Alexander Leitner

Probing Native Protein Structures by Chemical Cross-linking, Mass Spectrometry, and Bioinformatics

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.

Structural probing of a protein phosphatase 2A network by chemical cross-linking and mass spectrometry.

Dynamic Assembly Structural characterization of protein complexes has yielded significant insight into biological function; however, most structural techniques require stable, homogenous samples. This presents a challenge in characterizing transient signaling complexes. Herzog et al. (p. 1348) used chemical cross-linking and mass spectroscopy (XL-MS) to characterize the modular and dynamic interaction network involving phosphatase 2A (PP2A), which interacts with tens of regulatory and adaptor proteins in diverse signaling pathways. They found 176 interprotein and 569 intraprotein distance restraints that delineated the topology of the network. The study establishes the importance of XL-MS in the suite of structural methods used to characterize dynamic assemblies. Spatial restraints revealed by chemical cross-linking and mass spectrometry elucidate the topology of a dynamic signaling network. The identification of proximate amino acids by chemical cross-linking and mass spectrometry (XL-MS) facilitates the structural analysis of homogeneous protein complexes. We gained distance restraints on a modular interaction network of protein complexes affinity-purified from human cells by applying an adapted XL-MS protocol. Systematic analysis of human protein phosphatase 2A (PP2A) complexes identified 176 interprotein and 570 intraprotein cross-links that link specific trimeric PP2A complexes to a multitude of adaptor proteins that control their cellular functions. Spatial restraints guided molecular modeling of the binding interface between immunoglobulin binding protein 1 (IGBP1) and PP2A and revealed the topology of TCP1 ring complex (TRiC) chaperonin interacting with the PP2A regulatory subunit 2ABG. This study establishes XL-MS as an integral part of hybrid structural biology approaches for the analysis of endogenous protein complexes.

false discovery rate estimation for cross- linked peptides identified by mass spectrometry

The mass spectrometric identification of chemically cross-linked peptides (CXMS) specifies spatial restraints of protein complexes; these values complement data obtained from common structure-determination techniques. Generic methods for determining false discovery rates of cross-linked peptide assignments are currently lacking, thus making data sets from CXMS studies inherently incomparable. Here we describe an automated target-decoy strategy and the software tool xProphet, which solve this problem for large multicomponent protein complexes.

Determination of the metabolites of nitrofuran antibiotics in animal tissue by high-performance liquid chromatography–tandem mass spectrometry

A LC-MS-MS method is presented to analyse simultaneously the metabolites of four nitrofuran antibacterial agents, furazolidone, furaltadone, nitrofurazone and nitrofurantoin in animal muscle tissue. Sample clean-up and analyte enrichment was performed by solid-phase extraction (SPE) with a polystyrene sorbent following combined hydrolysis of the protein-bound drug metabolites and derivatisation of the homogenised tissue with 2-nitrobenzaldehyde. Limits of detection of 0.5-5 ng g(-1) tissue and limits of determination of 2.5-10 ng g(-1) tissue were achieved using electrospray ionisation in positive mode. Analyte identification and quantification was performed according to EU guidelines, using multiple reaction monitoring (MRM) with one precursor ion and two product ions as identifiers. The use of an internal standard in combination with the simplified sample preparation led to a sensitive and reliable analysis method. The yield of the derivatisation reaction was between 66 and 74% and the recovery of SPE reached 92-105% for all values between 10 and 500 ng g(-1). The developed analytical protocol has been applied to contaminated tissue samples of furazolidone- and furaltadone-treated pigs and allowed unequivocal identification and quantification of the metabolites.

The Molecular Architecture of the Eukaryotic Chaperonin TRiC/CCT

TRiC/CCT is a highly conserved and essential chaperonin that uses ATP cycling to facilitate folding of approximately 10% of the eukaryotic proteome. This 1 MDa hetero-oligomeric complex consists of two stacked rings of eight paralogous subunits each. Previously proposed TRiC models differ substantially in their subunit arrangements and ring register. Here, we integrate chemical crosslinking, mass spectrometry, and combinatorial modeling to reveal the definitive subunit arrangement of TRiC. In vivo disulfide mapping provided additional validation for the crosslinking-derived arrangement as the definitive TRiC topology. This subunit arrangement allowed the refinement of a structural model using existing X-ray diffraction data. The structure described here explains all available crosslink experiments, provides a rationale for previously unexplained structural features, and reveals a surprising asymmetry of charges within the chaperonin folding chamber.

