Publications by authors named "Andrea Sinz"

143 Publications

An anti-HER2 nanobody binds to its antigen HER2 via two independent paratopes.

Int J Biol Macromol 2021 Apr 14;182:502-511. Epub 2021 Apr 14.

Lab of Environmental and Life Sciences, University of Nova Gorica, Vipavska cesta 13, 5000 Rožna Dolina, Nova Gorica, Slovenia. Electronic address:

High-resolution structural data of complexes between antibodies and membrane receptors still represent a demanding task. In this study, we used complementary sets of experimental data to obtain a structural model of the complex formed by the human epidermal growth factor receptor 2 (HER2) and its specific nanobody A10. First we identified by NMR the residues that bind or rearrange as a consequence of the complex formation. In parallel, the complex was cross-linked, digested and the resulting peptides were characterized by mass-spectrometry to define maximal distance restraints between HER2 and A10 amino acids in their complex. These independent datasets guided a docking process, refined by molecular dynamics simulations, to develop a model of the complex and estimate per-residue free-energy contributions. Such a model explains the experimental data and identifies a second, non-canonical paratope, located in the region opposite to the conventional nanobody paratope, formed by the hypervariable loop regions LH1 and LH3. Both paratopes contributed substantially to the overall affinity by binding to independent HER2 epitopes. Nanobody mutants with substitution of key interaction residues, as indicated by the model, possess significantly lower affinity for HER2. This is the first described case of a "natural" biparatopic nanobody, directly selected by in-vitro panning.
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http://dx.doi.org/10.1016/j.ijbiomac.2021.04.032DOI Listing
April 2021

First 3D-Structural Data of Full-Length Guanylyl Cyclase 1 in Rod-Outer-Segment Preparations of Bovine Retina by Cross-Linking/Mass Spectrometry.

J Mol Biol 2021 Mar 17;433(10):166947. Epub 2021 Mar 17.

Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, Halle/Saale, Germany; Center for Structural Mass Spectrometry, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3, Halle/Saale, Germany. Electronic address:

The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 derived from bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data of native ROS-GC1 from rod-outer-segment preparations of bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment.
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http://dx.doi.org/10.1016/j.jmb.2021.166947DOI Listing
March 2021

Probing glycation potential of dietary sugars in human blood by an integrated in vitro approach.

Food Chem 2021 Jun 27;347:128951. Epub 2020 Dec 27.

Department of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Germany; Department of Biochemistry, St. Petersburg State University, Russia. Electronic address:

Glycation is referred to as the interaction of protein amino and guanidino groups with reducing sugars and carbonyl products of their degradation. Resulting advanced glycation end-products (AGEs) contribute to pathogenesis of diabetes mellitus and neurodegenerative disorders. Upon their intestinal absorption, dietary sugars and α-dicarbonyl compounds interact with blood proteins yielding AGEs. Although the differences in glycation potential of monosaccharides are well characterized, the underlying mechanisms are poorly understood. To address this question, d-glucose, d-fructose and l-ascorbic acid were incubated with human serum albumin (HSA). The sugars and α-dicarbonyl intermediates of their degradation were analyzed in parallel to protein glycation patterns (exemplified with hydroimidazolone modifications of arginine residues and products of their hydrolysis) by bottom-up proteomics and computational chemistry. Glycation of HSA with sugars revealed 9 glyoxal- and 14 methylglyoxal-derived modification sites. Their dynamics was sugar-specific and depended on concentrations of α-dicarbonyls, their formation kinetics, and presence of stabilizing residues in close proximity to the glycation sites.
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http://dx.doi.org/10.1016/j.foodchem.2020.128951DOI Listing
June 2021

Co-purification of nitrate reductase 1 with components of the cytochrome bcc-aa oxidase supercomplex from spores of Streptomyces coelicolor A3(2).

FEBS Open Bio 2021 Mar 14;11(3):652-669. Epub 2021 Feb 14.

Institute of Microbiology, Martin-Luther University Halle-Wittenberg, Halle (Saale), Germany.

In order to reduce nitrate in vivo, the spore-specific respiratory nitrate reductase, Nar1, of Streptomyces coelicolor relies on an active cytochrome bcc-aa oxidase supercomplex (bcc-aa supercomplex). This suggests that membrane-associated Nar1, comprising NarG1, NarH1, and NarI1 subunits, might not act as a classical menaquinol oxidase but could either receive electrons from the bcc-aa supercomplex, or require the supercomplex to stabilize the reductase in the membrane to allow it to function. To address the biochemical basis for this dependence on the bcc-aa supercomplex, we purified two different Strep-tagged variants of Nar1 and enriched the native enzyme complex from spore extracts using different chromatographic and electrophoretic procedures. Polypeptides associated with the isolated Nar1 complexes were identified using mass spectrometry and included components of the bcc-aa supercomplex, along with an alternative, spore-specific cytochrome b component, QcrB3. Surprisingly, we also co-enriched the Nar3 enzyme with Nar1 from the wild-type strain of S. coelicolor. Two differentially migrating active Nar1 complexes could be identified after clear native polyacrylamide gel electrophoresis; these had masses of approximately 450 and 250 kDa. The distribution of active Nar1 in these complexes was influenced by the presence of cytochrome bd oxidase and by QcrB3; the presence of the latter shifted Nar1 into the larger complex. Together, these data suggest that several respiratory complexes can associate in the spore membrane, including Nar1, Nar3, and the bcc-aa supercomplex. Moreover, these findings provide initial support for the hypothesis that Nar1 and the bcc-aa supercomplex physically associate.
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http://dx.doi.org/10.1002/2211-5463.13086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7931247PMC
March 2021

