Structural visualization of the tubulin folding pathway directed by human chaperonin TRiC/CCT.

The ATP-dependent ring-shaped chaperonin TRiC/CCT is essential for cellular proteostasis. To uncover why some eukaryotic proteins can only fold with TRiC assistance, we reconstituted the folding of ß-tubulin using human prefoldin and TRiC. We find unstructured ß-tubulin is delivered by prefoldin to the open TRiC chamber followed by ATP-dependent chamber closure. Cryo-EM resolves four near-atomic-resolution structures containing progressively folded ß-tubulin intermediates within the closed TRiC chamber, culminating in native tubulin. This substrate folding pathway appears closely guided by site-specific interactions with conserved regions in the TRiC chamber. Initial electrostatic interactions between the TRiC interior wall and both the folded tubulin N domain and its C-terminal E-hook tail establish the native substrate topology, thus enabling C-domain folding. Intrinsically disordered CCT C termini within the chamber promote subsequent folding of tubulin's core and middle domains and GTP-binding. Thus, TRiC's chamber provides chemical and topological directives that shape the folding landscape of its obligate substrates.

The Chaperonin TRiC/CCT Associates with Prefoldin through a Conserved Electrostatic Interface Essential for Cellular Proteostasis.

Maintaining proteostasis in eukaryotic protein folding involves cooperation of distinct chaperone systems. To understand how the essential ring-shaped chaperonin TRiC/CCT cooperates with the chaperone prefoldin/GIMc (PFD), we integrate cryoelectron microscopy (cryo-EM), crosslinking-mass-spectrometry and biochemical and cellular approaches to elucidate the structural and functional interplay between TRiC/CCT and PFD. We find these hetero-oligomeric chaperones associate in a defined architecture, through a conserved interface of electrostatic contacts that serves as a pivot point for a TRiC-PFD conformational cycle. PFD alternates between an open "latched" conformation and a closed "engaged" conformation that aligns the PFD-TRiC substrate binding chambers. PFD can act after TRiC bound its substrates to enhance the rate and yield of the folding reaction, suppressing non-productive reaction cycles. Disrupting the TRiC-PFD interaction in vivo is strongly deleterious, leading to accumulation of amyloid aggregates. The supra-chaperone assembly formed by PFD and TRiC is essential to prevent toxic conformations and ensure effective cellular proteostasis.

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.

High-resolution structural information of membrane-bound α-synuclein provides insight into the MoA of the anti-Parkinson drug UCB0599.

α-synuclein (αS) is an intrinsically disordered protein whose functional ambivalence and protein structural plasticity are iconic. Coordinated protein recruitment ensures proper vesicle dynamics at the synaptic cleft, while deregulated oligomerization on cellular membranes contributes to cell damage and Parkinson's disease (PD). Despite the protein's pathophysiological relevance, structural knowledge is limited. Here, we employ NMR spectroscopy and chemical cross-link mass spectrometry on 14N/15N-labeled αS mixtures to provide for the first time high-resolution structural information of the membrane-bound oligomeric state of αS and demonstrate that in this state, αS samples a surprisingly small conformational space. Interestingly, the study locates familial Parkinson's disease mutants at the interface between individual αS monomers and reveals different oligomerization processes depending on whether oligomerization occurs on the same membrane surface (cis) or between αS initially attached to different membrane particles (trans). The explanatory power of the obtained high-resolution structural model is used to help determine the mode-of-actionof UCB0599. Here, it is shown that the ligand changes the ensemble of membrane-bound structures, which helps to explain the success this compound, currently being tested in Parkinson's disease patients in a phase 2 trial, has had in animal models of PD.

Cross-linking mass spectrometric analysis of the endogenous TREX complex from Saccharomyces cerevisiae.

The conserved TREX complex has multiple functions in gene expression such as transcription elongation, 3' end processing, mRNP assembly and nuclear mRNA export as well as the maintenance of genomic stability. In Saccharomyces cerevisiae, TREX is composed of the pentameric THO complex, the DEAD-box RNA helicase Sub2, the nuclear mRNA export adaptor Yra1, and the SR-like proteins Gbp2 and Hrb1. Here, we present the structural analysis of the endogenous TREX complex of S. cerevisiae purified from its native environment. To this end, we used cross-linking mass spectrometry to gain structural information on regions of the complex that are not accessible to classical structural biology techniques. We also used negative-stain electron microscopy to investigate the organization of the cross-linked complex used for XL-MS by comparing our endogenous TREX complex with recently published structural models of recombinant THO-Sub2 complexes. According to our analysis, the endogenous yeast TREX complex preferentially assembles into a dimer.

