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1.
Nat Chem Biol ; 17(12): 1212, 2021 12.
Article in English | MEDLINE | ID: mdl-34799740
2.
Nat Chem Biol ; 17(8): 839, 2021 08.
Article in English | MEDLINE | ID: mdl-34312561
3.
Nat Chem Biol ; 17(6): 626, 2021 Jun.
Article in English | MEDLINE | ID: mdl-34035516
4.
Mol Cell Proteomics ; 20: 100070, 2021.
Article in English | MEDLINE | ID: mdl-33711480

ABSTRACT

Proteomics has exposed a plethora of posttranslational modifications, but demonstrating functional relevance requires new approaches. Top-down proteomics of intact proteins has the potential to fully characterize protein modifications in terms of amount, site(s), and the order in which they are deposited on the protein; information that so far has been elusive to extract by shotgun proteomics. Data acquisition and analysis of intact multimodified proteins have however been a major challenge, in particular for positional isomers that carry the same number of modifications at different sites. Solutions were previously proposed to extract this information from fragmentation spectra, but these have so far mainly been limited to peptides and have entailed a large degree of manual interpretation. Here, we apply high-resolution Orbitrap fusion top-down analyses in combination with bioinformatics approaches to attempt to characterize multiple modified proteins and quantify positional isomers. Automated covalent fragment ion type definition, detection of mass precision and accuracy, and extensive use of replicate spectra increase sequence coverage and drive down false fragment assignments from 10% to 1.5%. Such improved performance in fragment assignment is key to localize and quantify modifications from fragment spectra. The method is tested by investigating positional isomers of Ubiquitin mixed in known concentrations, which results in quantification of high ratios at very low standard errors of the mean (<5%), as well as with synthetic phosphorylated peptides. Application to multiphosphorylated Bora provides an estimation of the so far unknown stoichiometry of the known set of phosphosites and uncovers new sites from hyperphosphorylated Bora.


Subject(s)
Proteomics/methods , Isomerism , Mass Spectrometry , Protein Processing, Post-Translational
5.
Nat Methods ; 17(1): 28, 2020 01.
Article in English | MEDLINE | ID: mdl-31907475

Subject(s)
Proteome , Humans
6.
Mol Cell Proteomics ; 17(2): 216-232, 2018 02.
Article in English | MEDLINE | ID: mdl-29222160

ABSTRACT

Mitochondria exert an immense amount of cytophysiological functions, but the structural basis of most of these processes is still poorly understood. Here we use cross-linking mass spectrometry to probe the organization of proteins in native mouse heart mitochondria. Our approach provides the largest survey of mitochondrial protein interactions reported so far. In total, we identify 3,322 unique residue-to-residue contacts involving half of the mitochondrial proteome detected by bottom-up proteomics. The obtained mitochondrial protein interactome gives insights in the architecture and submitochondrial localization of defined protein assemblies, and reveals the mitochondrial localization of four proteins not yet included in the MitoCarta database. As one of the highlights, we show that the oxidative phosphorylation complexes I-V exist in close spatial proximity, providing direct evidence for supercomplex assembly in intact mitochondria. The specificity of these contacts is demonstrated by comparative analysis of mitochondria after high salt treatment, which disrupts the native supercomplexes and substantially changes the mitochondrial interactome.


Subject(s)
Mitochondria, Heart/metabolism , Mitochondrial Proteins/metabolism , Multiprotein Complexes/metabolism , Animals , Male , Mass Spectrometry , Mice, Inbred C57BL , Mitochondria, Heart/drug effects , Protein Interaction Maps , Proteomics , Sodium Chloride/pharmacology
7.
Nat Commun ; 8(1): 1708, 2017 11 22.
Article in English | MEDLINE | ID: mdl-29167428

ABSTRACT

Low pH-induced ligand release and receptor recycling are important steps for endocytosis. The transmembrane protein sortilin, a ß-propeller containing endocytosis receptor, internalizes a diverse set of ligands with roles in cell differentiation and homeostasis. The molecular mechanisms of pH-mediated ligand release and sortilin recycling are unresolved. Here we present crystal structures that show the sortilin luminal segment (s-sortilin) undergoes a conformational change and dimerizes at low pH. The conformational change, within all three sortilin luminal domains, provides an altered surface and the dimers sterically shield a large interface while bringing the two s-sortilin C-termini into close proximity. Biophysical and cell-based assays show that members of two different ligand families, (pro)neurotrophins and neurotensin, preferentially bind the sortilin monomer. This indicates that sortilin dimerization and conformational change discharges ligands and triggers recycling. More generally, this work may reveal a double mechanism for low pH-induced ligand release by endocytosis receptors.


