Protein-protein interactions are one of the main driving forces in highly complex cellular behavior; misbehavior at this level underlies many diseases.
The molecular details of these interactions are however in many cases still unknown and are difficult to solely uncover by structural biology techniques like Cryo-EM or crystallography due to size and flexibility constraints. We develop & apply structural proteomics techniques in close collaboration with research groups focusing on Cryo-EM and crystallography to uncover these structural details.
We are passionate about delivering answers to societal relevant clinical problems and improving patients lives. Currently, we focus on thrombosis for which we aim to uncover molecular details driving excessive clot formation. Based on these details novel therapeutics will be developed with the overarching aim of reversible anti-clotting agents.
We apply 3 mass spectrometry-based techniques to answer relevant structural biology questions.
Full characterization of protein sequence and PTMs for all proteins in the sample.
Uncovering distance constraints at ~20Å resolution within and between proteins.
Uncovering contact points between proteins and DNA/RNA at low resolution.
Richard obtained his PhD in 2010 in bioinformatics in the lab of prof. dr. Ritsert Jansen at the University of Groningen on bioinformatics solutions for the then just introduced Orbitrap platforms. Afterwards, he continued his post-doctoral studies in the lab. of prof. dr. Matthias Mann at the Max Planck Institute for Biochemistry focusing on shotgun proteomics development. During this time he discovered a passion for structural proteomics.
In 2015 Richard moved to the laboratory of prof. dr. Albert Heck to start his junior group focusing on structural proteomics. During this time he has developed the mass spectrometry assays, data analysis software and chemistry solutions to efficiently extract protein structure information by mass spectrometry.
For a full list please refer to google scholar.
Pascal Albanese, Sem Tamara, Guido Saracco, Cristina Pagliano, Richard A. Scheltema
Grana are a characteristic feature of higher plants’ thylakoid membranes, consisting of stacks of appressed membranes enriched in Photosystem II (PSII) and associated light-harvesting complex II (LHCII) proteins, together forming the PSII-LHCII supercomplex. Grana stacks undergo light-dependent structural changes, mainly by reorganizing the supramolecular structure of PSII-LHCII supercomplexes. LHCII is vital for grana formation, in which also PSII-LHCII supercomplexes are involved. By combining top-down and crosslinking mass spectrometry we uncover the spatial organization of paired PSII-LHCII supercomplexes within thylakoid membranes. The resulting model highlights a basic molecular mechanism whereby plants maintain grana stacking at changing light conditions. This mechanism relies on interactions between stroma-exposed N-terminal loops of LHCII trimers and Lhcb4 subunits facing each other in adjacent membranes. The combination of light-dependent LHCII N-terminal trimming and extensive N-terminal α-acetylation likely affects interactions between pairs of PSII-LHCII supercomplexes across the stromal gap, ultimately mediating membrane folding in grana stacks.
Barbara Steigenberger, Roland J. Pieters, Albert J. R. Heck, Richard A. Scheltema
Chemical cross-linking mass spectrometry is rapidly emerging as a prominent technique to study protein structures. Structural information is obtained by covalently connecting peptides in close proximity by small reagents and identifying the resulting peptide pairs by mass spectrometry. However, substoichiometric reaction efficiencies render routine detection of cross-linked peptides problematic. Here, we present a new trifunctional cross-linking reagent, termed PhoX, which is decorated with a stable phosphonic acid handle. This makes the cross-linked peptides amenable to the well-established immobilized metal affinity chromatography (IMAC) enrichment. The handle allows for 300× enrichment efficiency and 97% specificity. We exemplify the approach on various model proteins and protein complexes, e.g., resulting in a structural model of the LRP1/RAP complex. Almost completely removing linear peptides allows PhoX, although noncleavable, to be applied to complex lysates. Focusing the database search to the 1400 most abundant proteins, we were able to identify 1156 cross-links in a single 3 h measurement.