Cross-Linking Mass Spectrometry Uncovers Interactions Between High-Density Lipoproteins and the SARS-CoV-2 Spike Glycoprotein.

High-density lipoprotein (HDL) levels are reduced in patients with coronavirus disease 2019 (COVID-19), and the extent of this reduction is associated with poor clinical outcomes. While lipoproteins are known to play a key role during the life cycle of the hepatitis C virus, their influence on coronavirus (CoV) infections is poorly understood. In this study, we utilize cross-linking mass spectrometry (XL-MS) to determine circulating protein interactors of the severe acute respiratory syndrome (SARS)-CoV-2 spike glycoprotein. XL-MS of plasma isolated from patients with COVID-19 uncovered HDL protein interaction networks, dominated by acute-phase serum amyloid proteins, whereby serum amyloid A2 was shown to bind to apolipoprotein (Apo) D. XL-MS on isolated HDL confirmed ApoD to interact with SARS-CoV-2 spike but not SARS-CoV-1 spike. Other direct interactions of SARS-CoV-2 spike upon HDL included ApoA1 and ApoC3. The interaction between ApoD and spike was further validated in cells using immunoprecipitation-MS, which uncovered a novel interaction between both ApoD and spike with membrane-associated progesterone receptor component 1. Mechanistically, XL-MS coupled with data-driven structural modeling determined that ApoD may interact within the receptor-binding domain of the spike. However, ApoD overexpression in multiple cell-based assays had no effect upon viral replication or infectivity. Thus, SARS-CoV-2 spike can bind to apolipoproteins on HDL, but these interactions do not appear to alter infectivity.

DSBSO-Based XL-MS Analysis of Breast Cancer PDX Tissues to Delineate Protein Interaction Network in Clinical Samples.

Protein-protein interactions (PPIs) are fundamental to understanding biological systems as protein complexes are the active molecular modules critical for carrying out cellular functions. Dysfunctional PPIs have been associated with various diseases including cancer. Systems-wide PPI analysis not only sheds light on pathological mechanisms, but also represents a paradigm in identifying potential therapeutic targets. In recent years, cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for defining endogenous PPIs of cellular networks. While proteome-wide studies have been performed in cell lysates, intact cells and tissues, applications of XL-MS in clinical samples have not been reported. In this study, we adopted a DSBSO-based in vivo XL-MS platform to map interaction landscapes from two breast cancer patient-derived xenograft (PDX) models. As a result, we have generated a PDX interaction network comprising 2,557 human proteins and identified interactions unique to breast cancer subtypes. Interestingly, most of the observed differences in PPIs correlated well with protein abundance changes determined by TMT-based proteome quantitation. Collectively, this work has demonstrated the feasibility of XL-MS analysis in clinical samples, and established an analytical workflow for tissue cross-linking that can be generalized for mapping PPIs from patient samples in the future to dissect disease-relevant cellular networks.

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.

RNxQuest: An Extension to the xQuest Pipeline Enabling Analysis of Protein-RNA Cross-Linking/Mass Spectrometry Data.

Cross-linking and mass spectrometry (XL-MS) workflows are increasingly popular techniques for generating low-resolution structural information about interacting biomolecules. xQuest is an established software package for analysis of protein-protein XL-MS data, supporting stable isotope-labeled cross-linking reagents. Resultant paired peaks in mass spectra aid sensitivity and specificity of data analysis. The recently developed cross-linking of isotope-labeled RNA and mass spectrometry (CLIR-MS) approach extends the XL-MS concept to protein-RNA interactions, also employing isotope-labeled cross-link (XL) species to facilitate data analysis. Data from CLIR-MS experiments are broadly compatible with core xQuest functionality, but the required analysis approach for this novel data type presents several technical challenges not optimally served by the original xQuest package. Here we introduce RNxQuest, a Python package extension for xQuest, which automates the analysis approach required for CLIR-MS data, providing bespoke, state-of-the-art processing and visualization functionality for this novel data type. Using functions included with RNxQuest, we evaluate three false discovery rate control approaches for CLIR-MS data. We demonstrate the versatility of the RNxQuest-enabled data analysis pipeline by also reanalyzing published protein-RNA XL-MS data sets that lack isotope-labeled RNA. This study demonstrates that RNxQuest provides a sensitive and specific data analysis pipeline for detection of isotope-labeled XLs in protein-RNA XL-MS experiments.