Toward Increased Reliability, Transparency, and Accessibility in Cross-linking Mass Spectrometry.

Structure 2020 11 15;28(11):1259-1268. Epub 2020 Oct 15.

Wellcome Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3BF, Scotland, UK; Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, 13355 Berlin, Germany. Electronic address:

Cross-linking mass spectrometry (MS) has substantially matured as a method over the past 2 decades through parallel development in multiple labs, demonstrating its applicability to protein structure determination, conformation analysis, and mapping protein interactions in complex mixtures. Cross-linking MS has become a much-appreciated and routinely applied tool, especially in structural biology. Therefore, it is timely that the community commits to the development of methodological and reporting standards. This white paper builds on an open process comprising a number of events at community conferences since 2015 and identifies aspects of Cross-linking MS for which guidelines should be developed as part of a Cross-linking MS standards initiative.
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http://dx.doi.org/10.1016/j.str.2020.09.011DOI Listing
November 2020

Exploring GPCR-arrestin interfaces with genetically encoded crosslinkers.

EMBO Rep 2020 11 14;21(11):e50437. Epub 2020 Sep 14.

Institute of Biochemistry, Faculty of Life Sciences, University of Leipzig, Leipzig, Germany.

β-arrestins (βarr1 and βarr2) are ubiquitous regulators of G protein-coupled receptor (GPCR) signaling. Available data suggest that β-arrestins dock to different receptors in different ways. However, the structural characterization of GPCR-arrestin complexes is challenging and alternative approaches to study GPCR-arrestin complexes are needed. Here, starting from the finger loop as a major site for the interaction of arrestins with GPCRs, we genetically incorporate non-canonical amino acids for photo- and chemical crosslinking into βarr1 and βarr2 and explore binding topologies to GPCRs forming either stable or transient complexes with arrestins: the vasopressin receptor 2 (rhodopsin-like), the corticotropin-releasing factor receptor 1, and the parathyroid hormone receptor 1 (both secretin-like). We show that each receptor leaves a unique footprint on arrestins, whereas the two β-arrestins yield quite similar crosslinking patterns. Furthermore, we show that the method allows defining the orientation of arrestin with respect to the GPCR. Finally, we provide direct evidence for the formation of arrestin oligomers in the cell.
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http://dx.doi.org/10.15252/embr.202050437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645262PMC
November 2020

IGF2BP1 is a targetable SRC/MAPK-dependent driver of invasive growth in ovarian cancer.

RNA Biol 2021 Mar 2;18(3):391-403. Epub 2020 Sep 2.

Sect. Molecular Cell Biology, Inst. of Molecular Medicine, Charles Tanford Protein Center, Medical Faculty, Martin Luther University Halle-Wittenberg, Halle, Germany.

Epithelial-to-mesenchymal transition (EMT) is a hallmark of aggressive, mesenchymal-like high-grade serous ovarian carcinoma (HGSOC). The SRC kinase is a key driver of cancer-associated EMT promoting adherens junction (AJ) disassembly by phosphorylation-driven internalization and degradation of AJ proteins. Here, we show that the IGF2 mRNA-binding protein 1 (IGF2BP1) is up-regulated in mesenchymal-like HGSOC and promotes SRC activation by a previously unknown protein-ligand-induced, but RNA-independent mechanism. IGF2BP1-driven invasive growth of ovarian cancer cells essentially relies on the SRC-dependent disassembly of AJs. Concomitantly, IGF2BP1 enhances ERK2 expression in an RNA-binding dependent manner. Together this reveals a post-transcriptional mechanism of interconnected stimulation of SRC/ERK signalling in ovarian cancer cells. The IGF2BP1-SRC/ERK2 axis is targetable by the SRC-inhibitor saracatinib and MEK-inhibitor selumetinib. However, due to IGF2BP1-directed stimulation, only combinatorial treatment effectively overcomes the IGF2BP1-promoted invasive growth in 3D culture conditions as well as intraperitoneal mouse models. In conclusion, we reveal an unexpected role of IGF2BP1 in enhancing SRC/MAPK-driven invasive growth of ovarian cancer cells. This provides a rationale for the therapeutic benefit of combinatorial SRC/MEK inhibition in mesenchymal-like HGSOC.
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http://dx.doi.org/10.1080/15476286.2020.1812894DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7951963PMC
March 2021

Structural analysis of 70S ribosomes by cross-linking/mass spectrometry reveals conformational plasticity.

