 performs its biological functions. Here, we studied the dissociation process of the intrinsically disordered N-terminal transactivation domain 2 (TAD2) of p53 and the transcriptional adaptor zinc-binding 2 (Taz2) domain of transcriptional coactivator p300 using a combination of classical molecular dynamics, steered molecular dynamics, self-organizing maps, and time-resolved force distribution analysis (TRFDA). We observed two different dissociation pathways with different probabilities. One dissociation pathway starts from the TAD2 N-terminus and propagates to the α-helix and finally the C-terminus. The other dissociation pathway is in the opposite order. Subsequent TRFDA results reveal that key residues in TAD2 play critical roles. Besides the residues in agreement with previous experimental results, we also highlighted some other residues that play important roles in the disassociation process. In the dissociation process, non-native interactions were formed to partially compensate for the energy loss due to the breaking of surrounding native interactions. Moreover, our statistical analysis results of other experimentally determined complex structures involving either Taz2 or TAD2 suggest that the binding of the Taz2-TAD2 complex is mainly governed by the binding site of Taz2, which includes three main binding regions. Therefore, the complexes involving Taz2 may follow similar binding/unbinding behaviors, which could be studied together to generate common principles.

PathDetect-SOM: A Neural Network Approach for the Identification of Pathways in Ligand Binding Simulations.

Understanding the process of ligand-protein recognition is important to unveil biological mechanisms and to guide drug discovery and design. Enhanced-sampling molecular dynamics is now routinely used to simulate the ligand binding process, resulting in the need for suitable tools for the analysis of large data sets of binding events. Here, we designed, implemented, and tested PathDetect-SOM, a tool based on self-organizing maps to build concise visual models of the ligand binding pathways sampled along single simulations or replicas. The tool performs a geometric clustering of the trajectories and traces the pathways over an easily interpretable 2D map and, using an approximate transition matrix, it can build a graph model of concurrent pathways. The tool was tested on three study cases representing different types of problems and simulation techniques. A clear reconstruction of the sampled pathways was derived in all cases, and useful information on the energetic features of the processes was recovered. The tool is available at https://github.com/MottaStefano/PathDetect-SOM.

Dynamic Profiling of ß-Coronavirus 3CL Mpro Protease Ligand-Binding Sites.

ß-coronavirus (CoVs) alone has been responsible for three major global outbreaks in the 21st century. The current crisis has led to an urgent requirement to develop therapeutics. Even though a number of vaccines are available, alternative strategies targeting essential viral components are required as a backup against the emergence of lethal viral variants. One such target is the main protease (Mpro) that plays an indispensable role in viral replication. The availability of over 270 Mpro X-ray structures in complex with inhibitors provides unique insights into ligand-protein interactions. Herein, we provide a comprehensive comparison of all nonredundant ligand-binding sites available for SARS-CoV2, SARS-CoV, and MERS-CoV Mpro. Extensive adaptive sampling has been used to investigate structural conservation of ligand-binding sites using Markov state models (MSMs) and compare conformational dynamics employing convolutional variational auto-encoder-based deep learning. Our results indicate that not all ligand-binding sites are dynamically conserved despite high sequence and structural conservation across ß-CoV homologs. This highlights the complexity in targeting all three Mpro enzymes with a single pan inhibitor.

Machine Learning Prediction of Allosteric Drug Activity from Molecular Dynamics.

Allosteric drugs have been attracting increasing interest over the past few years. In this context, it is common practice to use high-throughput screening for the discovery of non-natural allosteric drugs. While the discovery stage is supported by a growing amount of biological information and increasing computing power, major challenges still remain in selecting allosteric ligands and predicting their effect on the target protein's function. Indeed, allosteric compounds can act both as inhibitors and activators of biological responses. Computational approaches to the problem have focused on variations on the theme of molecular docking coupled to molecular dynamics with the aim of recovering information on the (long-range) modulation typical of allosteric proteins.

Reconstruction of ARNT PAS-B Unfolding Pathways by Steered Molecular Dynamics and Artificial Neural Networks.

Several experimental studies indicated that large conformational changes, including partial domain unfolding, have a role in the functional mechanisms of the basic helix loop helix Per/ARNT/SIM (bHLH-PAS) transcription factors. Recently, single-molecule atomic force microscopy (AFM) revealed two distinct pathways for the mechanical unfolding of the ARNT PAS-B. In this work we used steered molecular dynamics simulations to gain new insights into this process at an atomistic level. To reconstruct and classify pathways sampled in multiple simulations, we designed an original approach based on the use of self-organizing maps (SOMs). This led us to identify two types of unfolding pathways for the ARNT PAS-B, which are in good agreement with the AFM findings. Analysis of average forces mapped on the SOM revealed a stable conformation of the PAS-B along one pathway, which represents a possible structural model for the intermediate state detected by AFM. The approach here proposed will facilitate the study of other signal transmission mechanisms involving the folding/unfolding of PAS domains.

