The molecular architecture of the nuclear basket.

The nuclear pore complex (NPC) is the sole mediator of nucleocytoplasmic transport. Despite great advances in understanding its conserved core architecture, the peripheral regions can exhibit considerable variation within and between species. One such structure is the cage-like nuclear basket. Despite its crucial roles in mRNA surveillance and chromatin organization, an architectural understanding has remained elusive. Using in-cell cryo-electron tomography and subtomogram analysis, we explored the NPC's structural variations and the nuclear basket across fungi (yeast; S. cerevisiae), mammals (mouse; M. musculus), and protozoa (T. gondii). Using integrative structural modeling, we computed a model of the basket in yeast and mammals that revealed how a hub of nucleoporins (Nups) in the nuclear ring binds to basket-forming Mlp/Tpr proteins: the coiled-coil domains of Mlp/Tpr form the struts of the basket, while their unstructured termini constitute the basket distal densities, which potentially serve as a docking site for mRNA preprocessing before nucleocytoplasmic transport.

A Structural Model of the Endogenous Human BAF Complex Informs Disease Mechanisms.

Mammalian SWI/SNF complexes are ATP-dependent chromatin remodeling complexes that regulate genomic architecture. Here, we present a structural model of the endogenously purified human canonical BAF complex bound to the nucleosome, generated using cryoelectron microscopy (cryo-EM), cross-linking mass spectrometry, and homology modeling. BAF complexes bilaterally engage the nucleosome H2A/H2B acidic patch regions through the SMARCB1 C-terminal α-helix and the SMARCA4/2 C-terminal SnAc/post-SnAc regions, with disease-associated mutations in either causing attenuated chromatin remodeling activities. Further, we define changes in BAF complex architecture upon nucleosome engagement and compare the structural model of endogenous BAF to those of related SWI/SNF-family complexes. Finally, we assign and experimentally interrogate cancer-associated hot-spot mutations localizing within the endogenous human BAF complex, identifying those that disrupt BAF subunit-subunit and subunit-nucleosome interfaces in the nucleosome-bound conformation. Taken together, this integrative structural approach provides important biophysical foundations for understanding the mechanisms of BAF complex function in normal and disease states.

A Pan-plant Protein Complex Map Reveals Deep Conservation and Novel Assemblies.

Plants are foundational for global ecological and economic systems, but most plant proteins remain uncharacterized. Protein interaction networks often suggest protein functions and open new avenues to characterize genes and proteins. We therefore systematically determined protein complexes from 13 plant species of scientific and agricultural importance, greatly expanding the known repertoire of stable protein complexes in plants. By using co-fractionation mass spectrometry, we recovered known complexes, confirmed complexes predicted to occur in plants, and identified previously unknown interactions conserved over 1.1 billion years of green plant evolution. Several novel complexes are involved in vernalization and pathogen defense, traits critical for agriculture. We also observed plant analogs of animal complexes with distinct molecular assemblies, including a megadalton-scale tRNA multi-synthetase complex. The resulting map offers a cross-species view of conserved, stable protein assemblies shared across plant cells and provides a mechanistic, biochemical framework for interpreting plant genetics and mutant phenotypes.

Modular Organization and Assembly of SWI/SNF Family Chromatin Remodeling Complexes.

Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are multi-subunit molecular machines that play vital roles in regulating genomic architecture and are frequently disrupted in human cancer and developmental disorders. To date, the modular organization and pathways of assembly of these chromatin regulators remain unknown, presenting a major barrier to structural and functional determination. Here, we elucidate the architecture and assembly pathway across three classes of mSWI/SNF complexes-canonical BRG1/BRM-associated factor (BAF), polybromo-associated BAF (PBAF), and newly defined ncBAF complexes-and define the requirement of each subunit for complex formation and stability. Using affinity purification of endogenous complexes from mammalian and Drosophila cells coupled with cross-linking mass spectrometry (CX-MS) and mutagenesis, we uncover three distinct and evolutionarily conserved modules, their organization, and the temporal incorporation of these modules into each complete mSWI/SNF complex class. Finally, we map human disease-associated mutations within subunits and modules, defining specific topological regions that are affected upon subunit perturbation.

Structural basis of a transcription pre-initiation complex on a divergent promoter.