Sci Rep 2020 07 28;10(1):12618. Epub 2020 Jul 28.

Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3a, 06120, Halle/Saale, Germany.

The ribosome is not only a highly complex molecular machine that translates the genetic information into proteins, but also an exceptional specimen for testing and optimizing cross-linking/mass spectrometry (XL-MS) workflows. Due to its high abundance, ribosomal proteins are frequently identified in proteome-wide XL-MS studies of cells or cell extracts. Here, we performed in-depth cross-linking of the E. coli ribosome using the amine-reactive cross-linker disuccinimidyl diacetic urea (DSAU). We analyzed 143 E. coli ribosomal structures, mapping a total of 10,771 intramolecular distances for 126 cross-link-pairs and 3,405 intermolecular distances for 97 protein pairs. Remarkably, 44% of intermolecular cross-links covered regions that have not been resolved in any high-resolution E. coli ribosome structure and point to a plasticity of cross-linked regions. We systematically characterized all cross-links and discovered flexible regions, conformational changes, and stoichiometric variations in bound ribosomal proteins, and ultimately remodeled 2,057 residues (15,794 atoms) in total. Our working model explains more than 95% of all cross-links, resulting in an optimized E. coli ribosome structure based on the cross-linking data obtained. Our study might serve as benchmark for conducting biochemical experiments on newly modeled protein regions, guided by XL-MS. Data are available via ProteomeXchange with identifier PXD018935.
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http://dx.doi.org/10.1038/s41598-020-69313-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7387497PMC
July 2020

Mass Spectrometric Identification of SARS-CoV-2 Proteins from Gargle Solution Samples of COVID-19 Patients.

J Proteome Res 2020 11 23;19(11):4389-4392. Epub 2020 Jun 23.

Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Charles Tanford Center, 06120 Halle, Germany.

Mass spectrometry (MS) can deliver valuable diagnostic data that complement genomic information and allow us to increase our current knowledge of the COVID-19 disease caused by the SARS-CoV-2 virus. We developed a simple, MS-based method to specifically detect SARS-CoV-2 proteins from gargle solution samples of COVID-19 patients. The protocol consists of an acetone precipitation and tryptic digestion of proteins contained within the gargle solution, followed by a targeted MS analysis. Our methodology identifies unique peptides originating from SARS-CoV-2 nucleoprotein. Building on these promising initial results, faster MS protocols can now be developed as routine diagnostic tools for COVID-19 patients. Data are available via ProteomeXchange with identifier PXD019423.
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http://dx.doi.org/10.1021/acs.jproteome.0c00280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7315851PMC
November 2020

The COVID-19 MS Coalition-accelerating diagnostics, prognostics, and treatment.

Lancet 2020 06 27;395(10239):1761-1762. Epub 2020 May 27.

Michael Barber Centre for Collaborative Mass Spectrometry, Manchester Institute of Biotechnology, University of Manchester, Manchester M1 7DN, UK; Department of Chemistry, University of Manchester, Manchester M1 7DN, UK. Electronic address:

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http://dx.doi.org/10.1016/S0140-6736(20)31211-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7255197PMC
June 2020

Cross-linking/mass spectrometry at the crossroads.

Anal Bioanal Chem 2020 Sep 29;412(24):5981-5987. Epub 2020 May 29.

Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Charles Tanford Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3a, 06120, Halle (Saale), Germany.

Cross-linking/mass spectrometry (XL-MS) has come a long way. Originally, XL-MS was used to study relatively small, purified proteins. Meanwhile, it is employed to investigate protein-protein interactions on a proteome-wide level, giving snapshots of cellular processes. Currently, XL-MS is at the intersection of a multitude of workflows and the impact this technique has in addressing specific biological questions is steadily growing. This article is intended to give a bird's-eye view of the current status of XL-MS, the benefits of using MS-cleavable cross-linkers, and the challenges posed in the future development of this powerful technology. We also illustrate how XL-MS can deliver valuable structural insights into protein complexes when used in combination with other structural techniques, such as electron microscopy. Graphical abstract.
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http://dx.doi.org/10.1007/s00216-020-02700-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442761PMC
September 2020

Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin co-receptor assemblies.

Nat Commun 2020 05 8;11(1):2277. Epub 2020 May 8.

Molecular Signal Processing Department, Leibniz Institute of Plant Biochemistry (IPB), Weinberg 3, 06120, Halle (Saale), Germany.

