Chemical cross-linking coupled with mass spectrometry is a popular technique for deriving structural information on proteins and protein complexes. Also, cross-linking has become a powerful tool for ...stabilizing macromolecular complexes for single-particle cryo-electron microscopy. However, an effect of cross-linking on protein structure and function should not be forgotten, and surprisingly, it has not been investigated in detail so far. Here, we used kinetic studies, mass spectrometry, and NMR spectroscopy to systematically investigate an impact of cross-linking on structure and function of human carbonic anhydrase and alcohol dehydrogenase 1 from Saccharomyces cerevisiae. We found that cross-linking induces rather local structural disturbances and the overall fold is preserved even at a higher cross-linker concentration. The results establish general experimental conditions for chemical cross-linking with minimal effect on protein structure and function.
The combination of chemical cross-linking and mass spectrometry is currently a progressive technology for deriving structural information of proteins and protein complexes. In addition, chemical ...cross-linking is a powerful tool for stabilizing macromolecular complexes for single particle cryo-electron microscopy. Broad pallets of cross-linking chemistry, currently available for the majority of cross-linking experiments, still rely on the amine-reactive N-hydroxysuccinimide esters targeting mainly N-termini and lysine side chains. These cross-linkers are divided into two groups: water soluble and water insoluble; and research teams prefer one or another speculating on the benefits of their choice. However, the effect of cross-linker polarity on the outcome of cross-linking reaction has never been studied. Herein, we use both polar (bis(sulfosuccinimidyl) glutarate) and non-polar (disuccinimidyl glutarate) cross-linkers and systematically investigated the impact of cross-linker hydrophobicity on resulting distance constraints, using bovine serum albumin as a model protein.
Even though the amine reactive BS2G and DSG cross-linkers have the same length of spacer and are based on N-hydroxysuccinimidic group, our data showed that each of them formed preferentially different cross-links. We demonstrated that the choice of cross-linker can have a significant impact on the output data for structural characterization of biomolecules. Using equimolar mixtures of DSG with d6-BS2G, and BS2G with d6-DSG, we established that the polar BS2G preferentially bound to polar regions of modified molecule, whereas non-polar DSG bound to hydrophobic regions. This phenomenon established that the mixture of polar and non-polar cross-linkers acted as an efficient tool for the determination of distance constraints in proteins.
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•The BS2G and DSG cross-linkers form different cross-links.•The polarity of cross-linker had a significant impact on outcome.•The mix of polar and non-polar cross-linkers is more efficient for protein studies.
Intermediate filaments (IFs) are essential constituents of the metazoan cytoskeleton. A vast family of cytoplasmic IF proteins are capable of self-assembly from soluble tetrameric species into ...typical 10-12 nm wide filaments. The primary structure of these proteins includes the signature central 'rod' domain of ~ 300 residues which forms a dimeric α-helical coiled coil composed of three segments (coil1A, coil1B and coil2) interconnected by non-helical, flexible linkers (L1 and L12). The rod is flanked by flexible terminal head and tail domains. At present, the molecular architecture of mature IFs is only poorly known, limiting our capacity to rationalize the effect of numerous disease-related mutations found in IF proteins. Here we addressed the molecular structure of soluble vimentin tetramers which are formed by two antiparallel, staggered dimers with coil1B domains aligned (A
tetramers). By examining a series of progressive truncations, we show that the presence of the coil1A domain is essential for the tetramer formation. In addition, we employed a novel chemical cross-linking pipeline including isotope labelling to identify intra- and interdimeric cross-links within the tetramer. We conclude that the tetramer is synergistically stabilized by the interactions of the aligned coil1B domains, the interactions between coil1A and the N-terminal portion of coil2, and the electrostatic attraction between the oppositely charged head and rod domains. Our cross-linking data indicate that, starting with a straight A
tetramer, flexibility of linkers L1 and L12 enables 'backfolding' of both the coil1A and coil2 domains onto the tetrameric core formed by the coil1B domains. Through additional small-angle X-ray scattering experiments we show that the elongated A
tetramers dominate in low ionic strength solutions, while there is also a significant structural flexibility especially in the terminal domains.
Mouse activating Nkrp1 proteins are commonly described as type II transmembrane receptors with disulfide-linked homodimeric structure. Their function and the manner in which Nkrp1 proteins of mouse ...strain (C57BL/6) oligomerize are still poorly understood. To assess the oligomerization state of Nkrp1 proteins, mouse activating EGFP-Nkrp1s were expressed in mammalian lymphoid cells and their oligomerization evaluated by Förster resonance energy transfer (FRET). Alternatively, Nkrp1s oligomers were detected by Western blotting to specify the ratio between monomeric and dimeric forms. We also performed structural characterization of recombinant ectodomains of activating Nkrp1 receptors. Nkrp1 isoforms c1, c2 and f were expressed prevalently as homodimers, whereas the Nkrp1a displays larger proportion of monomers on the cell surface. Cysteine-to-serine mutants revealed the importance of all stalk cysteines for protein dimerization in living cells with a major influence of cysteine at position 74 in two Nkrp1 protein isoforms. Our results represent a new insight into the oligomerization of Nkrp1 receptors on lymphoid cells, which will help to determine their function.