Subject(s)
Adaptor Proteins, Vesicular Transport/chemistry , Adaptor Proteins, Vesicular Transport/metabolism , Endocytosis/physiology , Adaptor Proteins, Vesicular Transport/genetics , Amino Acid Substitution , Animals , Binding Sites , Crystallography, X-Ray , HEK293 Cells , Humans , Hydrogen-Ion Concentration , Ligands , Mice , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation , Protein Interaction Domains and Motifs , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Static Electricity
8.
Nat Commun ; 8: 15473, 2017 05 19.
Article in English | MEDLINE | ID: mdl-28524877

ABSTRACT

We describe optimized fragmentation schemes and data analysis strategies substantially enhancing the depth and accuracy in identifying protein cross-links using non-restricted whole proteome databases. These include a novel hybrid data acquisition strategy to sequence cross-links at both MS2 and MS3 level and a new algorithmic design XlinkX v2.0 for data analysis. As proof-of-concept we investigated proteome-wide protein interactions in E. coli and HeLa cell lysates, respectively, identifying 1,158 and 3,301 unique cross-links at ∼1% false discovery rate. These protein interaction repositories provide meaningful structural information on many endogenous macromolecular assemblies, as we showcase on several protein complexes involved in translation, protein folding and carbohydrate metabolism.


Subject(s)
DNA Fragmentation , Proteome/chemistry , Proteomics , Algorithms , Carbohydrates/chemistry , Computational Biology , Databases, Protein , Escherichia coli/metabolism , False Positive Reactions , HeLa Cells , Humans , Models, Statistical , Protein Biosynthesis , Protein Conformation , Protein Folding , Reproducibility of Results , Tandem Mass Spectrometry
9.
Science ; 355(6330): 1181-1184, 2017 03 17.
Article in English | MEDLINE | ID: mdl-28302852

ABSTRACT

Cyanobacteria have a robust circadian oscillator, known as the Kai system. Reconstituted from the purified protein components KaiC, KaiB, and KaiA, it can tick autonomously in the presence of adenosine 5'-triphosphate (ATP). The KaiC hexamers enter a natural 24-hour reaction cycle of autophosphorylation and assembly with KaiB and KaiA in numerous diverse forms. We describe the preparation of stoichiometrically well-defined assemblies of KaiCB and KaiCBA, as monitored by native mass spectrometry, allowing for a structural characterization by single-particle cryo-electron microscopy and mass spectrometry. Our data reveal details of the interactions between the Kai proteins and provide a structural basis to understand periodic assembly of the protein oscillator.


Subject(s)
Bacterial Proteins/chemistry , Circadian Clocks , Circadian Rhythm Signaling Peptides and Proteins/chemistry , Circadian Rhythm , Cyanobacteria/physiology , Adenosine Triphosphate/chemistry , Bacterial Proteins/ultrastructure , Circadian Rhythm Signaling Peptides and Proteins/ultrastructure , Cryoelectron Microscopy , Mass Spectrometry , Models, Molecular , Protein Multimerization
10.
J Biol Chem ; 292(3): 912-924, 2017 01 20.
Article in English | MEDLINE | ID: mdl-27956551