Cullin RING-type E3 ubiquitin ligases SCF and their AUX/IAA targets perceive the phytohormone auxin. The F-box protein TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, by adopting biochemical, structural proteomics and in vivo approaches we unveil how flexibility in AUX/IAAs and regions in TIR1 affect their conformational ensemble allowing surface accessibility of degrons. We resolve TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degron's vicinity, cooperatively position AUX/IAAs on TIR1. We identify essential residues at the TIR1 N- and C-termini, which provide non-native interaction interfaces with IDRs and the folded PB1 domain of AUX/IAAs. We thereby establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation, and might provide conformational flexibility for a multiplicity of functional states.
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http://dx.doi.org/10.1038/s41467-020-16147-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7210949PMC
May 2020

The Isotope-Labeled, MS-Cleavable Cross-Linker Disuccinimidyl Dibutyric Urea for Improved Cross-Linking/Mass Spectrometry Studies.

J Am Soc Mass Spectrom 2020 Feb 21;31(2):183-189. Epub 2019 Nov 21.

Institute of Pharmacy , Martin Luther University Halle-Wittenberg, Charles Tanford Protein Center , Kurt-Mothes-Str. 3a , D-06120 Halle/Saale , Germany.

Previous studies have shown the benefits of the amine-reactive, CID-MS/MS-cleavable cross-linker disuccinimidyl dibutyric urea (DSBU) for structural proteomics studies via cross-linking/MS (XL-MS). To further facilitate the automation of XL-MS experiments, we synthesized a deuterated (D) version of the DSBU cross-linker combining the advantages of MS-cleavable linkers and isotope labeling. The rationale of conducting XL-MS with a mixture of unlabeled and stable isotope-labeled DSBU is to obtain characteristic mass differences at the MS level indicating cross-linked species. These cross-linked species can then be selected for fragmentation by collisional activation. At the MS/MS level, the characteristic 26-u doublets arising from cleavage of the central urea group in DSBU confirm the amino acid sequences of cross-linked peptides as well as the exact cross-linking sites. D-labeled DSBU was tested on three systems with increasing complexity: (i) bovine serum albumin as purified protein, (ii) ribosome as large, multimeric protein assembly, and (iii) embryo extract as complete proteome. We demonstrate the benefits arising from the use of isotope-labeled DSBU for an automated assignment of cross-linked products. Combining isotope labeling and MS cleavability in one cross-linker resulted in higher cross-link identification numbers especially for highly complex protein mixtures.
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http://dx.doi.org/10.1021/jasms.9b00008DOI Listing
February 2020

Delineating the Molecular Basis of the Calmodulin‒bMunc13-2 Interaction by Cross-Linking/Mass Spectrometry-Evidence for a Novel CaM Binding Motif in bMunc13-2.

Cells 2020 01 7;9(1). Epub 2020 Jan 7.

Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, D-06120 Halle/Saale, Germany.

Exploring the interactions between the Ca binding protein calmodulin (CaM) and its target proteins remains a challenging task. Members of the Munc13 protein family play an essential role in short-term synaptic plasticity, modulated via the interaction with CaM at the presynaptic compartment. In this study, we focus on the bMunc13-2 isoform expressed in the brain, as strong changes in synaptic transmission were observed upon its mutagenesis or deletion. The CaM‒bMunc13-2 interaction was previously characterized at the molecular level using short bMunc13-2-derived peptides only, revealing a classical 1‒5‒10 CaM binding motif. Using larger protein constructs, we have now identified for the first time a novel and unique CaM binding site in bMunc13-2 that contains an -terminal extension of a classical 1‒5‒10 CaM binding motif. We characterize this motif using a range of biochemical and biophysical methods and highlight its importance for the CaM‒bMunc13-2 interaction.
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http://dx.doi.org/10.3390/cells9010136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017353PMC
January 2020

Cross-linking/mass spectrometry to get a closer view on protein interaction networks.

Curr Opin Biotechnol 2020 06 28;63:48-53. Epub 2019 Dec 28.

Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Charles Tanford Center, Kurt-Mothes-Str. 3a, D-06120 Halle (Saale), Germany. Electronic address:

Just recently, chemical cross-linking combined with mass spectrometry (XL-MS) has emerged as valuable tool to study protein interaction networks on the system-wide level. The current challenges in XL-MS are to develop robust workflows enabling a comprehensive capture of dynamic biological assemblies in their native environment in a routine manner. In this review, we will highlight both the latest technological developments as well as selected applications of XL-MS for investigating protein networks in cells, organisms, and tissue. In addition, different bioinformatics tools for data analysis will be presented. In light of these exciting new developments, XL-MS can be expected to become one of the most versatile techniques to address important biological questions in a timely manner.
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http://dx.doi.org/10.1016/j.copbio.2019.12.009DOI Listing
June 2020

A biuret-derived, MS-cleavable cross-linking reagent for protein structural analysis: A proof-of-principle study.

J Mass Spectrom 2020 Jan 9;55(1):e4449. Epub 2019 Dec 9.

Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Charles Tanford Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3a, Halle (Saale), D-06120, Germany.