Most eukaryotic promoter regions are divergently transcribed. As the RNA polymerase II pre-initiation complex (PIC) is intrinsically asymmetric and responsible for transcription in a single direction, it is unknown how divergent transcription arises. Here, the Saccharomyces cerevisiae Mediator complexed with a PIC (Med-PIC) was assembled on a divergent promoter and analyzed by cryoelectron microscopy. The structure reveals two distinct Med-PICs forming a dimer through the Mediator tail module, induced by a homodimeric activator protein localized near the dimerization interface. The tail dimer is associated with ∼80-bp upstream DNA, such that two flanking core promoter regions are positioned and oriented in a suitable form for PIC assembly in opposite directions. Also, cryoelectron tomography visualized the progress of the PIC assembly on the two core promoter regions, providing direct evidence for the role of the Med-PIC dimer in divergent transcription.

Nuclear mRNPs are compact particles packaged with a network of proteins promoting RNA-RNA interactions.

Messenger RNAs (mRNAs) are at the center of the central dogma of molecular biology. In eukaryotic cells, these long ribonucleic acid polymers do not exist as naked transcripts; rather, they associate with mRNA-binding proteins to form messenger ribonucleoprotein (mRNP) complexes. Recently, global proteomic and transcriptomic studies have provided comprehensive inventories of mRNP components. However, knowledge of the molecular features of distinct mRNP populations has remained elusive. We purified endogenous nuclear mRNPs from Saccharomyces cerevisiae by harnessing the mRNP biogenesis factors THO and Sub2 in biochemical procedures optimized to preserve the integrity of these transient ribonucleoprotein assemblies. We found that these mRNPs are compact particles that contain multiple copies of Yra1, an essential protein with RNA-annealing properties. To investigate their molecular and architectural organization, we used a combination of proteomics, RNA sequencing, cryo-electron microscopy, cross-linking mass spectrometry, structural models, and biochemical assays. Our findings indicate that yeast nuclear mRNPs are packaged around an intricate network of interconnected proteins capable of promoting RNA-RNA interactions via their positively charged intrinsically disordered regions. The evolutionary conservation of the major mRNA-packaging factor (yeast Yra1 and Aly/REF in metazoans) points toward a general paradigm governing nuclear mRNP packaging.

Yeast TLDc domain proteins regulate assembly state and subcellular localization of the V-ATPase.

Yeast vacuoles perform crucial cellular functions as acidic degradative organelles, storage compartments, and signaling hubs. These functions are mediated by important protein complexes, including the vacuolar-type H+-ATPase (V-ATPase), responsible for organelle acidification. To gain a more detailed understanding of vacuole function, we performed cross-linking mass spectrometry on isolated vacuoles, detecting many known as well as novel protein-protein interactions. Among these, we identified the uncharacterized TLDc-domain-containing protein Rtc5 as a novel interactor of the V-ATPase. We further analyzed the influence of Rtc5 and of Oxr1, the only other yeast TLDc-domain-containing protein, on V-ATPase function. We find that both Rtc5 and Oxr1 promote the disassembly of the vacuolar V-ATPase in vivo, counteracting the role of the RAVE complex, a V-ATPase assembly chaperone. Furthermore, Oxr1 is necessary for the retention of a Golgi-specific subunit of the V-ATPase in this compartment. Collectively, our results shed light on the in vivo roles of yeast TLDc-domain proteins as regulators of the V-ATPase, highlighting the multifaceted regulation of this crucial protein complex.

An integrated structural model of the DNA damage-responsive H3K4me3 binding WDR76:SPIN1 complex with the nucleosome.

Serial capture affinity purification (SCAP) is a powerful method to isolate a specific protein complex. When combined with cross-linking mass spectrometry and computational approaches, one can build an integrated structural model of the isolated complex. Here, we applied SCAP to dissect a subpopulation of WDR76 in complex with SPIN1, a histone reader that recognizes trimethylated histone H3 lysine4 (H3K4me3). In contrast to a previous SCAP analysis of the SPIN1:SPINDOC complex, histones and the H3K4me3 mark were enriched with the WDR76:SPIN1 complex. Next, interaction network analysis of copurifying proteins and microscopy analysis revealed a potential role of the WDR76:SPIN1 complex in the DNA damage response. Since we detected 149 pairs of cross-links between WDR76, SPIN1, and histones, we then built an integrated structural model of the complex where SPIN1 recognized the H3K4me3 epigenetic mark while interacting with WDR76. Finally, we used the powerful Bayesian Integrative Modeling approach as implemented in the Integrative Modeling Platform to build a model of WDR76 and SPIN1 bound to the nucleosome.

Cross-linking mass spectrometry discovers, evaluates, and corroborates structures and protein-protein interactions in the human cell.