ABSTRACT

LDL receptor-related protein 1 (LRP1) is a highly modular protein and the largest known mammalian endocytic receptor. LRP1 binds and internalizes many plasma components, playing multiple crucial roles as a scavenger and signaling molecule. One major challenge to studying LRP1 has been that it is difficult to express such a large, highly glycosylated, and cysteine-rich protein, limiting structural studies to LRP1 fragments. Here, we report the first recombinant expression of the complete 61 domains of the full-length LRP1 ectodomain. This advance was achieved with a multistep cloning approach and by using DNA dilutions to improve protein yields. We investigated the binding properties of LRP1 using receptor-associated protein (RAP) as a model ligand due to its tight binding interaction. The LRP1 conformation was studied in its bound and unbound state using mass spectrometry, small-angle X-ray scattering, and negative-stain electron microscopy at neutral and acidic pH. Our findings revealed a pH-dependent release of the ligand associated with a conformational change of the receptor. In summary, this investigation of the complete LRP1 ectodomain significantly advances our understanding of this important receptor and provides the basis for further elucidating the mechanism of action of LRP1 in a whole and integrated system.


Subject(s)
Low Density Lipoprotein Receptor-Related Protein-1/chemistry , Glycosylation , HEK293 Cells , Humans , Hydrogen-Ion Concentration , Low Density Lipoprotein Receptor-Related Protein-1/genetics , Low Density Lipoprotein Receptor-Related Protein-1/metabolism , Protein Domains , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship , X-Ray Diffraction
11.
EMBO J ; 35(24): 2634-2657, 2016 12 15.
Article in English | MEDLINE | ID: mdl-27797822

ABSTRACT

The emergence of proteomics has led to major technological advances in mass spectrometry (MS). These advancements not only benefitted MS-based high-throughput proteomics but also increased the impact of mass spectrometry on the field of structural and molecular biology. Here, we review how state-of-the-art MS methods, including native MS, top-down protein sequencing, cross-linking-MS, and hydrogen-deuterium exchange-MS, nowadays enable the characterization of biomolecular structures, functions, and interactions. In particular, we focus on the role of mass spectrometry in integrated structural and molecular biology investigations of biological macromolecular complexes and cellular machineries, highlighting work on CRISPR-Cas systems and eukaryotic transcription complexes.


Subject(s)
Mass Spectrometry/methods , Molecular Biology/trends , Macromolecular Substances/chemistry
12.
ACS Cent Sci ; 2(7): 445-55, 2016 Jul 27.
Article in English | MEDLINE | ID: mdl-27504491

ABSTRACT

Multisite phosphorylation is a common pathway to regulate protein function, activity, and interaction pattern in vivo, but routine biochemical analysis is often insufficient to identify the number and order of individual phosphorylation reactions and their mechanistic impact on the protein behavior. Here, we integrate complementary mass spectrometry (MS)-based approaches to characterize a multisite phosphorylation-regulated protein system comprising Polo-like kinase 1 (Plk1) and its coactivators Aurora kinase A (Aur-A) and Bora, the interplay of which is essential for mitotic entry after DNA damage-induced cell cycle arrest. Native MS and cross-linking-MS revealed that Aur-A/Bora-mediated Plk1 activation is accompanied by the formation of Aur-A/Bora and Plk1/Bora heterodimers. We found that the Aur-A/Bora interaction is independent of the Bora phosphorylation state, whereas the Plk1/Bora interaction is dependent on extensive Bora multisite phosphorylation. Bottom-up and top-down proteomics analyses showed that Bora multisite phosphorylation proceeds via a well-ordered sequence of site-specific phosphorylation reactions, whereby we could reveal the involvement of up to 16 phosphorylated Bora residues. Ion mobility spectrometry-MS demonstrated that this multisite phosphorylation primes a substantial structural rearrangement of Bora, explaining the interdependence between extensive Bora multisite phosphorylation and Plk1/Bora complex formation. These results represent a first benchmark of our multipronged MS strategy, highlighting its potential to elucidate the mechanistic and structural implications of multisite protein phosphorylation.

13.
FEBS Lett ; 590(5): 595-604, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26849035

ABSTRACT

The ubiquitous AAA-ATPase p97 segregates ubiquitylated proteins from their molecular environment. Previous studies of the nucleotide-dependent conformational changes of p97 were inconclusive. Here, we determined its structure in the presence of ADP, AMP-PNP, or ATP-γS at 6.1-7.4 Šresolution using single particle cryo-electron microscopy. Both AAA domains, D1 and D2, assemble into essentially six-fold symmetrical rings. The pore of the D1-ring remains essentially closed under all nucleotide conditions, whereas the D2-ring shows an iris-like opening for ADP. The largest conformational changes of p97 are 'swinging motions' of the N-terminal domains, which may enable segregation of ubiquitylated substrates from their environment.