Chemical cross-linking combined with mass spectrometry (XL-MS) and computational modeling has evolved as an alternative method to derive protein 3D structures and to map protein interaction networks. Special focus has been laid recently on the development and application of cross-linkers that are cleavable by collisional activation as they yield distinct signatures in tandem mass spectra. Building on our experiences with cross-linkers containing an MS-labile urea group, we now present the biuret-based, CID-MS/MS-cleavable cross-linker imidodicarbonyl diimidazole (IDDI) and demonstrate its applicability for protein cross-linking studies based on the four model peptides angiotensin II, MRFA, substance P, and thymopentin.
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http://dx.doi.org/10.1002/jms.4449DOI Listing
January 2020

Oligomeric state, hydrodynamic properties and target recognition of human Calcium and Integrin Binding protein 2 (CIB2).

Sci Rep 2019 10 21;9(1):15058. Epub 2019 Oct 21.

Department of Neurosciences, Biomedicine and Movement Sciences, Section of Biological Chemistry, University of Verona, Verona, Italy.

Calcium- and Integrin-Binding protein 2 (CIB2) is a small and ubiquitously expressed protein with largely unknown biological function but ascertained role in hearing physiology and disease. Recent studies found that CIB2 binds Ca with moderate affinity and dimerizes under conditions mimicking the physiological ones. Here we provided new lines of evidence on CIB2 oligomeric state and the mechanism of interaction with the α7B integrin target. Based on a combination of native mass spectrometry, chemical cross-linking/mass spectrometry, analytical gel filtration, dynamic light scattering and molecular dynamics simulations we conclude that CIB2 is monomeric under all tested conditions and presents uncommon hydrodynamic properties, most likely due to the high content of hydrophobic solvent accessible surface. Surface plasmon resonance shows that the interaction with α7B occurs with relatively low affinity and is limited to the cytosolic region proximal to the membrane, being kinetically favored in the presence of physiological Mg and in the absence of Ca. Although CIB2 binds to an α7B peptide in a 1:1 stoichiometry, the formation of the complex might induce binding of another CIB2 molecule.
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http://dx.doi.org/10.1038/s41598-019-51573-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6803640PMC
October 2019

A Simple Cross-Linking/Mass Spectrometry Workflow for Studying System-wide Protein Interactions.

Anal Chem 2019 08 19;91(15):10236-10244. Epub 2019 Jul 19.

Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Charles Tanford Protein Center , Martin Luther University Halle-Wittenberg , Kurt-Mothes-Strasse 3a , D-06120 Halle (Saale) , Germany.

We present a cross-linking/mass spectrometry workflow for performing proteome-wide cross-linking analyses within 1 week. The workflow is based on the commercially available mass spectrometry-cleavable cross-linker disuccinimidyl dibutyric urea and can be employed by every lab having access to a mass spectrometer with tandem mass spectrometry capabilities. We provide an updated version 2.0 of the freeware software tool MeroX, available at www.StavroX.com , that allows us to conduct fully automated and reliable studies delivering insights into protein-protein interaction networks and protein conformations at the proteome level. We exemplify our optimized workflow for mapping protein-protein interaction networks in embryos on a system-wide level. From cross-linked embryo extracts, we detected 29931 cross-link spectrum matches corresponding to 7436 unique cross-linked residues in biological triplicate experiments at a 1% false discovery rate. Among these, 1611 interprotein cross-linking sites were identified and yielded valuable information about protein-protein interactions. The 5825 remaining intraprotein cross-links yield information about the conformational landscape of proteins in their cellular environment.
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http://dx.doi.org/10.1021/acs.analchem.9b02372DOI Listing
August 2019

First Community-Wide, Comparative Cross-Linking Mass Spectrometry Study.

Anal Chem 2019 06 22;91(11):6953-6961. Epub 2019 May 22.

Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Charles Tanford Protein Center , Martin Luther University Halle-Wittenberg , Kurt-Mothes-Strasse 3a , 06120 Halle/Saale , Germany.

The number of publications in the field of chemical cross-linking combined with mass spectrometry (XL-MS) to derive constraints for protein three-dimensional structure modeling and to probe protein-protein interactions has increased during the last years. As the technique is now becoming routine for in vitro and in vivo applications in proteomics and structural biology there is a pressing need to define protocols as well as data analysis and reporting formats. Such consensus formats should become accepted in the field and be shown to lead to reproducible results. This first, community-based harmonization study on XL-MS is based on the results of 32 groups participating worldwide. The aim of this paper is to summarize the status quo of XL-MS and to compare and evaluate existing cross-linking strategies. Our study therefore builds the framework for establishing best practice guidelines to conduct cross-linking experiments, perform data analysis, and define reporting formats with the ultimate goal of assisting scientists to generate accurate and reproducible XL-MS results.
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http://dx.doi.org/10.1021/acs.analchem.9b00658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6625963PMC
June 2019

Profiling of Seed Proteome in Pea ( L.) Lines Characterized with High and Low Responsivity to Combined Inoculation with Nodule Bacteria and Arbuscular Mycorrhizal Fungi.

