Chemical cross-linking coupled with mass spectrometry (XL-MS) has become a powerful tool for probing residue-level proximities within macromolecular assemblies. By providing sparse but informative distance restraints, XL-MS can be integrated with electron microscopy and domain-level high-resolution structures to model the architecture of protein complexes. Unlike X-ray crystallography, electron microscopy, or solid-state Nuclear Magnetic Resonance (NMR), XL-MS can be applied under near-physiological conditions, scaled to large modular systems, and performed at higher throughput. In this review, we highlight recent advances in the field, with particular emphasis on the impact of AI-driven structure prediction. As an illustration, we describe a hybrid protocol that combines the Integrative Modeling Platform (IMP) with the deep neural network Chai-1 to dock and refine the helicase Dbp10 on a transient ribosome biogenesis intermediate using XL-MS restraints.

The current richness of sequence data needs efficient methodologies to display and analyze the complexity of the information in a compact and readable manner. Traditionally, phylogenetic trees and sequence similarity networks have been used to display and analyze sequences of protein families. These methods aim to shed light on key computational biology problems such as sequence classification and functional inference. Here, we present a new methodology, AlignScape, based on self-organizing maps. AlignScape is applied to three large families of proteins: the kinases and GPCRs from human, and bacterial T6SS proteins. AlignScape provides a map of the similarity landscape and a tree representation of multiple sequence alignments These representations are useful to display, cluster, and classify sequences as well as identify functional trends. The efficient GPU implementation of AlignScape allows the analysis of large MSAs in a few minutes. Furthermore, we show how the AlignScape analysis of proteins belonging to the T6SS complex can be used to predict coevolving partners.

Chemical modification of aptamers is an important step to improve their performance and stability in biological media. This can be performed either during their identification (mod-SELEX) or after the in vitro selection process (post-SELEX). In order to reduce the complexity and workload of the post-SELEX modification of aptamers, we have evaluated the possibility of improving a previously reported, chemically modified aptamer by combining enzymatic synthesis and nucleotides bearing bioisosteres of the parent cubane side-chains or substituted cubane moieties. This method lowers the synthetic burden often associated with post-SELEX approaches and allowed to identify one additional sequence that maintains binding to the PvLDH target protein, albeit with reduced specificity. In addition, while bioisosteres often improve the potency of small molecule drugs, this does not extend to chemically modified aptamers. Overall, this versatile method can be applied for the post-SELEX modification of other aptamers and functional nucleic acids. Post-SELEX modification of aptamers allows to improve their performance but comes at the cost of uncertain and labor-intensive structure activity relationship studies. Here, we have evaluated the possibility of improving a previously reported, chemically modified aptamer by combining enzymatic synthesis and nucleotides bearing bioisosteres of the parent cubane side-chains or substituted cubane moieties. This method lowers the synthetic burden associated with post-SELEX approaches and allowed to identify one additional sequence that maintains binding to the PvLDH target protein, albeit with reduced specificity.+image

The type VI secretion system (T6SS) of Gram-negative bacteria inhibits competitor cells through contact-dependent translocation of toxic effector proteins. In Proteobacteria, the T6SS is anchored to the cell envelope through a megadalton-sized membrane complex (MC). However, the genomes of Bacteroidota with T6SSs appear to lack genes encoding homologs of canonical MC components. Here, we identify five genes in Bacteroides fragilis ( tssNQOPR ) that are essential for T6SS function and encode a Bacteroidota-specific MC. We purify this complex, reveal its dimensions using electron microscopy, and identify a protein-protein interaction network underlying the assembly of the MC including the stoichiometry of the five TssNQOPR components. Protein TssN mediates the connection between the Bacteroidota MC and the conserved baseplate. Although MC gene content and organization varies across the phylum Bacteroidota, no MC homologs are detected outside of T6SS loci, suggesting ancient co-option and functional convergence with the non-homologous MC of Pseudomonadota.