Subject(s)
Adenosine Triphosphatases/chemistry , Nuclear Proteins/chemistry , Nucleotides/pharmacology , Adenosine Triphosphatases/metabolism , Hydrolysis , Models, Molecular , Nuclear Proteins/metabolism , Protein Structure, Tertiary/drug effects
14.
Methods Mol Biol ; 1394: 109-127, 2016.
Article in English | MEDLINE | ID: mdl-26700045

ABSTRACT

During the last 15 years, the combination of chemical cross-linking and high-resolution mass spectrometry (MS) has matured into an alternative approach for analyzing 3D-structures of proteins and protein complexes. Using the distance constraints imposed by the cross-links, models of the protein or protein complex under investigation can be created. The majority of cross-linking studies are currently conducted with homobifunctional amine-reactive cross-linkers. We extend this "traditional" cross-linking/MS strategy by adding complementary photo-cross-linking data. For this, the diazirine-containing unnatural amino acids photo-leucine and photo-methionine are incorporated into the proteins and cross-link formation is induced by UV-A irradiation. The advantage of the photo-cross-linking strategy is that it is not restricted to lysine residues and that hydrophobic regions in proteins can be targeted, which is advantageous for investigating membrane proteins. We consider the strategy of combining cross-linkers with orthogonal reactivities and distances to be ideally suited for maximizing the amount of structural information that can be gained from a cross-linking experiment.


Subject(s)
Amines/chemistry , Amino Acids/chemistry , Cross-Linking Reagents/chemistry , Models, Molecular , Molecular Conformation , Multiprotein Complexes/chemistry , Proteins/chemistry , Computational Biology , HEK293 Cells , Humans , Proteome , Proteomics/methods , Software
15.
Proc Natl Acad Sci U S A ; 112(35): 11018-23, 2015 Sep 01.
Article in English | MEDLINE | ID: mdl-26283364

ABSTRACT

Toll-like receptors (TLRs) are crucial in innate recognition of invading micro-organisms and their subsequent clearance. Bacteria are not passive bystanders and have evolved complex evasion mechanisms. Staphylococcus aureus secretes a potent TLR2 antagonist, staphylococcal superantigen-like protein 3 (SSL3), which prevents receptor stimulation by pathogen-associated lipopeptides. Here, we present crystal structures of SSL3 and its complex with TLR2. The structure reveals that formation of the specific inhibitory complex is predominantly mediated by hydrophobic contacts between SSL3 and TLR2 and does not involve interaction of TLR2-glycans with the conserved Lewis(X) binding site of SSL3. In the complex, SSL3 partially covers the entrance to the lipopeptide binding pocket in TLR2, reducing its size by ∼50%. We show that this is sufficient to inhibit binding of agonist Pam2CSK4 effectively, yet allows SSL3 to bind to an already formed TLR2-Pam2CSK4 complex. The binding site of SSL3 overlaps those of TLR2 dimerization partners TLR1 and TLR6 extensively. Combined, our data reveal a robust dual mechanism in which SSL3 interferes with TLR2 activation at two stages: by binding to TLR2, it blocks ligand binding and thus inhibits activation. Second, by interacting with an already formed TLR2-lipopeptide complex, it prevents TLR heterodimerization and downstream signaling.