Molecules 2019 Apr 23;24(8). Epub 2019 Apr 23.

Department of Biotechnology, All-Russia Research Institute for Agricultural Microbiology, 196608 St. Petersburg, Russia.

Legume crops represent the major source of food protein and contribute to human nutrition and animal feeding. An essential improvement of their productivity can be achieved by symbiosis with beneficial soil microorganisms-rhizobia (Rh) and arbuscular mycorrhizal (AM) fungi. The efficiency of these interactions depends on plant genotype. Recently, we have shown that, after simultaneous inoculation with Rh and AM, the productivity gain of pea ( L) line K-8274, characterized by high efficiency of interaction with soil microorganisms (EIBSM), was higher in comparison to a low-EIBSM line K-3358. However, the molecular mechanisms behind this effect are still uncharacterized. Therefore, here, we address the alterations in pea seed proteome, underlying the symbiosis-related productivity gain, and identify 111 differentially expressed proteins in the two lines. The high-EIBSM line K-8274 responded to inoculation by prolongation of seed maturation, manifested by up-regulation of proteins involved in cellular respiration, protein biosynthesis, and down-regulation of late-embryogenesis abundant (LEA) proteins. In contrast, the low-EIBSM line K-3358 demonstrated lower levels of the proteins, related to cell metabolism. Thus, we propose that the EIBSM trait is linked to prolongation of seed filling that needs to be taken into account in pulse crop breeding programs. The raw data have been deposited to the ProteomeXchange with identifier PXD013479.
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http://dx.doi.org/10.3390/molecules24081603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6514749PMC
April 2019

Cytochrome bcc-aa3 Oxidase Supercomplexes in the Aerobic Respiratory Chain of Streptomyces coelicolor A3(2).

J Mol Microbiol Biotechnol 2018 12;28(6):255-268. Epub 2019 Mar 12.

Institute of Microbiology, Martin-Luther University Halle-Wittenberg, Halle (Saale), Germany,

Streptomyces coelicolor A3(2), an obligately aerobic, oxidase-positive, and filamentous soil bacterium, lacks a soluble cytochrome c in its respiratory chain, having instead a membrane-associated diheme c-type cytochrome, QcrC. This necessitates complex formation to allow electron transfer between the cytochrome bcc and aa3 oxidase respiratory complexes. Combining genetic complementation studies with in-gel cytochrome oxidase activity staining, we demonstrate that the complete qcrCAB-ctaCDFE gene locus on the chromosome, encoding, respectively, the bcc and aa3 complexes, is required to manifest a cytochrome oxidase enzyme activity in both spores and mycelium of a qcr-cta deletion mutant. Blue-native-PAGE identified a cytochrome aa3 oxidase complex of approximately 270 kDa, which catalyzed oxygen-dependent diaminobenzidine oxidation without the requirement for exogenously supplied cytochrome c, indicating association with QcrC. Furthermore, higher molecular mass complexes were identified upon addition of soluble cytochrome c, suggesting the supercomplex is unstable and readily dissociates into subcomplexes lacking QcrC. Immunological and mass spectrometric analyses of active, high-molecular mass oxidase-containing complexes separated by clear-native PAGE identified key subunits of both the bcc complex and the aa3 oxidase, supporting supercomplex formation. Our data also indicate that the cytochrome b QcrB of the bcc complex is less abundant in spores compared with mycelium.
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http://dx.doi.org/10.1159/000496390DOI Listing
April 2021

Structural Investigation of Proteins and Protein Complexes by Chemical Cross-Linking/Mass Spectrometry.

Adv Exp Med Biol 2018;1105:101-121

Department of Pharmaceutical Chemistry & Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.

During the last two decades, cross-linking combined with mass spectrometry (MS) has evolved as a valuable tool to gain structural insights into proteins and protein assemblies. Structural information is obtained by introducing covalent connections between amino acids that are in spatial proximity in proteins and protein complexes. The distance constraints imposed by the cross-linking reagent provide information on the three-dimensional arrangement of the covalently connected amino acid residues and serve as basis for de-novo or homology modeling approaches. As cross-linking/MS allows investigating protein 3D-structures and protein-protein interactions not only in-vitro, but also in-vivo, it is especially appealing for studying protein systems in their native environment. In this chapter, we describe the principles of cross-linking/MS and illustrate its value for investigating protein 3D-structures and for unraveling protein interaction networks.
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http://dx.doi.org/10.1007/978-981-13-2200-6_8DOI Listing
July 2019

A cross-linking/mass spectrometry workflow based on MS-cleavable cross-linkers and the MeroX software for studying protein structures and protein-protein interactions.

Nat Protoc 2018 12;13(12):2864-2889

Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle, Germany.