Proteins play a central role in most biological processes within the cell, and deciphering how they interact is key to understand their function. Cross- linking coupled with mass spectrometry is an essential tool for elucidating protein-protein interactions (PPIs). Despite its importance, we still know surprisingly little about the principles that underlie the process of chemical cross- link formation itself and how it is influenced by different physicochemical factors. To understand the molecular details of cross- link formation, we have set up a comprehensive kinetic model and carried out simulations of protein cross- linking on large protein complexes. We dissect the contribution on the cross- link yield of parameters such as amino acid reactivity, cross- linker concentration, and hydrolysis rate. Our model can compute cross- link formation based solely on the structure of a protein complex, thereby enabling realistic predictions for a diverse set of systems. We quantitatively show how cross- links and mono- links are in direct competition and how the hydrolysis rate and abundance of cross- linker and proteins directly influence their relative formation. We show how cross- links and mono- links exist in an "all- against- all" competition due to their simultaneous formation, resulting in a nonintuitive network of interdependence. We show that this interdependence is locally confined and mainly limited to direct neighbors or residues in direct vicinity. These results enable us to identify the optimal cross- linker concentration at which the maximal number of cross- links is formed. Taken together, our study establishes a comprehensive kinetic model to quantitatively describe cross- link formation for PPIs.

Proteolysis targeting chimeras (PROTACs) are heterobifunctional ligands that mediate the interaction between a protein target and an E3 ligase, resulting in a ternary complex, whose interaction with the ubiquitination machinery leads to target degradation. This technology is emerging as an exciting new avenue for therapeutic development, with several PROTACs currently undergoing clinical trials targeting cancer. Here, we describe a general and computationally efficient methodology combining restraint-based docking, energy-based rescoring, and a filter based on the minimal solvent-accessible surface distance to produce PROTAC-compatible PPIs suitable for when there is no a priori known PROTAC ligand. In a benchmark employing a manually curated data set of 13 ternary complex crystals, we achieved an accuracy of 92% when starting from bound structures and 77% when starting from unbound structures, respectively. Our method only requires that the ligand-bound structures of the monomeric forms of the E3 ligase and target proteins be given to run, making it general, accurate, and highly efficient, with the ability to impact early-stage PROTAC-based drug design campaigns where no structural information about the ternary complex structure is available.

Tar file containing the dataset used for the "Rational prediction of PROTAC compatible ligase-target interfaces" manuscript by Pereira et al.

A. baumannii can rapidly acquire new resistance mechanisms and persist on abiotic surface, enabling the colonization of asymptomatic human host. In Acinetobacter the type VI secretion system (T6SS) is involved in twitching, surface motility and is used for interbacterial competition allowing the bacteria to uptake DNA. A. baumannii possesses a T6SS that has been well studied for its regulation and specific activity, but little is known concerning its assembly and architecture. The T6SS nanomachine is built from three architectural sub-complexes. Unlike the baseplate (BP) and the tail-tube complex (TTC), which are inherited from bacteriophages, the membrane complex (MC) originates from bacteria. The MC is the most external part of the T6SS and, as such, is subjected to evolution and adaptation. One unanswered question on the MC is how such a gigantesque molecular edifice is inserted and crosses the bacterial cell envelope. The A. baumannii MC lacks an essential component, the TssJ lipoprotein, which anchors the MC to the outer membrane. In this work, we studied how A. baumannii compensates the absence of a TssJ. We have characterized for the first time the A. baumannii's specific T6SS MC, its unique characteristic, its membrane localization, and assembly dynamics. We also defined its composition, demonstrating that its biogenesis employs three Acinetobacter-specific envelope-associated proteins that define an intricate network leading to the assembly of a five-proteins membrane super-complex. Our data suggest that A. baumannii has divided the function of TssJ by (1) co-opting a new protein TsmK that stabilizes the MC and by (2) evolving a new domain in TssM for homo-oligomerization, a prerequisite to build the T6SS channel. We believe that the atypical species-specific features we report in this study will have profound implication in our understanding of the assembly and evolutionary diversity of different T6SSs, that warrants future investigation.

The vibrating reed method is a recognized technique for the mechanical analysis of materials and offers a number of advantages over other types of test. However, little detailed evaluation of the method has been reported, particularly those aspects which affect accuracy and reproducibility. In this investigation, a vibrating reed apparatus was constructed which incorporated automatic detection of the reed displacement. A number of clamp designs were assessed and a range of materials, including some of biomedical interest, was examined. It was found that significant experimental inaccuracies were present and that these led to errors of approximately 15% in the measured properties. The most important contribution to this error arose from the clamping mechanism.