Comparative Analysis of Chemical Cross-Linking Mass Spectrometry Data Indicates That Protein STY Residues Rarely React with N-Hydroxysuccinimide Ester Cross-Linkers.

When it comes to mass spectrometry data analysis for identification of peptide pairs linked by N-hydroxysuccinimide (NHS) ester cross-linkers, search engines bifurcate in their setting of cross-linkable sites. Some restrict NHS ester cross-linkable sites to lysine (K) and protein N-terminus, referred to as K only for short, whereas others additionally include serine (S), threonine (T), and tyrosine (Y) by default. Here, by setting amino acids with chemically inert side chains such as glycine (G), valine (V), and leucine (L) as cross-linkable sites, which serves as a negative control, we show that software-identified STY-cross-links are only as reliable as GVL-cross-links. This is true across different NHS ester cross-linkers including DSS, DSSO, and DSBU, and across different search engines including MeroX, xiSearch, and pLink. Using a published data set originated from synthetic peptides, we demonstrate that STY-cross-links indeed have a high false discovery rate. Further analysis revealed that depending on the data and the search engine used to analyze the data, up to 65% of the STY-cross-links identified are actually K-K cross-links of the same peptide pairs, up to 61% are actually K-mono-links, and the rest tend to contain short peptides at high risk of false identification.

MS Annika 2.0 Identifies Cross-Linked Peptides in MS2-MS3-Based Workflows at High Sensitivity and Specificity.

Cross-linking mass spectrometry has become a powerful tool for the identification of protein-protein interactions and for gaining insight into the structures of proteins. We previously published MS Annika, a cross-linking search engine which can accurately identify cross-linked peptides in MS2 spectra from a variety of different MS-cleavable cross-linkers. In this publication, we present MS Annika 2.0, an updated version implementing a new search algorithm that, in addition to MS2 level, only supports the processing of data from MS2-MS3-based approaches for the identification of peptides from MS3 spectra, and introduces a novel scoring function for peptides identified across multiple MS stages. Detected cross-links are validated by estimating the false discovery rate (FDR) using a target-decoy approach. We evaluated the MS3-search-capabilities of MS Annika 2.0 on five different datasets covering a variety of experimental approaches and compared it to XlinkX and MaXLinker, two other cross-linking search engines. We show that MS Annika detects up to 4 times more true unique cross-links while simultaneously yielding less false positive hits and therefore a more accurate FDR estimation than the other two search engines. All mass spectrometry proteomics data along with result files have been deposited to the ProteomeXchange consortium via the PRIDE partner repository with the dataset identifier PXD041955.

Integrated Analysis of Cross-Links and Dead-End Peptides for Enhanced Interpretation of Quantitative XL-MS.

Chemical cross-linking with mass spectrometry provides low-resolution structural information on proteins in cells and tissues. Combined with quantitation, it can identify changes in the interactome between samples, for example, control and drug-treated cells or young and old mice. A difference can originate from protein conformational changes that alter the solvent-accessible distance separating the cross-linked residues. Alternatively, a difference can result from conformational changes localized to the cross-linked residues, for example, altering the solvent exposure or reactivity of those residues or post-translational modifications of the cross-linked peptides. In this manner, cross-linking is sensitive to a variety of protein conformational features. Dead-end peptides are cross-links attached only at one end to a protein with the other terminus being hydrolyzed. As a result, changes in their abundance reflect only conformational changes localized to the attached residue. For this reason, analyzing both quantified cross-links and their corresponding dead-end peptides can help elucidate the likely conformational changes giving rise to observed differences in cross-link abundance. We describe analysis of dead-end peptides in the XLinkDB public cross-link database and, with quantified mitochondrial data isolated from failing heart versus healthy mice, show how a comparison of abundance ratios between cross-links and their corresponding dead-end peptides can be leveraged to reveal possible conformational explanations.

Improved Analysis of Cross-Linking Mass Spectrometry Data with Kojak 2.0, Advanced by Integration into the Trans-Proteomic Pipeline.