Chemical cross-linking in combination with mass spectrometric analysis of the created cross-linked products is an emerging technology aimed at deriving valuable structural information from proteins and protein complexes. The goal of our protocol is to obtain distance constraints for structure determination of proteins and to investigate protein-protein interactions. We present an integrated workflow for cross-linking/mass spectrometry (MS) based on protein cross-linking with MS-cleavable reagents, followed by enzymatic digestion, enrichment of cross-linked peptides by strong cation-exchange chromatography (SCX), and LC/MS/MS analysis. To exploit the full potential of MS-cleavable cross-linkers, we developed an updated version of the freely available MeroX software for automated data analysis. The commercially available, MS-cleavable cross-linkers (DSBU and CDI) used herein possess different lengths and react with amine as well as hydroxy groups. Owing to the formation of two characteristic 26-u doublets in their MS/MS spectra, many fewer false positives are found than when using classic, non-cleavable cross-linkers. The protocol, exemplified herein for BSA and the whole Escherichia coli ribosome, is robust and widely applicable, and it allows facile identification of cross-links for deriving spatial constraints from purified proteins and protein complexes. The cross-linking/MS procedure takes 2-3 days to complete.
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http://dx.doi.org/10.1038/s41596-018-0068-8DOI Listing
December 2018

Molecular Details of Retinal Guanylyl Cyclase 1/GCAP-2 Interaction.

Front Mol Neurosci 2018 19;11:330. Epub 2018 Sep 19.

Department of Pharmaceutical Chemistry and Bioanalytics, Charles Tanford Protein Center, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle, Germany.

The rod outer segment guanylyl cyclase 1 (ROS-GC1) is an essential component of photo-transduction in the retina. In the light-induced signal cascade, membrane-bound ROS-GC1 restores cGMP levels in the dark in a calcium-dependent manner. With decreasing calcium concentration in the intracellular compartment, ROS-GC1 is activated via the intracellular site by guanylyl cyclase-activating proteins (GCAP-1/-2). Presently, the exact activation mechanism is elusive. To obtain structural insights into the ROS-GC1 regulation by GCAP-2, chemical cross-linking/mass spectrometry studies using GCAP-2 and three ROS-GC1 peptides were performed in the presence and absence of calcium. The majority of cross-links were identified with the -terminal lobe of GCAP-2 and a peptide comprising parts of ROS-GC1's catalytic domain and -terminal extension. Consistently with the cross-linking results, surface plasmon resonance and fluorescence measurements confirmed specific binding of this ROS-GC peptide to GCAP-2 with a dissociation constant in the low micromolar range. These results imply that a region of the catalytic domain of ROS-GC1 can participate in the interaction with GCAP-2. Additional binding surfaces upstream of the catalytic domain, in particular the juxtamembrane domain, can currently not be excluded.
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http://dx.doi.org/10.3389/fnmol.2018.00330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156451PMC
September 2018

Reconstitution of mammalian cleavage factor II involved in 3' processing of mRNA precursors.

RNA 2018 12 23;24(12):1721-1737. Epub 2018 Aug 23.

Institute of Biochemistry and Biotechnology, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, 06099 Halle, Germany.

Cleavage factor II (CF II) is a poorly characterized component of the multiprotein complex catalyzing 3' cleavage and polyadenylation of mammalian mRNA precursors. We have reconstituted CF II as a heterodimer of hPcf11 and hClp1. The heterodimer is active in partially reconstituted cleavage reactions, whereas hClp1 by itself is not. Pcf11 moderately stimulates the RNA 5' kinase activity of hClp1; the kinase activity is dispensable for RNA cleavage. CF II binds RNA with nanomolar affinity. Binding is mediated mostly by the two zinc fingers in the C-terminal region of hPcf11. RNA is bound without pronounced sequence-specificity, but extended G-rich sequences appear to be preferred. We discuss the possibility that CF II contributes to the recognition of cleavage/polyadenylation substrates through interaction with G-rich far-downstream sequence elements.
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http://dx.doi.org/10.1261/rna.068056.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6239180PMC
December 2018

Structure and mechanism of the two-component α-helical pore-forming toxin YaxAB.

Nat Commun 2018 05 4;9(1):1806. Epub 2018 May 4.

Center for Integrated Protein Science Munich (CIPSM), Department of Chemistry, Chair of Biochemistry, Technische Universität München, Lichtenbergstrasse 4, 85747, Garching, Germany.