Oleg Klykov, Carmen van der Zwaan, Albert J.R. Heck, Alexander B. Meijer, Richard A. Scheltema
Fibrinogen hexamers are major components of blood clots. After release of fibrinopeptides resulting in fibrin monomers, clot formation occurs through fibrin oligomerization followed by lateral aggregation, packing into fibrin fibers, and consequent branching. Shedding light on fibrin clots by in situ cross-linking mass spectrometry and structural modeling extends our current knowledge of the structure of fibrin with regard to receptor-binding hotspots. Further restraint-driven molecular docking reveals how fibrin oligomers laterally aggregate into clots and uncovers the molecular architecture of the clot to albumin interaction. We hypothesize this interaction is involved in the prevention of clot degradation. Mapping known mutations validates the generated structural model and, for a subset, brings their molecular mechanisms into view.
Oleg Klykov, Barbara Steigenberger, Sibel Pektaş, Domenico Fasci, Albert J. R. Heck, Richard A. Scheltema
Cross-linking mass spectrometry (XL-MS) has received considerable interest, owing to its potential to investigate protein–protein interactions (PPIs) in an unbiased fashion in complex protein mixtures. Recent developments have enabled the detection of thousands of PPIs from a single experiment. A unique strength of XL-MS, in comparison with other methods for determining PPIs, is that it provides direct spatial information for the detected interactions. This is accomplished by the use of bifunctional cross-linking molecules that link two amino acids in close proximity with a covalent bond. Upon proteolytic digestion, this results in two newly linked peptides, which are identifiable by MS. XL-MS has received the required boost to tackle more-complex samples with recent advances in cross-linking chemistry with MS-cleavable or reporter-based cross-linkers and faster, more sensitive and more versatile MS platforms. This protocol provides a detailed description of our optimized conditions for a full-proteome native protein preparation followed by cross-linking using the gas-phase cleavable cross-linking reagent disuccinimidyl sulfoxide (DSSO). Following cross-linking, we demonstrate extensive sample fractionation and substantially simplified data analysis with XlinkX in Proteome Discoverer, as well as subsequent protein structure investigations with DisVis and HADDOCK. This protocol produces data of high confidence and can be performed within ~10 d, including structural investigations.
Robert D. Fagerlund, Max E. Wilkinson, Oleg Klykov, Arjan Barendregt, F. Grant Pearce, Sebastian N. Kieper, Howard W. R. Maxwell, Angela Capolupo, Albert J. R. Heck, Kurt L. Krause, Mihnea Bostina, Richard A. Scheltema, Raymond H. J. Staals, and Peter C. Fineran
CRISPR-Cas systems provide prokaryotic adaptive immunity against invading genetic elements. For immunity, fragments of invader DNA are integrated into CRISPR arrays by Cas1 and Cas2 proteins. Type I-F systems contain a unique fusion of Cas2 to Cas3, the enzyme responsible for destruction of invading DNA. Structural, biophysical, and biochemical analyses of Cas1 and Cas2-3 from Pectobacterium atrosepticum demonstrated that they form a 400-kDa complex with a Cas14:Cas2-32 stoichiometry. Cas1–Cas2-3 binds, processes, and catalyzes the integration of DNA into CRISPR arrays independent of Cas3 activity. The arrangement of Cas3 in the complex, together with its redundant role in processing and integration, supports a scenario where Cas3 couples invader destruction with immunization—a process recently demonstrated in vivo.
Christian Benda, Judith Ebert, Richard A. Scheltema, Herbert B. Schiller, Marc Baumgärtner, Fabien Bonneau, Matthias Mann, Elena Conti
The Cmr complex is an RNA-guided endonuclease that cleaves foreign RNA targets as part of the CRISPR prokaryotic defense system. We investigated the molecular architecture of the P. furiosus Cmr complex using an integrative structural biology approach. We determined crystal structures of P. furiosus Cmr1, Cmr2, Cmr4, and Cmr6 and combined them with known structural information to interpret the cryo-EM map of the complex. To support structure determination, we obtained residue-specific interaction data using protein crosslinking and mass spectrometry. The resulting pseudoatomic model reveals how the superhelical backbone of the complex is defined by the polymerizing principles of Cmr4 and Cmr5 and how it is capped at the extremities by proteins of similar folds. The inner surface of the superhelix exposes conserved residues of Cmr4 that we show are required for target-cleavage activity. The structural and biochemical data thus identify Cmr4 as the conserved endoribonuclease of the Cmr complex.
We gratefully acknowledge funding from the following sources