Allosteric Priming of E. coli CheY by the Flagellar Motor Protein FliM.

Phosphorylation of Escherichia coli CheY protein transduces chemoreceptor stimulation to a highly cooperative flagellar motor response. CheY binds to the N-terminal peptide of the FliM motor protein (FliMN). Constitutively active D13K-Y106W CheY has been an important tool for motor physiology. The crystal structures of CheY and CheY ⋅ FliMN with and without D13K-Y106W have shown FliMN-bound CheY contains features of both active and inactive states. We used molecular dynamics (MD) simulations to characterize the CheY conformational landscape accessed by FliMN and D13K-Y106W. Mutual information measures identified the central features of the long-range CheY allosteric network between D57 phosphorylation site and the FliMN interface, namely the closure of the α4-ß4 hinge and inward rotation of Y- or W106 with W58. We used hydroxy-radical foot printing with mass spectroscopy (XFMS) to track the solvent accessibility of these and other side chains. The solution XFMS oxidation rate correlated with the solvent-accessible area of the crystal structures. The protection of allosteric relay side chains reported by XFMS confirmed the intermediate conformation of the native CheY ⋅ FliMN complex, the inactive state of free D13K-Y106W CheY, and the MD-based network architecture. We extended the MD analysis to determine temporal coupling and energetics during activation. Coupled aromatic residue rotation was a graded rather than a binary switch, with Y- or W106 side-chain burial correlated with increased FliMN affinity. Activation entrained CheY fold stabilization to FliMN affinity. The CheY network could be partitioned into four dynamically coordinated sectors. Residue substitutions mapped to sectors around D57 or the FliMN interface according to phenotype. FliMN increased sector size and interactions. These sectors fused between the substituted K13-W106 residues to organize a tightly packed core and novel surfaces that may bind additional sites to explain the cooperative motor response. The community maps provide a more complete description of CheY priming than proposed thus far.

Structure, Dynamics and Cellular Insight Into Novel Substrates of the Legionella pneumophila Type II Secretion System.

Legionella pneumophila is a Gram-negative bacterium that is able to replicate within a broad range of aquatic protozoan hosts. L. pneumophila is also an opportunistic human pathogen that can infect macrophages and epithelia in the lung and lead to Legionnaires' disease. The type II secretion system is a key virulence factor of L. pneumophila and is used to promote bacterial growth at low temperatures, regulate biofilm formation, modulate host responses to infection, facilitate bacterial penetration of mucin gels and is necessary for intracellular growth during the initial stages of infection. The L. pneumophila type II secretion system exports at least 25 substrates out of the bacterium and several of these, including NttA to NttG, contain unique amino acid sequences that are generally not observed outside of the Legionella genus. NttA, NttC, and NttD are required for infection of several amoebal species but it is unclear what influence other novel substrates have within their host. In this study, we show that NttE is required for optimal infection of Acanthamoeba castellanii and Vermamoeba vermiformis amoeba and is essential for the typical colony morphology of L. pneumophila. In addition, we report the atomic structures of NttA, NttC, and NttE and through a combined biophysical and biochemical hypothesis driven approach we propose novel functions for these substrates during infection. This work lays the foundation for future studies into the mechanistic understanding of novel type II substrate functions and how these relate to L. pneumophila ecology and disease.

Structure-based enzyme engineering improves donor-substrate recognition of Arabidopsis thaliana glycosyltransferases.

Glycosylation of secondary metabolites involves plant UDP-dependent glycosyltransferases (UGTs). UGTs have shown promise as catalysts in the synthesis of glycosides for medical treatment. However, limited understanding at the molecular level due to insufficient biochemical and structural information has hindered potential applications of most of these UGTs. In the absence of experimental crystal structures, we employed advanced molecular modeling and simulations in conjunction with biochemical characterization to design a workflow to study five Group H Arabidopsis thaliana (76E1, 76E2, 76E4, 76E5, 76D1) UGTs. Based on our rational structural manipulation and analysis, we identified key amino acids (P129 in 76D1; D374 in 76E2; K275 in 76E4), which when mutated improved donor substrate recognition than wildtype UGTs. Molecular dynamics simulations and deep learning analysis identified structural differences, which drive substrate preferences. The design of these UGTs with broader substrate specificity may play important role in biotechnological and industrial applications. These findings can also serve as basis to study other plant UGTs and thereby advancing UGT enzyme engineering.

Conformational coupling by trans-phosphorylation in calcium calmodulin dependent kinase II.