Fragmentation ion spectral analysis of chemically cross-linked proteins is an established technology in the proteomics research repertoire for determining protein interactions, spatial orientation, and structure. Here we present Kojak version 2.0, a major update to the original Kojak algorithm, which was developed to identify cross-linked peptides from fragment ion spectra using a database search approach. A substantially improved algorithm with updated scoring metrics, support for cleavable cross-linkers, and identification of cross-links between 15N-labeled homomultimers are among the newest features of Kojak 2.0 presented here. Kojak 2.0 is now integrated into the Trans-Proteomic Pipeline, enabling access to dozens of additional tools within that suite. In particular, the PeptideProphet and iProphet tools for validation of cross-links improve the sensitivity and accuracy of correct cross-link identifications at user-defined thresholds. These new features improve the versatility of the algorithm, enabling its use in a wider range of experimental designs and analysis pipelines. Kojak 2.0 remains open-source and multiplatform.

Enabling Photoactivated Cross-Linking Mass Spectrometric Analysis of Protein Complexes by Novel MS-Cleavable Cross-Linkers.

Cross-linking mass spectrometry (XL-MS) is a powerful tool for studying protein-protein interactions and elucidating architectures of protein complexes. While residue-specific XL-MS studies have been very successful, accessibility of interaction regions nontargetable by specific chemistries remain difficult. Photochemistry has shown great potential in capturing those regions because of nonspecific reactivity, but low yields and high complexities of photocross-linked products have hindered their identification, limiting current studies predominantly to single proteins. Here, we describe the development of three novel MS-cleavable heterobifunctional cross-linkers, namely SDASO (Succinimidyl diazirine sulfoxide), to enable fast and accurate identification of photocross-linked peptides by MSn. The MSn-based workflow allowed SDASO XL-MS analysis of the yeast 26S proteasome, demonstrating the feasibility of photocross-linking of large protein complexes for the first time. Comparative analyses have revealed that SDASO cross-linking is robust and captures interactions complementary to residue-specific reagents, providing the foundation for future applications of photocross-linking in complex XL-MS studies.

Recent progress in mass spectrometry-based strategies for elucidating protein-protein interactions.

Protein-protein interactions are fundamental to various aspects of cell biology with many protein complexes participating in numerous fundamental biological processes such as transcription, translation and cell cycle. MS-based proteomics techniques are routinely applied for characterising the interactome, such as affinity purification coupled to mass spectrometry that has been used to selectively enrich and identify interacting partners of a bait protein. In recent years, many orthogonal MS-based techniques and approaches have surfaced including proximity-dependent labelling of neighbouring proteins, chemical cross-linking of two interacting proteins, as well as inferring PPIs from the co-behaviour of proteins such as the co-fractionating profiles and the thermal solubility profiles of proteins. This review discusses the underlying principles, advantages, limitations and experimental considerations of these emerging techniques. In addition, a brief account on how MS-based techniques are used to investigate the structural and functional properties of protein complexes, including their topology, stoichiometry, copy number and dynamics, are discussed.

OpenPepXL: An Open-Source Tool for Sensitive Identification of Cross-Linked Peptides in XL-MS.

Cross-linking MS (XL-MS) has been recognized as an effective source of information about protein structures and interactions. In contrast to regular peptide identification, XL-MS has to deal with a quadratic search space, where peptides from every protein could potentially be cross-linked to any other protein. To cope with this search space, most tools apply different heuristics for search space reduction. We introduce a new open-source XL-MS database search algorithm, OpenPepXL, which offers increased sensitivity compared with other tools. OpenPepXL searches the full search space of an XL-MS experiment without using heuristics to reduce it. Because of efficient data structures and built-in parallelization OpenPepXL achieves excellent runtimes and can also be deployed on large compute clusters and cloud services while maintaining a slim memory footprint. We compared OpenPepXL to several other commonly used tools for identification of noncleavable labeled and label-free cross-linkers on a diverse set of XL-MS experiments. In our first comparison, we used a data set from a fraction of a cell lysate with a protein database of 128 targets and 128 decoys. At 5% FDR, OpenPepXL finds from 7% to over 50% more unique residue pairs (URPs) than other tools. On data sets with available high-resolution structures for cross-link validation OpenPepXL reports from 7% to over 40% more structurally validated URPs than other tools. Additionally, we used a synthetic peptide data set that allows objective validation of cross-links without relying on structural information and found that OpenPepXL reports at least 12% more validated URPs than other tools. It has been built as part of the OpenMS suite of tools and supports Windows, macOS, and Linux operating systems. OpenPepXL also supports the MzIdentML 1.2 format for XL-MS identification results. It is freely available under a three-clause BSD license at https://openms.org/openpepxl.

MS Annika: A New Cross-Linking Search Engine.