The structure and biochemical properties of protease inhibitors from the thyropin family are poorly understood in parasites and pathogens. Here, we introduce a novel family member, Ir-thyropin ...(IrThy), which is secreted in the saliva of
ticks, vectors of Lyme borreliosis and tick-borne encephalitis. The IrThy molecule consists of two consecutive thyroglobulin type-1 (Tg1) domains with an unusual disulfide pattern. Recombinant IrThy was found to inhibit human host-derived cathepsin proteases with a high specificity for cathepsins V, K, and L among a wide range of screened cathepsins exhibiting diverse endo- and exopeptidase activities. Both Tg1 domains displayed inhibitory activities, but with distinct specificity profiles. We determined the spatial structure of one of the Tg1 domains by solution NMR spectroscopy and described its reactive center to elucidate the unique inhibitory specificity. Furthermore, we found that the inhibitory potency of IrThy was modulated in a complex manner by various glycosaminoglycans from host tissues. IrThy was additionally regulated by pH and proteolytic degradation. This study provides a comprehensive structure-function characterization of IrThy-the first investigated thyropin of parasite origin-and suggests its potential role in host-parasite interactions at the tick bite site.
The limited information available on the structure of complexes involving transcription factors and cognate DNA response elements represents a major obstacle in the quest to understand their ...mechanism of action at the molecular level. We implemented a concerted structural proteomics approach, which combined hydrogen-deuterium exchange (HDX), quantitative protein-protein and protein-nucleic acid cross-linking (XL), and homology analysis, to model the structure of the complex between the full-length DNA binding domain (DBD) of Forkhead box protein O4 (FOXO4) and its DNA binding element (DBE). The results confirmed that FOXO4-DBD assumes the characteristic forkhead topology shared by these types of transcription factors, but its binding mode differs significantly from those of other members of the family. The results showed that the binding interaction stabilized regions that were rather flexible and disordered in the unbound form. Surprisingly, the conformational effects were not limited only to the interface between bound components, but extended also to distal regions that may be essential to recruiting additional factors to the transcription machinery. In addition to providing valuable new insights into the binding mechanism, this project provided an excellent evaluation of the merits of structural proteomics approaches in the investigation of systems that are not directly amenable to traditional high-resolution techniques.
Fast photochemical oxidation of proteins (FPOP) footprinting is a structural mass spectrometry method that maps proteins by fast and irreversible chemical reactions. The position of oxidative ...modification reflects solvent accessibility and site reactivity and thus provides information about protein conformation, structural dynamics, and interactions. Bottom-up mass spectrometry is an established standard method to analyze FPOP samples. In the bottom-up approach, all forms of the protein are digested together by a protease of choice, which results in a mixture of peptides from various subpopulations of proteins with varying degrees of photochemical oxidation. Here, we investigate the possibility to analyze a specifically selected population of only singly oxidized proteins. This requires utilization of more specific top-down mass spectrometry approaches. The key element of any top-down experiment is the selection of a suitable method of ion isolation, excitation, and fragmentation. Here, we employ and compare collision-induced dissociation, electron-transfer dissociation, and electron-capture dissociation combined with multi-continuous accumulation of selected ions. A singly oxidized subpopulation of FPOP-labeled ubiquitin was used to optimize the method. The top-down approach in FPOP is limited to smaller proteins, but its usefulness was demonstrated by using it to visualize structural changes induced by co-factor removal from the holo/apo myoglobin system. The top-down data were compared with the literature and with the bottom-up data set obtained on the same samples. The top-down results were found to be in good agreement, which indicates that monitoring a singly oxidized FPOP ion population by the top-down approach is a functional workflow for oxidative protein footprinting.
Covalent labeling of proteins in combination with mass spectrometry has been established as a complementary technique to classical structural methods, such as X-ray, NMR, or cryogenic electron ...microscopy (Cryo-EM), used for protein structure determination. Although the current covalent labeling techniques enable the protein solvent accessible areas with sufficient spatial resolution to be monitored, there is still high demand for alternative, less complicated, and inexpensive approaches. Here, we introduce a new covalent labeling method based on fast fluoroalkylation of proteins (FFAP). FFAP uses fluoroalkyl radicals formed by reductive decomposition of Togni reagents with ascorbic acid to label proteins on a time scale of seconds. The feasibility of FFAP to effectively label proteins was demonstrated by monitoring the differential amino acids modification of native horse heart apomyoglobin/holomyoglobin and the human haptoglobin–hemoglobin complex. The obtained data confirmed the Togni reagent-mediated FFAP is an advantageous alternative method for covalent labeling in applications such as protein footprinting and epitope mapping of proteins (and their complexes) in general. Data are accessible via the ProteomeXchange server with the data set identifier PXD027310.
Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent ...labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues. This report further demonstrates the benefits of FFAP as a new method for structural characterization of therapeutic antibodies and interaction interfaces of antigen–antibody complexes. The results obtained from human trastuzumab and its complex with human epidermal growth factor receptor 2 (HER2) correlate well with previously published structural data and demonstrate the potential of FFAP in structural biology.
A combination of covalent labeling techniques and mass spectrometry (MS) is currently a progressive approach for deriving insights related to the mapping of protein surfaces or protein–ligand ...interactions. In this study, we mapped an interaction interface between the DNA binding domain (DBD) of FOXO4 protein and the DNA binding element (DAF16) using fast photochemical oxidation of proteins (FPOP). Residues involved in protein–DNA interaction were identified using the bottom-up approach. To confirm the findings and avoid a misinterpretation of the obtained data, caused by possible multiple radical oxidations leading to the protein surface alteration and oxidation of deeply buried amino acid residues, a top-down approach was employed for the first time in FPOP analysis. An isolation of singly oxidized ions enabled their gas-phase separation from multiply oxidized species followed by CID and ECD fragmentation. Application of both fragmentation techniques allowed generation of complementary fragment sets, out of which the regions shielded in the presence of DNA were deduced. The findings obtained by bottom-up and top-down approaches were highly consistent. Finally, FPOP results were compared with those of the HDX study of the FOXO4-DBD·DAF16 complex. No contradictions were found between the methods. Moreover, their combination provides complementary information related to the structure and dynamics of the protein–DNA complex. Data are available via ProteomeXchange with identifier PXD027624.
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