Cross-linking mass spectrometry (XL-MS) has become an indispensable tool for the emerging field of systems structural biology over the recent years. However, the confidence in individual protein-protein interactions (PPIs) depends on the correct assessment of individual inter-protein cross-links. In this article, we describe a mono- and intralink filter (mi-filter) that is applicable to any kind of cross-linking data and workflow. It stipulates that only proteins for which at least one monolink or intra-protein cross-link has been identified within a given data set are considered for an inter-protein cross-link and therefore participate in a PPI. We show that this simple and intuitive filter has a dramatic effect on different types of cross-linking data ranging from individual protein complexes over medium-complexity affinity enrichments to proteome-wide cell lysates and significantly reduces the number of false-positive identifications for inter-protein links in all these types of XL-MS data.

Chemical cross-linking (XL) coupled to mass spectrometry (MS) has become a powerful approach to probe the structure of protein assemblies. Although most of the applications concerned purified complexes, latest developments focus on large-scale in vivo studies. Pushing in this direction, we developed an advanced in vivo cross-linking mass spectrometry platform to study the cellular interactome of living bacterial cells. It is based on in vivo labeling and involves a one-step enrichment by click chemistry on a solid support. Our approach shows an impressive efficiency on Neisseria meningitidis, leading to the identification of about 3300 cross-links for the LC-MS/MS analysis of a biological triplicate using a benchtop high-resolution Orbitrap mass spectrometer. Highly dynamic multiprotein complexes were successfully captured and characterized in all bacterial compartments, showing the great potential and precision of our proteome-wide approach. Our workflow paves new avenues for the large-scale and nonbiased analysis of protein-protein interactions. All raw data, databases, and processing parameters are available on ProteomeXchange via PRIDE repository (data set identifier PXD021553).

Human health is threatened by bacterial infections that are increasingly resistant to multiple drugs. A recently emerged strategy consists of disarming pathogenic bacteria by targeting and blocking their virulence factors. The type VI secretion system (T6SS) is a widespread secretion nanomachine encoded and employed by pathogenic strains to establish their virulence process during host invasion. Given the conservation of T6SS in several human bacterial pathogens, the discovery of an effective broad-spectrum T6SS virulence blocker represents an attractive target for development of antivirulence therapies. Here, we identified and validated a protein protein interaction interface, TssK-TssG, as a key factor in the assembly of the T6SS baseplate (BP) complex in the pathogen enteroaggregative Escherichia coli (EAEC). In silico and biochemical studies revealed that the determinants of the interface are broadly conserved among pathogenic species, suggesting a role for this interface as a target for T6SS inhibition. Based on the high-resolution structure of the TssKFGE wedge complex, we rationally designed a biomimetic cyclic peptide (BCP) that blocks the assembly of the EAEC BP complex and inhibits the function of T6SS in bacterial cultures. Our BCP is the first compound completely designed from prior structural knowledge with anti-T6SS activity that can be used as a model to target human pathogens. IMPORTANCE New therapeutic options are urgently needed to fight drug-resistant and life-threatening infections. In contrast to antibiotics that inhibit the growth pathways of bacteria, the antivirulence strategy is a promising approach to disarm pathogens by interfering with bacterial virulence factors without exerting evolutionary pressure. The type VI secretion system (T6SS) is used by many pathogens, including members of the antibiotic-resistant ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), to establish their virulence during the invasion of the human host. Although the T6SS is undoubtedly involved in pathogenesis, strategies targeting this virulence factor are crucially lacking. Here, we used a combination of genetics, microbiology, biochemical, biophysics, and bioinformatics approaches to rationally design a biomimetic peptide that interferes with T6SS assembly and functioning. This study represents a novel proof of concept for an antivirulence strategy which aims to interfere with the assembly of the T6SS.

Electron cryo-microscopy (cryo-EM) has emerged as a powerful method to obtain three-dimensional (3D) structures of macromolecular complexes at atomic or near-atomic resolution. However, de novo building of atomic models from near-atomic resolution (3-5 Å) cryo-EM density maps is a challenging task, in particular since poorly resolved side-chain densities hamper sequence assignment by automatic procedures at a lower resolution. Furthermore, segmentation of EM density maps into individual subunits remains a difficult problem when no three-dimensional structures of these subunits exist, or when significant conformational changes occur between the isolated and complexed form of the subunits. To tackle these issues, we have developed a graph-based method to thread most of the C- α trace of the protein backbone into the EM density map. The EM density is described as a weighted graph such that the resulting minimum spanning tree encompasses the high-density regions of the map. A pruning algorithm cleans the tree and finds the most probable positions of the C- α atoms, using side-chain density when available, as a collection of C- α trace fragments. By complementing experimental EM maps with contact predictions from sequence co-evolutionary information, we demonstrate that our approach can correctly segment EM maps into individual subunits and assign amino acids sequence to backbone traces to generate full-atom models.