Subject(s)
Endotoxins/physiology , Staphylococcus aureus/physiology , Toll-Like Receptor 2/antagonists & inhibitors , Dimerization , Endotoxins/chemistry , Endotoxins/genetics , Molecular Structure , Mutagenesis , Protein Binding , Toll-Like Receptor 2/chemistry
16.
Anal Chem ; 87(8): 4152-8, 2015 Apr 21.
Article in English | MEDLINE | ID: mdl-25803405

ABSTRACT

Top-down analysis of intact proteins by mass spectrometry provides an ideal platform for comprehensive proteoform characterization, in particular, for the identification and localization of post-translational modifications (PTM) co-occurring on a protein. One of the main bottlenecks in top-down proteomics is insufficient protein sequence coverage caused by incomplete protein fragmentation. Based on previous work on peptides, increasing sequence coverage and PTM localization by combining sequential ETD and HCD fragmentation in a single fragmentation event, we hypothesized that protein sequence coverage and phospho-proteoform characterization could be equally improved by this new dual fragmentation method termed EThcD, recently been made available on the Orbitrap Fusion. Here, we systematically benchmark the performance of several (hybrid) fragmentation methods for intact protein analysis on an Orbitrap Fusion, using as a model system a 17.5 kDa N-terminal fragment of the mitotic regulator Bora. During cell division Bora becomes multiply phosphorylated by a variety of cell cycle kinases, including Aurora A and Plk1, albeit at distinctive sites. Here, we monitor the phosphorylation of Bora by Aurora A and Plk1, analyzing the generated distinctive phospho-proteoforms by top-down fragmentation. We show that EThcD and ETciD on a Fusion are feasible and capable of providing richer fragmentation spectra compared to HCD or ETD alone, increasing protein sequence coverage, and thereby facilitating phosphosite localization and the determination of kinase specific phosphorylation sites in these phospho-proteoforms. Data are available via ProteomeXchange with identifier PXD001845.


Subject(s)
Aurora Kinase B/analysis , Proteomics , Aurora Kinase B/metabolism , Mass Spectrometry , Phosphorylation
17.
Proteomics ; 15(16): 2756-65, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25641908

ABSTRACT

Antibody-drug conjugates (ADCs) are a novel class of biopharmaceuticals several of which are now being investigated in clinical studies. In ADCs, potent cytotoxic drugs are coupled via a linker to reactive residues in IgG monoclonal antibodies. Linkage to lysine residues in the IgGs, using N-hydroxysuccinimide ester based chemistry, is one of the possible options. To control drug load and specificity, proper knowledge is required about which lysine residues are most accessible and reactive. Here, we combine native MS and bottom-up proteomics to monitor the overall drug load and site-specific lysine reactivity, using N-hydroxysuccinimide-based tandem mass tags. High-resolution Orbitrap native MS enables us to monitor and quantify, due to the achieved baseline resolution, the sequential incorporation of up to 69 tandem mass tag molecules into human IgGs. Complementary, bottom-up proteomics facilitates the identification of some very reactive "hot-spot" conjugation sites. However, we also identify lysine residues that are highly resistant to chemical labeling. Our integrated approach gives insight into the conjugation properties of IgGs at both the intact protein and residue levels, providing fundamental information for controlling drug load and specificity in lysine-linked ADCs.


Subject(s)
Immunoconjugates/chemistry , Immunoglobulin G/chemistry , Lysine/chemistry , Mass Spectrometry/methods , Proteomics/methods , Amino Acid Sequence , Humans , Models, Molecular , Molecular Sequence Data
18.
PLoS One ; 9(11): e112886, 2014.
Article in English | MEDLINE | ID: mdl-25387007

ABSTRACT

We describe the detailed structural investigation of nidogen-1/laminin γ1 complexes using full-length nidogen-1 and a number of laminin γ1 variants. The interactions of nidogen-1 with laminin variants γ1 LEb2-4, γ1 LEb2-4 N836D, γ1 short arm, and γ1 short arm N836D were investigated by applying a combination of (photo-)chemical cross-linking, high-resolution mass spectrometry, and computational modeling. In addition, surface plasmon resonance and ELISA studies were used to determine kinetic constants of the nidogen-1/laminin γ1 interaction. Two complementary cross-linking strategies were pursued to analyze solution structures of laminin γ1 variants and nidogen-1. The majority of distance information was obtained with the homobifunctional amine-reactive cross-linker bis(sulfosuccinimidyl)glutarate. In a second approach, UV-induced cross-linking was performed after incorporation of the diazirine-containing unnatural amino acids photo-leucine and photo-methionine into laminin γ1 LEb2-4, laminin γ1 short arm, and nidogen-1. Our results indicate that Asn-836 within laminin γ1 LEb3 domain is not essential for complex formation. Cross-links between laminin γ1 short arm and nidogen-1 were found in all protein regions, evidencing several additional contact regions apart from the known interaction site. Computational modeling based on the cross-linking constraints indicates the existence of a conformational ensemble of both the individual proteins and the nidogen-1/laminin γ1 complex. This finding implies different modes of interaction resulting in several distinct protein-protein interfaces.