Significant recent advances in structural biology, particularly in the field of cryoelectron microscopy, have dramatically expanded our ability to create structural models of proteins and protein complexes. However, many proteins remain refractory to these approaches because of their low abundance, low stability, or-in the case of complexes-simply not having yet been analyzed. Here, we demonstrate the power of using cross-linking mass spectrometry (XL-MS) for the high-throughput experimental assessment of the structures of proteins and protein complexes. This included those produced by high-resolution but in vitro experimental data, as well as in silico predictions based on amino acid sequence alone. We present the largest XL-MS dataset to date, describing 28,910 unique residue pairs captured across 4,084 unique human proteins and 2,110 unique protein-protein interactions. We show that models of proteins and their complexes predicted by AlphaFold2, and inspired and corroborated by the XL-MS data, offer opportunities to deeply mine the structural proteome and interactome and reveal mechanisms underlying protein structure and function.

Higher-Order Structural Organization of the Mitochondrial Proteome Charted by In Situ Cross-Linking Mass Spectrometry.

Mitochondria are densely packed with proteins, of which most are involved physically or more transiently in protein-protein interactions (PPIs). Mitochondria host among others all enzymes of the Krebs cycle and the oxidative phosphorylation pathway and are foremost associated with cellular bioenergetics. However, mitochondria are also important contributors to apoptotic cell death and contain their own genome indicating that they play additionally an eminent role in processes beyond bioenergetics. Despite intense efforts in identifying and characterizing mitochondrial protein complexes by structural biology and proteomics techniques, many PPIs have remained elusive. Several of these (membrane embedded) PPIs are less stable in vitro hampering their characterization by most contemporary methods in structural biology. Particularly in these cases, cross-linking mass spectrometry (XL-MS) has proven valuable for the in-depth characterization of mitochondrial protein complexes in situ. Here, we highlight experimental strategies for the analysis of proteome-wide PPIs in mitochondria using XL-MS. We showcase the ability of in situ XL-MS as a tool to map suborganelle interactions and topologies and aid in refining structural models of protein complexes. We describe some of the most recent technological advances in XL-MS that may benefit the in situ characterization of PPIs even further, especially when combined with electron microscopy and structural modeling.

Intensity and retention time prediction improves the rescoring of protein-nucleic acid cross-links.

In protein-RNA cross-linking mass spectrometry, UV or chemical cross-linking introduces stable bonds between amino acids and nucleic acids in protein-RNA complexes that are then analyzed and detected in mass spectra. This analytical tool delivers valuable information about RNA-protein interactions and RNA docking sites in proteins, both in vitro and in vivo. The identification of cross-linked peptides with oligonucleotides of different length leads to a combinatorial increase in search space. We demonstrate that the peptide retention time prediction tasks can be transferred to the task of cross-linked peptide retention time prediction using a simple amino acid composition encoding, yielding improved identification rates when the prediction error is included in rescoring. For the more challenging task of including fragment intensity prediction of cross-linked peptides in the rescoring, we obtain, on average, a similar improvement. Further improvement in the encoding and fine-tuning of retention time and intensity prediction models might lead to further gains, and merit further research.

RawVegetable 2.0: Refining XL-MS Data Acquisition through Enhanced Quality Control.

We present RawVegetable 2.0, a software tailored for assessing mass spectrometry data quality and fine-tuned for cross-linking mass spectrometry (XL-MS) applications. Building upon the capabilities of its predecessor, RawVegetable 2.0 introduces four main modules, each providing distinct and new functionalities: 1) Pair Finder, which identifies ion doublets characteristic of cleavable cross-linking experiments; 2) Diagnostic Peak Finder, which locates potential reporter ions associated with a specific cross-linker; 3) Precursor Signal Ratio, which computes the ratio between precursor intensity and the total signal in an MS/MS scan; and 4) Xrea, which evaluates spectral quality by analyzing the heterogeneity of peak intensities within a spectrum. These modules collectively streamline the process of optimizing mass spectrometry data acquisition for both Proteomics and XL-MS experiments. RawVegetable 2.0, along with a comprehensive tutorial is freely accessible for academic use at: http://patternlabforproteomics.org/rawvegetable2.

PhoXplex: Combining Phospho-enrichable Cross-Linking with Isobaric Labeling for Quantitative Proteome-Wide Mapping of Protein Interfaces.

