In recent years, chemical crosslinking of protein complexes and the identification of crosslinked residues by mass spectrometry (XL-MS; sometimes abbreviated as CX-MS) has become an important ...technique bridging mass spectrometry (MS) and structural biology. By now, XL-MS is well established and supported by publicly available resources as a convenient and versatile part of the structural biologist's toolbox. The combination of XL-MS with cryo-electron microscopy (cryo-EM) and/or integrative modeling is particularly promising to study the topology and structure of large protein assemblies. Among the targets studied so far are proteasomes, ribosomes, polymerases, chromatin remodelers, and photosystem complexes. Here we provide an overview of recent advances in XL-MS, the current state of the field, and a cursory outlook on future challenges.
Chemical crosslinking followed by the mass spectrometric analysis of cross-linked peptides (XL-MS) identifies contact sites between residues within a single or between multiple proteins.
The application of XL-MS to many biologically relevant molecular machines has been shown, with a rapidly growing number of successful studies reported in the past 2–3 years.
Crosslinking data are useful in integrative modeling workflows by providing distance restraints on the surface of folded proteins and complexes. XL-MS has been shown to be particularly powerful in combination with 3D cryo-electron microscopy.
Chemical cross-linking in combination with LC-MS/MS (XL-MS) is an emerging technology to obtain low-resolution structural (distance) restraints of proteins and protein complexes. These restraints can ...also be used to characterize protein complexes by integrative modeling of the XL-MS data, either in combination with other types of structural information or by themselves, to establish spatial relationships of subunits in protein complexes. Here we present a protocol that has been successfully used to generate XL-MS data from a multitude of native proteins and protein complexes. It includes the experimental steps for performing the cross-linking reaction using disuccinimidyl suberate (a homobifunctional, lysine-reactive cross-linking reagent), the enrichment of cross-linked peptides by peptide size-exclusion chromatography (SEC; to remove smaller, non-cross-linked peptides), instructions for tandem MS analysis and the analysis of MS data via the open-source computational software pipeline xQuest and xProphet (available from http://proteomics.ethz.ch). Once established, this robust protocol should take ∼4 d to complete, and it is generally applicable to purified proteins and protein complexes.
Proteins constitute a key class of molecular components that perform essential biochemical reactions in living cells. Whether the aim is to extensively characterize a given protein or to perform ...high-throughput qualitative and quantitative analysis of the proteome content of a sample, liquid chromatography coupled to tandem mass spectrometry has become the technology of choice. In this review, we summarize the current state of mass spectrometry applied to bottom-up proteomics, the approach that focuses on analyzing peptides obtained from proteolytic digestion of proteins. With the recent advances in instrumentation and methodology, we show that the field is moving away from providing qualitative identification of long lists of proteins to delivering highly consistent and accurate quantification values for large numbers of proteins across large numbers of samples. We believe that this shift will have a profound impact for the field of proteomics and life science research in general.
Muscle contraction is initiated by the release of calcium (Ca(2+)) from the sarcoplasmic reticulum into the cytoplasm of myocytes through ryanodine receptors (RyRs). RyRs are homotetrameric channels ...with a molecular mass of more than 2.2 megadaltons that are regulated by several factors, including ions, small molecules and proteins. Numerous mutations in RyRs have been associated with human diseases. The molecular mechanism underlying the complex regulation of RyRs is poorly understood. Using electron cryomicroscopy, here we determine the architecture of rabbit RyR1 at a resolution of 6.1 Å. We show that the cytoplasmic moiety of RyR1 contains two large α-solenoid domains and several smaller domains, with folds suggestive of participation in protein-protein interactions. The transmembrane domain represents a chimaera of voltage-gated sodium and pH-activated ion channels. We identify the calcium-binding EF-hand domain and show that it functions as a conformational switch allosterically gating the channel.
► Mass spectrometry based methods provide structural information of protein complexes. ► Focus on chemical cross-linking mass spectrometry (CX-MS) and mass spectrometry of intact assemblies (native ...MS). ► Architecture of the 26S proteasome revealed by an integrative approach. ► Topology of the TRiC/CCT chaperonin probed by CX-MS. ► Mechanism of lipid binding to V-Type ATPases probed by native MS.
Virtually all the biological processes are controlled and catalyzed by proteins which are, in many cases, in complexes with other proteins. Therefore, understanding the architecture and structure of protein complexes is critical to understanding their biological role and function. Traditionally, high-resolution data for structural analysis of proteins or protein complexes have been generated by the powerful methods of X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. More recently, mass spectrometry (MS)-based methods have been developed that provide low-resolution structural information, which contributes to the determination of the native structure of protein complexes that have remained refractory to the high-resolution methods. Native MS and affinity purification coupled with MS (AP-MS) have been used to characterize the composition, stoichiometry and connectivity of protein complexes. Chemical cross-linking MS (CX-MS) provides protein–protein interaction data supplemented with distance information that indicates residues that are in close spatial proximity in the native protein structure. Hydrogen-deuterium exchange combined with MS has been used to map protein–protein binding sites. Here, we focus on recent developments in CX-MS and native MS and their application to challenging problems in structural biology.