Pore-forming toxins (PFT) are virulence factors that transform from soluble to membrane-bound states. The Yersinia YaxAB system represents a family of binary α-PFTs with orthologues in human, insect, and plant pathogens, with unknown structures. YaxAB was shown to be cytotoxic and likely involved in pathogenesis, though the molecular basis for its two-component lytic mechanism remains elusive. Here, we present crystal structures of YaxA and YaxB, together with a cryo-electron microscopy map of the YaxAB complex. Our structures reveal a pore predominantly composed of decamers of YaxA-YaxB heterodimers. Both subunits bear membrane-active moieties, but only YaxA is capable of binding to membranes by itself. YaxB can subsequently be recruited to membrane-associated YaxA and induced to present its lytic transmembrane helices. Pore formation can progress by further oligomerization of YaxA-YaxB dimers. Our results allow for a comparison between pore assemblies belonging to the wider ClyA-like family of α-PFTs, highlighting diverse pore architectures.
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http://dx.doi.org/10.1038/s41467-018-04139-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5935710PMC
May 2018

The First MS-Cleavable, Photo-Thiol-Reactive Cross-Linker for Protein Structural Studies.

J Am Soc Mass Spectrom 2019 Jan 20;30(1):139-148. Epub 2018 Apr 20.

Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Str. 3a, 06120, Halle (Saale), Germany.

Cleavable cross-linkers are gaining increasing importance for chemical cross-linking/mass spectrometry (MS) as they permit a reliable and automated data analysis in structural studies of proteins and protein assemblies. Here, we introduce 1,3-diallylurea (DAU) as the first CID-MS/MS-cleavable, photo-thiol-reactive cross-linker. DAU is a commercially available, inexpensive reagent that efficiently undergoes an anti-Markovnikov hydrothiolation with cysteine residues in the presence of a radical initiator upon UV-A irradiation. Radical cysteine cross-linking proceeds via an orthogonal "click reaction" and yields stable alkyl sulfide products. DAU reacts at physiological pH and cross-linking reactions with peptides, and proteins can be performed at temperatures as low as 4 °C. The central urea bond is efficiently cleaved upon collisional activation during tandem MS experiments generating characteristic product ions. This improves the reliability of automated cross-link identification. Different radical initiators have been screened for the cross-linking reaction of DAU using the thiol-containing compounds cysteine and glutathione. Our concept has also been exemplified for the biologically relevant proteins bMunc13-2 and retinal guanylyl cyclase-activating protein-2. Graphical abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-018-1952-8DOI Listing
January 2019

Free radical-initiated peptide sequencing (FRIPS)-based cross-linkers for improved peptide and protein structure analysis.

Mass Spectrom Rev 2019 03 16;38(2):187-201. Epub 2018 Apr 16.

Department of Pharmaceutical Chemistry and Bioanalytics, Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale), D-06120, Germany.

Free radical-initiated peptide sequencing (FRIPS) has recently been introduced as an analytical strategy to create peptide radical ions in a predictable and effective way by collisional activation of specifically modified peptides ions. FRIPS is based on the unimolecular dissociation of open-shell ions and yields fragments that resemble those obtained by electron capture dissociation (ECD) or electron transfer dissociation (ETD). In this review article, we describe the fundamentals of FRIPS and highlight its fruitful combination with chemical cross-linking/mass spectrometry (MS) as a highly promising option to derive complementary structural information of peptides and proteins. FRIPS does not only yield exhaustive sequence information of cross-linked peptides, but also defines the exact cross-linking sites of the connected peptides. The development of more advanced FRIPS cross-linkers that extend the FRIPS-based cross-linking/MS approach to the study of large protein assemblies and protein interaction networks can be eagerly anticipated.
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http://dx.doi.org/10.1002/mas.21568DOI Listing
March 2019

The effect of simulated microgravity on the Brassica napus seedling proteome.

Funct Plant Biol 2018 Mar;45(4):440-452

Department of Plant Physiology and Biochemistry, St. Petersburg State University, RU 199034, St. Petersburg, Russian Federation.

The magnitude and the direction of the gravitational field represent an important environmental factor affecting plant development. In this context, the absence or frequent alterations of the gravity field (i.e. microgravity conditions) might compromise extraterrestrial agriculture and hence space inhabitation by humans. To overcome the deleterious effects of microgravity, a complete understanding of the underlying changes on the macromolecular level is necessary. However, although microgravity-related changes in gene expression are well characterised on the transcriptome level, proteomic data are limited. Moreover, information about the microgravity-induced changes in the seedling proteome during seed germination and the first steps of seedling development is completely missing. One of the valuable tools to assess gravity-related issues is 3D clinorotation (i.e. rotation in two axes). Therefore, here we address the effects of microgravity, simulated by a two-axial clinostat, on the proteome of 24- and 48-h-old seedlings of oilseed rape (Brassica napus L.). The liquid chromatography-MS-based proteomic analysis and database search revealed 95 up- and 38 downregulated proteins in the tryptic digests obtained from the seedlings subjected to simulated microgravity, with 42 and 52 annotations detected as being unique for 24- and 48-h treatment times, respectively. The polypeptides involved in protein metabolism, transport and signalling were annotated as the functional groups most strongly affected by 3-D clinorotation.
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http://dx.doi.org/10.1071/FP16378DOI Listing
March 2018