Integrating cross-linking mass spectrometry (XL-MS) into structural biology workflows provides valuable information about the spatial arrangement of amino acid stretches, which can guide elucidation of protein assembly architecture. Additionally, the combination of XL-MS with peptide quantitation techniques is a powerful approach to delineate protein interface dynamics across diverse conditions. While XL-MS is increasingly effective with isolated proteins or small complexes, its application to whole-cell samples poses technical challenges related to analysis depth and throughput. The use of enrichable cross-linkers has greatly improved the detectability of protein interfaces in a proteome-wide scale, facilitating global protein-protein interaction mapping. Therefore, bringing together enrichable cross-linking and multiplexed peptide quantification is an appealing approach to enable comparative characterization of structural attributes of proteins and protein interactions. Here, we combined phospho-enrichable cross-linking with TMT labeling to develop a streamline workflow (PhoXplex) for the detection of differential structural features across a panel of cell lines in a global scale. We achieved deep coverage with quantification of over 9000 cross-links and long loop-links in total including potentially novel interactions. Overlaying AlphaFold predictions and disorder protein annotations enables exploration of the quantitative cross-linking data set, to reveal possible associations between mutations and protein structures. Lastly, we discuss current shortcomings and perspectives for deep whole-cell profiling of protein interfaces at large-scale.

Leveraging Cross-Linking Mass Spectrometry for Modeling Antibody-Antigen Complexes.

Elucidating antibody-antigen complexes at the atomic level is of utmost interest for understanding immune responses and designing better therapies. Cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for mapping protein-protein interactions, suggesting valuable structural insights. However, the use of XL-MS studies to enable epitope/paratope mapping of antibody-antigen complexes is still limited up to now. XL-MS data can be used to drive integrative modeling of antibody-antigen complexes, where cross-links information serves as distance restraints for the precise determination of binding interfaces. In this approach, XL-MS data are employed to identify connections between binding interfaces of the antibody and the antigen, thus informing molecular modeling. Current literature provides minimal input about the impact of XL-MS data on the integrative modeling of antibody-antigen complexes. Here, we applied XL-MS to retrieve information about binding interfaces of three antibody-antigen complexes. We leveraged XL-MS data to perform integrative modeling using HADDOCK (active-passive residues and distance restraints strategies) and AlphaLink2. We then compared these three approaches with initial predictions of investigated antibody-antigen complexes by AlphaFold Multimer. This work emphasizes the importance of cross-linking data in resolving conformational dynamics of antibody-antigen complexes, ultimately enhancing the design of better protein therapeutics and vaccines.

Notch-Jagged signaling complex defined by an interaction mosaic.

The Notch signaling system links cellular fate to that of its neighbors, driving proliferation, apoptosis, and cell differentiation in metazoans, whereas dysfunction leads to debilitating developmental disorders and cancers. Other than a five-by-five domain complex, it is unclear how the 40 extracellular domains of the Notch1 receptor collectively engage the 19 domains of its canonical ligand, Jagged1, to activate Notch1 signaling. Here, using cross-linking mass spectrometry (XL-MS), biophysical, and structural techniques on the full extracellular complex and targeted sites, we identify five distinct regions, two on Notch1 and three on Jagged1, that form an interaction network. The Notch1 membrane-proximal regulatory region individually binds to the established Notch1 epidermal growth factor (EGF) 8-EGF13 and Jagged1 C2-EGF3 activation sites as well as to two additional Jagged1 regions, EGF8-EGF11 and cysteine-rich domain. XL-MS and quantitative interaction experiments show that the three Notch1-binding sites on Jagged1 also engage intramolecularly. These interactions, together with Notch1 and Jagged1 ectodomain dimensions and flexibility, determined by small-angle X-ray scattering, support the formation of nonlinear architectures. Combined, the data suggest that critical Notch1 and Jagged1 regions are not distal but engage directly to control Notch1 signaling, thereby redefining the Notch1-Jagged1 activation mechanism and indicating routes for therapeutic applications.

In-cell structures of conserved supramolecular protein arrays at the mitochondria-cytoskeleton interface in mammalian sperm.

Mitochondria-cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information defining mitochondria-cytoskeleton interfaces in any cell type. Here, we use cryofocused ion beam milling-enabled cryoelectron tomography to image mammalian sperm, where mitochondria wrap around the flagellar cytoskeleton. We find that mitochondria are tethered to their neighbors through intermitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage-dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types in which mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.

Molecular characterization of a complex of apoptosis-inducing factor 1 with cytochrome c oxidase of the mitochondrial respiratory chain.