Determining structures of protein complexes is crucial for understanding cellular functions. Here, we describe an integrative structure determination approach that relies on in vivo measurements of genetic interactions. We construct phenotypic profiles for point mutations crossed against gene deletions or exposed to environmental perturbations, followed by converting similarities between two profiles into an upper bound on the distance between the mutated residues. We determine the structure of the yeast histone H3-H4 complex based on ~500,000 genetic interactions of 350 mutants. We then apply the method to subunits Rpb1-Rpb2 of yeast RNA polymerase II and subunits RpoB-RpoC of bacterial RNA polymerase. The accuracy is comparable to that based on chemical cross-links; using restraints from both genetic interactions and cross-links further improves model accuracy and precision. The approach provides an efficient means to augment integrative structure determination with in vivo observations.

This repository contains the input experimental data used in a tutorial on modeling of RNA Polymerase III and the largest cluster of output models.

Mapping structure-function relationships of macromolecular assemblies plays a key role in understanding the workings of living cells. Structure determination is typically carried out in vitro and requires pure samples of the studied assemblies. Here, we describe an integrative structure determination approach based on in vivo measurements of genetic interactions of point mutants of the assembly proteins. Using the point-mutant epistatic miniarray profiling (pE-MAP) to measure growth phenotypes (Braberg et al. Cell, 2013), we construct phenotypic profiles for point mutations crossed against single gene deletions, other point mutations, and/or chemical or physical perturbations. We quantify the similarity between pE-MAP profiles, allowing us to convert the phenotypic data into upper distance bounds on pairs of mutated residues. We parametrized these distance restraints using a pE-MAP dataset of 305 point mutations in histones H3 and H4 crossed against an array of ∼1,400 gene deletion alleles. Using integrative structure modeling based on this data, we reconstructed the structure of the H3-H4 dimer with an accuracy better than 3.0 Å RMSD for Ca-atoms. To assess the applicability of the approach, we determined the structures of two additional protein assemblies from pE-Map datasets not used in the parametrization: (1) subunits Rpb1 and Rpb2 of yeast RNA polymerase using a dataset that includes 53 single point mutants and a library of ∼1,200 gene-deletion alleles, and (2) subunits RpoB and RpoC of a bacterial RNA polymerase using a dataset of 49 point mutations subject to 139 different conditions (e.g., treatments with chemicals and temperature shocks). The resulting model accuracy is comparable to that obtained with chemical cross-linking data. The approach is implemented in our open-source Integrative Modeling Platform software (http://integrativemodeling.org), thus allowing us to model structures based on other types of structural datasets as well as genetic interactions data.

Bacteria have evolved macromolecular machineries that secrete effectors and toxins to survive and thrive in diverse environments. The type VI secretion system (T6SS) is a contractile machine that is related to Myoviridae phages. It is composed of a phage tail‐like structure inserted in the bacterial cell envelope by a membrane complex (MC) comprising the TssJ, TssL and TssM proteins. We previously reported the low‐resolution negative‐stain electron microscopy structure of the enteroaggregative Escherichia coli MC and proposed a rotational 5‐fold symmetry with a TssJ:TssL:TssM stoichiometry of 2:2:2. Here, cryo‐electron tomography analyses of the T6SS MC confirm the 5‐fold symmetry in situ and identify the regions of the structure that insert into the bacterial membranes. A high‐resolution model obtained by single‐particle cryo‐electron microscopy highlights new features: five additional copies of TssJ, yielding a TssJ:TssL:TssM stoichiometry of 3:2:2, an 11‐residue loop in TssM, protruding inside the lumen of the MC and constituting a functionally important periplasmic gate, and hinge regions. Based on these data, we propose an updated model on MC structure and dynamics during T6SS assembly and function. The type VI secretion system (T6SS) is a phage tail‐related contractile machine used by bacteria to deliver a wide range of effector proteins into bacterial or eukaryotic cells. This study reveals the detailed structure of the T6SS membrane complex from enteroaggregative Escherichia coli. The detailed structure of the membrane‐embedded T6SS complex from enteroaggregative E. coli reveals novel structural features essential for its assembly.