The complete structure of the 55S mammalian mitochondrial ribosome

Resolving whole mitoribosomes Mitochondria probably evolved from a prokaryotic cell living within a proto-eukaryotic cell. Consequently, mitochondria have lost much of their genomic DNA, except for a few genes that require highly divergent mitoribosomes for protein translation. Greber et al. and Amunts et al. have used cryo–electron microscopy to uncover the structure of this complex (see the Perspective by Beckmann and Hermann) and reveal an unusual mRNA binding channel. The structure supplies clues for how aminoglycoside antibiotics might inhibit mitoribosomes and how mutations in mitoribosomes might cause human disease. Science, this issue p. 303, p. 288; see also A. Amunts et al., Science, 3 April, p. 95 The protein-synthesizing machinery of mammalian mitochondria differs substantially from bacterial and eukaryotic ribosomes. [Also see Perspective by Beckmann and Hermann] Mammalian mitochondrial ribosomes (mitoribosomes) synthesize mitochondrially encoded membrane proteins that are critical for mitochondrial function. Here we present the complete atomic structure of the porcine 55S mitoribosome at 3.8 angstrom resolution by cryo–electron microscopy and chemical cross-linking/mass spectrometry. The structure of the 28S subunit in the complex was resolved at 3.6 angstrom resolution by focused alignment, which allowed building of a detailed atomic structure including all of its 15 mitoribosomal-specific proteins. The structure reveals the intersubunit contacts in the 55S mitoribosome, the molecular architecture of the mitoribosomal messenger RNA (mRNA) binding channel and its interaction with transfer RNAs, and provides insight into the highly specialized mechanism of mRNA recruitment to the 28S subunit. Furthermore, the structure contributes to a mechanistic understanding of aminoglycoside ototoxicity.

Expanding the Chemical Cross-Linking Toolbox by the Use of Multiple Proteases and Enrichment by Size Exclusion Chromatography

Chemical cross-linking in combination with mass spectrometric analysis offers the potential to obtain low-resolution structural information from proteins and protein complexes. Identification of peptides connected by a cross-link provides direct evidence for the physical interaction of amino acid side chains, information that can be used for computational modeling purposes. Despite impressive advances that were made in recent years, the number of experimentally observed cross-links still falls below the number of possible contacts of cross-linkable side chains within the span of the cross-linker. Here, we propose two complementary experimental strategies to expand cross-linking data sets. First, enrichment of cross-linked peptides by size exclusion chromatography selects cross-linked peptides based on their higher molecular mass, thereby depleting the majority of unmodified peptides present in proteolytic digests of cross-linked samples. Second, we demonstrate that the use of proteases in addition to trypsin, such as Asp-N, can additionally boost the number of observable cross-linking sites. The benefits of both SEC enrichment and multiprotease digests are demonstrated on a set of model proteins and the improved workflow is applied to the characterization of the 20S proteasome from rabbit and Schizosaccharomyces pombe.

Epoxycyclopentenone-Containing Oxidized Phospholipids Restore Endothelial Barrier Function via Cdc42 and Rac

After an acute phase of inflammation or injury, restoration of the endothelial barrier is important to regain vascular integrity and to prevent edema formation. However, little is known about mediators that control restoration of endothelial barrier function. We show here that oxidized phospholipids that accumulate at sites of inflammation and tissue damage are potent regulators of endothelial barrier function. Oxygenated epoxyisoprostane-containing phospholipids, but not fragmented oxidized phospholipids, exhibited barrier-protective effects mediated by small GTPases Cdc42 and Rac and their cytoskeletal, focal adhesion, and adherens junction effector proteins. Oxidized phospholipid-induced cytoskeletal rearrangements resulted in a unique peripheral actin rim formation, which was mimicked by coexpression of constitutively active Cdc42 and Rac, and abolished by coexpression of dominant-negative Rac and Cdc42. Thus, oxidative modification of phospholipids during inflammation leads to the formation of novel regulators that may be critically involved in restoration of vascular barrier function.

Architecture of the large subunit of the mammalian mitochondrial ribosome

Mitochondrial ribosomes synthesize a number of highly hydrophobic proteins encoded on the genome of mitochondria, the organelles in eukaryotic cells that are responsible for energy conversion by oxidative phosphorylation. The ribosomes in mammalian mitochondria have undergone massive structural changes throughout their evolution, including ribosomal RNA shortening and acquisition of mitochondria-specific ribosomal proteins. Here we present the three-dimensional structure of the 39S large subunit of the porcine mitochondrial ribosome determined by cryo-electron microscopy at 4.9 Å resolution. The structure, combined with data from chemical crosslinking and mass spectrometry experiments, reveals the unique features of the 39S subunit at near-atomic resolution and provides detailed insight into the architecture of the polypeptide exit site. This region of the mitochondrial ribosome has been considerably remodelled compared to its bacterial counterpart, providing a specialized platform for the synthesis and membrane insertion of the highly hydrophobic protein components of the respiratory chain.

Crosslinking and Mass Spectrometry : An Integrated Technology to Understand the Structure and Function of Molecular Machines

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 biologists 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.