The calcium calmodulin-dependent protein kinase II (CaMKII) is a dodecameric holoenzyme important for encoding memory. Its activation, triggered by binding of calcium-calmodulin, persists autonomously after calmodulin dissociation. One (receiver) kinase captures and subsequently phosphorylates the regulatory domain peptide of a donor kinase forming a chained dimer as the first stage of autonomous activation. Protein dynamics simulations examined the conformational changes triggered by dimer formation and phosphorylation, aimed to provide a molecular rationale for human mutations that result in learning disabilities. Ensembles generated from X-ray crystal structures were characterized by network centrality and community analysis. Mutual information related collective motions to local fragment dynamics encoded with a structural alphabet. Implicit solvent tCONCOORD conformational ensembles revealed the dynamic architecture of inactive kinase domains was co-opted in the activated dimer but the network hub shifted from the nucleotide binding cleft to the captured peptide. Explicit solvent molecular dynamics (MD) showed nucleotide and substrate binding determinants formed coupled nodes in long-range signal relays between regulatory peptides in the dimer. Strain in the extended captured peptide was balanced by reduced flexibility of the receiver kinase C-lobe core. The relays were organized around a hydrophobic patch between the captured peptide and a key binding helix. The human mutations aligned along the relays. Thus, these mutations could disrupt the allosteric network alternatively, or in addition, to altered binding affinities. Non-binding protein sectors distant from the binding sites mediated the allosteric signalling; providing possible targets for inhibitor design. Phosphorylation of the peptide modulated the dielectric of its binding pocket to strengthen the patch, non-binding sectors, domain interface and temporal correlations between parallel relays. These results provide the molecular details underlying the reported positive kinase cooperativity to enrich the discussion on how autonomous activation by phosphorylation leads to long-term behavioural effects.

Ligand-induced perturbation of the HIF-2α:ARNT dimer dynamics.

Hypoxia inducible factors (HIFs) are transcription factors belonging to the basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) protein family with a role in sensing oxygen levels in the cell. Under hypoxia, the HIF-α degradation pathway is blocked and dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT) makes HIF-α transcriptionally active. Due to the common hypoxic environment of tumors, inhibition of this mechanism by destabilization of HIF-α:ARNT dimerization has been proposed as a promising therapeutic strategy. Following the discovery of a druggable cavity within the PAS-B domain of HIF-2α, research efforts have been directed to identify artificial ligands that can impair heterodimerization. Although the crystallographic structures of the HIF-2α:ARNT complex have elucidated the dimer architecture and the 0X3-inhibitor placement within the HIF-2α PAS-B, unveiling the inhibition mechanism requires investigation of how ligand-induced perturbations could dynamically propagate through the structure and affect dimerization. To this end, we compared evolutionary features, intrinsic dynamics and energetic properties of the dimerization interfaces of HIF-2α:ARNT in both the apo and holo forms. Residue conservation analysis highlighted inter-domain connecting elements that have a role in dimerization. Analysis of domain contributions to the dimerization energy demonstrated the importance of bHLH and PAS-A of both partners and of HIF-2α PAS-B domain in dimer stabilization. Among quaternary structure oscillations revealed by Molecular Dynamics simulations, the hinge-bending motion of the ARNT PAS-B domain around the flexible PAS-A/PAS-B linker supports a general model for ARNT dimerization in different heterodimers. Comparison of the HIF-2α:ARNT dynamics in the apo and 0X3-bound forms indicated a model of inhibition where the HIF-2α-PAS-B interfaces are destabilised as a result of water-bridged ligand-protein interactions and these local effects allosterically propagate to perturb the correlated motions of the domains and inter-domain communication. These findings will guide the design of improved inhibitors to contrast cell survival in tumor masses.

In silico identification of rescue sites by double force scanning.

Motivation: A deleterious amino acid change in a protein can be compensated by a second-site rescue mutation. These compensatory mechanisms can be mimicked by drugs. In particular, the location of rescue mutations can be used to identify protein regions that can be targeted by small molecules to reactivate a damaged mutant. Results: We present the first general computational method to detect rescue sites. By mimicking the effect of mutations through the application of forces, the double force scanning (DFS) method identifies the second-site residues that make the protein structure most resilient to the effect of pathogenic mutations. We tested DFS predictions against two datasets containing experimentally validated and putative evolutionary-related rescue sites. A remarkably good agreement was found between predictions and experimental data. Indeed, almost half of the rescue sites in p53 was correctly predicted by DFS, with 65% of remaining sites in contact with DFS predictions. Similar results were found for other proteins in the evolutionary dataset. Availability and implementation: The DFS code is available under GPL at https://fornililab.github.io/dfs/. Supplementary information: Supplementary data are available at Bioinformatics online.

Binding of Myomesin to Obscurin-Like-1 at the Muscle M-Band Provides a Strategy for Isoform-Specific Mechanical Protection.