Combining mass spectrometry-based chemical cross-linking and complexome profiling, we analyzed the interactome of heart mitochondria. We focused on complexes of oxidative phosphorylation and found that dimeric apoptosis-inducing factor 1 (AIFM1) forms a defined complex with ∼10% of monomeric cytochrome c oxidase (COX) but hardly interacts with respiratory chain supercomplexes. Multiple AIFM1 intercross-links engaging six different COX subunits provided structural restraints to build a detailed atomic model of the COX-AIFM12 complex (PDBDEV_00000092). An application of two complementary proteomic approaches thus provided unexpected insight into the macromolecular organization of the mitochondrial complexome. Our structural model excludes direct electron transfer between AIFM1 and COX. Notably, however, the binding site of cytochrome c remains accessible, allowing formation of a ternary complex. The discovery of the previously overlooked COX-AIFM12 complex and clues provided by the structural model hint at potential roles of AIFM1 in oxidative phosphorylation biogenesis and in programmed cell death.

Protein interaction landscapes revealed by advanced in vivo cross-linking-mass spectrometry.

Defining protein-protein interactions (PPIs) in their native environment is crucial to understanding protein structure and function. Cross-linking-mass spectrometry (XL-MS) has proven effective in capturing PPIs in living cells; however, the proteome coverage remains limited. Here, we have developed a robust in vivo XL-MS platform to facilitate in-depth PPI mapping by integrating a multifunctional MS-cleavable cross-linker with sample preparation strategies and high-resolution MS. The advancement of click chemistry-based enrichment significantly enhanced the detection of cross-linked peptides for proteome-wide analyses. This platform enabled the identification of 13,904 unique lysine-lysine linkages from in vivo cross-linked HEK 293 cells, permitting construction of the largest in vivo PPI network to date, comprising 6,439 interactions among 2,484 proteins. These results allowed us to generate a highly detailed yet panoramic portrait of human interactomes associated with diverse cellular pathways. The strategy presented here signifies a technological advancement for in vivo PPI mapping at the systems level and can be generalized for charting protein interaction landscapes in any organisms.

ECL 3.0: a sensitive peptide identification tool for cross-linking mass spectrometry data analysis.

BACKGROUND: Cross-linking mass spectrometry (XL-MS) is a powerful technique for detecting protein-protein interactions (PPIs) and modeling protein structures in a high-throughput manner. In XL-MS experiments, proteins are cross-linked by a chemical reagent (namely cross-linker), fragmented, and then fed into a tandem mass spectrum (MS/MS). Cross-linkers are either cleavable or non-cleavable, and each type requires distinct data analysis tools. However, both types of cross-linkers suffer from imbalanced fragmentation efficiency, resulting in a large number of unidentifiable spectra that hinder the discovery of PPIs and protein conformations. To address this challenge, researchers have sought to improve the sensitivity of XL-MS through invention of novel cross-linking reagents, optimization of sample preparation protocols, and development of data analysis algorithms. One promising approach to developing new data analysis methods is to apply a protein feedback mechanism in the analysis. It has significantly improved the sensitivity of analysis methods in the cleavable cross-linking data. The application of the protein feedback mechanism to the analysis of non-cleavable cross-linking data is expected to have an even greater impact because the majority of XL-MS experiments currently employs non-cleavable cross-linkers. RESULTS: In this study, we applied the protein feedback mechanism to the analysis of both non-cleavable and cleavable cross-linking data and observed a substantial improvement in cross-link spectrum matches (CSMs) compared to conventional methods. Furthermore, we developed a new software program, ECL 3.0, that integrates two algorithms and includes a user-friendly graphical interface to facilitate wider applications of this new program. CONCLUSIONS: ECL 3.0 source code is available at https://github.com/yuweichuan/ECL-PF.git . A quick tutorial is available at https://youtu.be/PpZgbi8V2xI .

Exhaustive Cross-Linking Search with Protein Feedback.

Improving the sensitivity of protein-protein interaction detection and protein structure probing is a principal challenge in cross-linking mass spectrometry (XL-MS) data analysis. In this paper, we propose an exhaustive cross-linking search method with protein feedback (ECL-PF) for cleavable XL-MS data analysis. ECL-PF adopts an optimized α/ß mass detection scheme and establishes protein-peptide association during the identification of cross-linked peptides. Existing major scoring functions can all benefit from the ECL-PF workflow to a great extent. In comparisons using synthetic data sets and hybrid simulated data sets, ECL-PF achieved 3-fold higher sensitivity over standard techniques. In experiments using real data sets, it also identified 65.6% more cross-link spectrum matches and 48.7% more unique cross-links.