Chemical cross-linking of reactive groups in native proteins and protein complexes in combination with the identification of cross-linked sites by mass spectrometry has been in use for more than a ...decade. Recent advances in instrumentation, cross-linking protocols, and analysis software have led to a renewed interest in this technique, which promises to provide important information about native protein structure and the topology of protein complexes. In this article, we discuss the critical steps of chemical cross-linking and its implications for (structural) biology: reagent design and cross-linking protocols, separation and mass spectrometric analysis of cross-linked samples, dedicated software for data analysis, and the use of cross-linking data for computational modeling. Finally, the impact of protein cross-linking on various biological disciplines is highlighted.
Mitochondrial ribosomes (mitoribosomes) are extensively modified ribosomes of bacterial descent specialized for the synthesis and insertion of membrane proteins that are critical for energy ...conversion and ATP production inside mitochondria. Mammalian mitoribosomes, which comprise 39S and 28S subunits, have diverged markedly from the bacterial ribosomes from which they are derived, rendering them unique compared to bacterial, eukaryotic cytosolic and fungal mitochondrial ribosomes. We have previously determined at 4.9 Å resolution the architecture of the porcine (Sus scrofa) 39S subunit, which is highly homologous to the human mitoribosomal large subunit. Here we present the complete atomic structure of the porcine 39S large mitoribosomal subunit determined in the context of a stalled translating mitoribosome at 3.4 Å resolution by cryo-electron microscopy and chemical crosslinking/mass spectrometry. The structure reveals the locations and the detailed folds of 50 mitoribosomal proteins, shows the highly conserved mitoribosomal peptidyl transferase active site in complex with its substrate transfer RNAs, and defines the path of the nascent chain in mammalian mitoribosomes along their idiosyncratic exit tunnel. Furthermore, we present evidence that a mitochondrial tRNA has become an integral component of the central protuberance of the 39S subunit where it architecturally substitutes for the absence of the 5S ribosomal RNA, a ubiquitous component of all cytoplasmic ribosomes.
The study of proteins and protein complexes using chemical cross-linking followed by the MS identification of the cross-linked peptides has found increasingly widespread use in recent years. Thus ...far, such analyses have used almost exclusively homobifunctional, amine-reactive cross-linking reagents. Here we report the development and application of an orthogonal cross-linking chemistry specific for carboxyl groups. Chemical cross-linking of acidic residues is achieved using homobifunctional dihydrazides as cross-linking reagents and a coupling chemistry at neutral pH that is compatible with the structural integrity of most protein complexes. In addition to cross-links formed through insertion of the dihydrazides with different spacer lengths, zero-length cross-link products are also obtained, thereby providing additional structural information. We demonstrate the application of the reaction and the MS identification of the resulting cross-linked peptides for the chaperonin TRiC/CCT and the 26S proteasome. The results indicate that the targeting of acidic residues for cross-linking provides distance restraints that are complementary and orthogonal to those obtained from lysine cross-linking, thereby expanding the yield of structural information that can be obtained from cross-linking studies and used in hybrid modeling approaches.
Characterizing cell surface receptors mediating viral infection is critical for understanding viral tropism and developing antiviral therapies. Nevertheless, due to challenges associated with ...detecting protein interactions on the cell surface, the host receptors of many human pathogens remain unknown. Here, we build a library consisting of most single transmembrane human receptors and implement a workflow for unbiased and high-sensitivity detection of receptor-ligand interactions. We apply this technology to elucidate the long-sought receptor of human cytomegalovirus (HCMV), the leading viral cause of congenital birth defects. We identify neuropilin-2 (Nrp2) as the receptor for HCMV-pentamer infection in epithelial/endothelial cells and uncover additional HCMV interactors. Using a combination of biochemistry, cell-based assays, and electron microscopy, we characterize the pentamer-Nrp2 interaction and determine the architecture of the pentamer-Nrp2 complex. This work represents an important approach to the study of host-pathogen interactions and provides a framework for understanding HCMV infection, neutralization, and the development of novel anti-HCMV therapies.
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•High-throughput screen for unbiased detection of low-affinity pathogen-host interaction•Identification of novel interactors for HCMV entry complexes•Nrp2 is the HCMV pentamer receptor in epithelial and endothelial cells•Pentamer-specific HCMV neutralizing antibodies block Nrp2 binding
A high-throughput screen for high-sensitivity detection of receptor-ligand interactions is used to identify long-sought cell-surface receptors responsible for human cytomegalovirus (HCMV) infection.
Chemical cross-links identified by mass spectrometry generate distance restraints that reveal low-resolution structural information on proteins and protein complexes. The technology to reliably ...generate such data has become mature and robust enough to shift the focus to the question of how these distance restraints can be best integrated into molecular modeling calculations. Here, we introduce three workflows for incorporating distance restraints generated by chemical cross-linking and mass spectrometry into ROSETTA protocols for comparative and de novo modeling and protein-protein docking. We demonstrate that the cross-link validation and visualization software Xwalk facilitates successful cross-link data integration. Besides the protocols we introduce XLdb, a database of chemical cross-links from 14 different publications with 506 intra-protein and 62 inter-protein cross-links, where each cross-link can be mapped on an experimental structure from the Protein Data Bank. Finally, we demonstrate on a protein-protein docking reference data set the impact of virtual cross-links on protein docking calculations and show that an inter-protein cross-link can reduce on average the RMSD of a docking prediction by 5.0 Å. The methods and results presented here provide guidelines for the effective integration of chemical cross-link data in molecular modeling calculations and should advance the structural analysis of particularly large and transient protein complexes via hybrid structural biology methods.
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