The sarcomeric cytoskeleton is a network of modular proteins that integrate mechanical and signaling roles. Obscurin, or its homolog obscurin-like-1, bridges the giant ruler titin and the myosin crosslinker myomesin at the M-band. Yet, the molecular mechanisms underlying the physical obscurin(-like-1):myomesin connection, important for mechanical integrity of the M-band, remained elusive. Here, using a combination of structural, cellular, and single-molecule force spectroscopy techniques, we decode the architectural and functional determinants defining the obscurin(-like-1):myomesin complex. The crystal structure reveals a trans-complementation mechanism whereby an incomplete immunoglobulin-like domain assimilates an isoform-specific myomesin interdomain sequence. Crucially, this unconventional architecture provides mechanical stability up to forces of ∼135 pN. A cellular competition assay in neonatal rat cardiomyocytes validates the complex and provides the rationale for the isoform specificity of the interaction. Altogether, our results reveal a novel binding strategy in sarcomere assembly, which might have implications on muscle nanomechanics and overall M-band organization.

The Gearbox of the Bacterial Flagellar Motor Switch.

Switching of flagellar motor rotation sense dictates bacterial chemotaxis. Multi-subunit FliM-FliG rotor rings couple signal protein binding in FliM with reversal of a distant FliG C-terminal (FliGC) helix involved in stator contacts. Subunit dynamics were examined in conformer ensembles generated by molecular simulations from the X-ray structures. Principal component analysis extracted collective motions. Interfacial loop immobilization by complex formation coupled elastic fluctuations of the FliM middle (FliMM) and FliG middle (FliGM) domains. Coevolved mutations captured interfacial dynamics as well as contacts. FliGM rotation was amplified via two central hinges to the FliGC helix. Intrinsic flexibility, reported by the FliGMC ensembles, reconciled conformers with opposite FliGC helix orientations. FliG domain stacking deformed the inter-domain linker and reduced flexibility; but conformational changes were not triggered by engineered linker deletions that cause a rotation-locked phenotype. These facts suggest that binary rotation states arise from conformational selection by stacking interactions.

Using Local States To Drive the Sampling of Global Conformations in Proteins.

Conformational changes associated with protein function often occur beyond the time scale currently accessible to unbiased molecular dynamics (MD) simulations, so that different approaches have been developed to accelerate their sampling. Here we investigate how the knowledge of backbone conformations preferentially adopted by protein fragments, as contained in precalculated libraries known as structural alphabets (SA), can be used to explore the landscape of protein conformations in MD simulations. We find that (a) enhancing the sampling of native local states in both metadynamics and steered MD simulations allows the recovery of global folded states in small proteins; (b) folded states can still be recovered when the amount of information on the native local states is reduced by using a low-resolution version of the SA, where states are clustered into macrostates; and (c) sequences of SA states derived from collections of structural motifs can be used to sample alternative conformations of preselected protein regions. The present findings have potential impact on several applications, ranging from protein model refinement to protein folding and design.

Coevolved Mutations Reveal Distinct Architectures for Two Core Proteins in the Bacterial Flagellar Motor.

Switching of bacterial flagellar rotation is caused by large domain movements of the FliG protein triggered by binding of the signal protein CheY to FliM. FliG and FliM form adjacent multi-subunit arrays within the basal body C-ring. The movements alter the interaction of the FliG C-terminal (FliGC) "torque" helix with the stator complexes. Atomic models based on the Salmonella entrovar C-ring electron microscopy reconstruction have implications for switching, but lack consensus on the relative locations of the FliG armadillo (ARM) domains (amino-terminal (FliGN), middle (FliGM) and FliGC) as well as changes during chemotaxis. The generality of the Salmonella model is challenged by the variation in motor morphology and response between species. We studied coevolved residue mutations to determine the unifying elements of switch architecture. Residue interactions, measured by their coevolution, were formalized as a network, guided by structural data. Our measurements reveal a common design with dedicated switch and motor modules. The FliM middle domain (FliMM) has extensive connectivity most simply explained by conserved intra and inter-subunit contacts. In contrast, FliG has patchy, complex architecture. Conserved structural motifs form interacting nodes in the coevolution network that wire FliMM to the FliGC C-terminal, four-helix motor module (C3-6). FliG C3-6 coevolution is organized around the torque helix, differently from other ARM domains. The nodes form separated, surface-proximal patches that are targeted by deleterious mutations as in other allosteric systems. The dominant node is formed by the EHPQ motif at the FliMMFliGM contact interface and adjacent helix residues at a central location within FliGM. The node interacts with nodes in the N-terminal FliGc α-helix triad (ARM-C) and FliGN. ARM-C, separated from C3-6 by the MFVF motif, has poor intra-network connectivity consistent with its variable orientation revealed by structural data. ARM-C could be the convertor element that provides mechanistic and species diversity.