LinX: A Software Tool for Uncommon Cross-Linking Chemistry

Kukačka, Zdeněk; Rosůlek, Michal; Jelı́nek, Jan; Slavata, Lukáš; Kavan, Daniel; Novák, Petr

doi:10.1021/acs.jproteome.0c00858

Cited by 5 publications

(3 citation statements)

References 32 publications

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“…After cross-linking, proteolytic digestion and LC–MS analysis, the data for the 14 N/ 15 N sample were processed by the LinX software 25 . In total, 40 unique XLs within the rod domain were identified (Supplementary Table 2 ).…”

Section: Resultsmentioning

confidence: 99%

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Molecular structure of soluble vimentin tetramers

Vermeire,

Lilina,

Hashim

et al. 2023

Sci Rep

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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 (A11 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 A11 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 A11 tetramers dominate in low ionic strength solutions, while there is also a significant structural flexibility especially in the terminal domains.

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Section: Resultsmentioning

confidence: 99%

“…The sample was cross-linked at a concentration of 0.3 mg/mL with DSG, DSS, DSPU and DSBU cross-linkers in 50 × molar excess. After trypsinolysis, the samples were measured by LC–MS (Agilent 1290 and 15 T solariX XR FT-ICR), obtaining high-resolution data suitable for data processing by in-house developed software LinX 25 .…”

Section: Methodsmentioning

confidence: 99%

Molecular structure of soluble vimentin tetramers

Vermeire,

Lilina,

Hashim

et al. 2023

Sci Rep

Self Cite

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“…The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms25042240/s1 References [6,24,68,[75][76][77][78][79] are cited in the Supplementary Materials.…”

Section: Supplementary Materialsmentioning

confidence: 99%

An Unusual Two-Domain Thyropin from Tick Saliva: NMR Solution Structure and Highly Selective Inhibition of Cysteine Cathepsins Modulated by Glycosaminoglycans

Matoušková,

Orsághová,

Srb

et al. 2024

IJMS

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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 Ixodes ricinus 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.

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Progress in methodologies and quality‐control strategies in protein cross‐linking mass spectrometry

et al. 2021

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Deciphering the interaction networks and structural dynamics of proteins is pivotal to better understanding their biological functions. Cross‐linking mass spectrometry (XL‐MS) is a powerful and increasingly popular technology that provides information about protein‐protein interactions and their structural constraints for individual proteins and multiprotein complexes on a proteome‐scale. In this review, we first assess the coverage and depth of the XL‐MS technique by utilizing publicly available datasets. We then delve into the progress in XL‐MS experimental and computational methodologies and examine different quality‐control strategies reported in the literature. Finally, we discuss the progress in XL‐MS applications along with the scope for future improvements.

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LinX: A Software Tool for Uncommon Cross-Linking Chemistry

Cited by 5 publications

References 32 publications

Molecular structure of soluble vimentin tetramers

Molecular structure of soluble vimentin tetramers

An Unusual Two-Domain Thyropin from Tick Saliva: NMR Solution Structure and Highly Selective Inhibition of Cysteine Cathepsins Modulated by Glycosaminoglycans

Progress in methodologies and quality‐control strategies in protein cross‐linking mass spectrometry

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