Subject(s)
Laminin/chemistry , Laminin/metabolism , Membrane Glycoproteins/metabolism , Animals , Cross-Linking Reagents/chemistry , HEK293 Cells , Humans , Kinetics , Laminin/genetics , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Mice , Models, Molecular , Protein Structure, Tertiary , Surface Plasmon Resonance , Tandem Mass Spectrometry
19.
Angew Chem Int Ed Engl ; 53(36): 9660-4, 2014 Sep 01.
Article in English | MEDLINE | ID: mdl-25044833

ABSTRACT

Protein phosphorylation is a widespread process forming the mechanistic basis of cellular signaling. Up to now, different aspects, for example, site-specificity, kinetics, role of co-factors, and structure-function relationships have been typically investigated by multiple techniques that are incompatible with one another. The approach introduced here maximizes the amount of information gained on protein (complex) phosphorylation while minimizing sample handling. Using high-resolution native mass spectrometry on intact protein (assemblies) up to 150 kDa we track the sequential incorporation of phosphate groups and map their localization by peptide LC-MS/MS. On two model systems, the protein kinase G and the interplay between Aurora kinase A and Bora, we demonstrate the simultaneous monitoring of various aspects of the phosphorylation process, namely the effect of different cofactors on PKG autophosphorylation and the interaction of AurA and Bora as both an enzyme-substrate pair and physical binding partners.


Subject(s)
Phosphorylation/physiology , Proteins/metabolism , Animals , Aurora Kinase A/metabolism , Cyclic GMP-Dependent Protein Kinases/metabolism , Humans , Kinetics , Mass Spectrometry , Substrate Specificity
20.
J Bacteriol ; 196(19): 3410-20, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25022854

ABSTRACT

Mycobacterium tuberculosis is dependent on cysteine biosynthesis, and reduced sulfur compounds such as mycothiol synthesized from cysteine serve in first-line defense mechanisms against oxidative stress imposed by macrophages. Two biosynthetic routes to l-cysteine, each with its own specific cysteine synthase (CysK1 and CysM), have been described in M. tuberculosis, but the function of a third putative sulfhydrylase in this pathogen, CysK2, has remained elusive. We present biochemical and biophysical evidence that CysK2 is an S-sulfocysteine synthase, utilizing O-phosphoserine (OPS) and thiosulfate as substrates. The enzyme uses a mechanism via a central aminoacrylate intermediate that is similar to that of other members of this pyridoxal phosphate-dependent enzyme family. The apparent second-order rate of the first half-reaction with OPS was determined as kmax/Ks = (3.97 × 10(3)) ± 619 M(-1) s(-1), which compares well to the OPS-specific mycobacterial cysteine synthase CysM with a kmax/Ks of (1.34 × 10(3)) ± 48.2. Notably, CysK2 does not utilize thiocarboxylated CysO as a sulfur donor but accepts thiosulfate and sulfide as donor substrates. The specificity constant kcat/Km for thiosulfate is 40-fold higher than for sulfide, suggesting an annotation as S-sulfocysteine synthase. Mycobacterial CysK2 thus provides a third metabolic route to cysteine, either directly using sulfide as donor or indirectly via S-sulfocysteine. Hypothetically, S-sulfocysteine could also act as a signaling molecule triggering additional responses in redox defense in the pathogen upon exposure to reactive oxygen species during dormancy.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Lyases/chemistry , Lyases/metabolism , Mycobacterium tuberculosis/enzymology , Serine/metabolism , Bacterial Proteins/genetics , Kinetics , Lyases/genetics , Mycobacterium tuberculosis/chemistry , Mycobacterium tuberculosis/genetics , Serine/analogs & derivatives , Substrate Specificity
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