Cross-linking mass spectrometry (XL-MS) has become a powerful technique that enables insights into protein structures and protein interactions. The development of cleavable cross-linkers has further promoted XL-MS through search space reduction, thereby allowing for proteome-wide studies. These new analysis possibilities foster the development of new cross-linkers, which not every search engine can deal with out of the box. In addition, some search engines for XL-MS data also struggle with the validation of identified cross-linked peptides, that is, false discovery rate (FDR) estimation, as FDR calculation is hampered by the fact that not only one but two peptides in a single spectrum have to be correct. We here present our new search engine, MS Annika, which can identify cross-linked peptides in MS2 spectra from a wide variety of cleavable cross-linkers. We show that MS Annika provides realistic estimates of FDRs without the need of arbitrary score cutoffs, being able to provide on average 44% more identifications at a similar or better true FDR than comparable tools. In addition, MS Annika can be used on proteome-wide studies due to fast, parallelized processing and provides a way to visualize the identified cross-links in protein 3D structures.

A Diazirine-Modified Membrane Lipid to Study Peptide/Lipid Interactions - Chances and Challenges.

Although incorporation of photo-activatable lipids into membranes potentially opens up novel avenues for investigating interactions with proteins, the question of whether diazirine-modified lipids are suitable for such studies, remains under debate. Focusing on the potential for studying lipid/peptide interactions by cross-linking mass spectrometry (XL-MS), we developed a diazirine-modified lipid (DiazPC), and examined its behaviour in membranes incorporating the model α-helical peptide LAVA20. We observed an unexpected backfolding of the diazirine-containing stearoyl chain of the lipid. This surprising behaviour challenges the potential application of DiazPC for future XL-MS studies of peptide and protein/lipid interactions. The observations made for DiazPC most likely represent a general phenomenon for any type of membrane lipids with a polar moiety incorporated into the alkyl chain. Our finding is therefore of importance for future protein/lipid interaction studies relying on modified lipid probes.

Mechanisms for restraining cAMP-dependent protein kinase revealed by subunit quantitation and cross-linking approaches.

Protein phosphorylation by cyclic AMP-dependent protein kinase (PKA) underlies key cellular processes, including sympathetic stimulation of heart cells, and potentiation of synaptic strength in neurons. Unrestrained PKA activity is pathological, and an enduring challenge is to understand how the activity of PKA catalytic subunits is directed in cells. We developed a light-activated cross-linking approach to monitor PKA subunit interactions with temporal precision in living cells. This enabled us to refute the recently proposed theory that PKA catalytic subunits remain tethered to regulatory subunits during cAMP elevation. Instead, we have identified other features of PKA signaling for reducing catalytic subunit diffusion and increasing recapture rate. Comprehensive quantitative immunoblotting of protein extracts from human embryonic kidney cells and rat organs reveals that regulatory subunits are always in large molar excess of catalytic subunits (average ∼17-fold). In the majority of organs tested, type II regulatory (RII) subunits were found to be the predominant PKA subunit. We also examined the architecture of PKA complexes containing RII subunits using cross-linking coupled to mass spectrometry. Quantitative comparison of cross-linking within a complex of RIIß and Cß, with or without the prototypical anchoring protein AKAP18α, revealed that the dimerization and docking domain of RIIß is between its second cAMP binding domains. This architecture is compatible with anchored RII subunits directing the myristylated N terminus of catalytic subunits toward the membrane for release and recapture within the plane of the membrane.

Integrative biology of native cell extracts: a new era for structural characterization of life processes.

Advances in electron microscopy have provided unprecedented access to the structural characterization of large, flexible and heterogeneous complexes. Until recently, cryo-electron microscopy (cryo-EM) has been applied to understand molecular organization in either highly purified, isolated biomolecules or in situ. An emerging field is developing, bridging the gap between the two approaches, and focuses on studying molecular organization in native cell extracts. This field has demonstrated its potential by resolving the structure of fungal fatty acid synthase (FAS) at 4.7 Å [Fourier shell correlation (FSC) = 0.143]; FAS was not only less than 50% enriched, but also retained higher-order binders, previously unknown. Although controversial in the sense that the lysis step might introduce artifacts, cell extracts preserve aspects of cellular function. In addition, cell extracts are accessible, besides cryo-EM, to modern proteomic methods, chemical cross-linking, network biology and biophysical modeling. We expect that automation in imaging cell extracts, along with the integration of molecular/cell biology approaches, will provide remarkable achievements in the study of closer-to-life biomolecular states of pronounced biotechnological and medical importance. Such steps will, eventually, bring us a step closer to the biophysical description of cellular processes in an integrative, holistic approach.