SecScan: a general approach for mapping disulfide bonds in synthetic and recombinant peptides and proteins

Denisov, Stepan S.; Ippel, Hans; Mans, Ben J.; Dijkgraaf, Ingrid; Hackeng, Tilman M.

doi:10.1039/c8cc08777f

Cited by 14 publications

(26 citation statements)

References 23 publications

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“…Our X-ray crystallographic and homology modeling studies indicate that EVA3 and its homologs have a topology referred to as a cystine knot (23) and belong to the class of proteins known as knottins. The disulfide-bonding pattern (but not the cystine knot topology) of EVA3 was recently predicted using selenocysteine scanning (37), and our X-ray crystallographic analyses are consistent with this previous report. Knottins form the largest inhibitor cystine-knot subgroup and are characterized by the knot-forming disulfide bond between Cys 3 and Cys 6 traversing a macrocycle created by disulfide bonds between Cys 1 and Cys 4 and between Cys 2 and Cys 5 .…”

Section: Discussionsupporting

confidence: 92%

A knottin scaffold directs the CXC-chemokine–binding specificity of tick evasins

Lee

Déruaz²,

Lynch

et al. 2019

Journal of Biological Chemistry

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Tick evasins (EVAs) bind either CC- or CXC-chemokines by a poorly understood promiscuous or “one-to-many” mechanism to neutralize inflammation. Because EVAs potently inhibit inflammation in many preclinical models, highlighting their potential as biological therapeutics for inflammatory diseases, we sought to further unravel the CXC-chemokine–EVA interactions. Using yeast surface display, we identified and characterized 27 novel CXC-chemokine–binding evasins homologous to EVA3 and defined two functional classes. The first, which included EVA3, exclusively bound ELR + CXC-chemokines, whereas the second class bound both ELR + and ELR − CXC-chemokines, in several cases including C X C-motif chemokine ligand 10 (CXCL10) but, surprisingly, not CXCL8. The X-ray crystal structure of EVA3 at a resolution of 1.79 Å revealed a single antiparallel β-sheet with six conserved cysteine residues forming a disulfide-bonded knottin scaffold that creates a contiguous solvent-accessible surface. Swapping analyses identified distinct knottin scaffold segments necessary for different CXC-chemokine–binding activities, implying that differential ligand positioning, at least in part, plays a role in promiscuous binding. Swapping segments also transferred chemokine-binding activity, resulting in a hybrid EVA with dual CXCL10- and CXCL8-binding activities. The solvent-accessible surfaces of the knottin scaffold segments have distinctive shape and charge, which we suggest drives chemokine-binding specificity. These studies provide structural and mechanistic insight into how CXC-chemokine–binding tick EVAs achieve class specificity but also engage in promiscuous binding.

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Section: Discussionsupporting

confidence: 92%

A knottin scaffold directs the CXC-chemokine–binding specificity of tick evasins

Lee

Déruaz²,

Lynch

et al. 2019

Journal of Biological Chemistry

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“…To study the relative importance of the flexible unstructured N and C termini of met-Evasin-3 on binding CXCL8, the Leu 1 –Asn 16 and Leu 57 –Arg 66 regions were removed, leading to a truncated Evasin-3 variant, designated as tEv3 17–56 (31). Truncation resulted in largely diminished broadening 1 H NMR resonances, gaining a higher number of NOE peaks necessary for calculation of a better resolved structure.…”

Section: Resultsmentioning

confidence: 99%

Tick saliva protein Evasin-3 modulates chemotaxis by disrupting CXCL8 interactions with glycosaminoglycans and CXCR2

Denisov

Ippel

Heinzmann

et al. 2019

Journal of Biological Chemistry

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Chemokines are a group of chemotaxis proteins that regulate cell trafficking and play important roles in immune responses and inflammation. Ticks are blood-sucking parasites that secrete numerous immune-modulatory agents in their saliva to evade host immune responses. Evasin-3 is a small salivary protein that belongs to a class of chemokine-binding proteins isolated from the brown dog tick, Rhipicephalus sanguineus . Evasin-3 has been shown to have a high affinity for chemokines CXCL1 and CXCL8 and to diminish inflammation in mice. In the present study, solution NMR spectroscopy was used to investigate the structure of Evasin-3 and its CXCL8–Evasin-3 complex. Evasin-3 is found to disrupt the glycosaminoglycan-binding site of CXCL8 and inhibit the interaction of CXCL8 with CXCR2. Structural data were used to design two novel CXCL8-binding peptides. The linear tEv3 17–56 and cyclic tcEv3 16–56 dPG Evasin-3 variants were chemically synthesized by solid-phase peptide synthesis. The affinity of these newly synthesized variants to CXCL8 was measured by surface plasmon resonance biosensor analysis. The K d values of tEv3 17–56 and tcEv3 16–56 dPG were 27 and 13 n m , respectively. Both compounds effectively inhibited CXCL8-induced migration of polymorphonuclear neutrophils. The present results suggest utility of synthetic Evasin-3 variants as scaffolds for designing and fine-tuning new chemokine-binding agents that suppress immune responses and inflammation.

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“…Lately, combining seleno-cysteine scanning and NMR analysis was shown to be a reliable approach for mapping disulfide bonds in cysteine-rich peptides and proteins (Denisov et al, 2019). The structurally conservative selenium substitution causes selective chemical shift changes of cysteine carbons involved in the mixed S-Se bond allowing identification by visual comparison of [ 1 H, 13 C]-HSQC spectra of native and Sec-mutants.…”

Section: Nmr Spectroscopy and Prediction Techniquesmentioning

confidence: 99%

Cysteines and Disulfide Bonds as Structure-Forming Units: Insights From Different Domains of Life and the Potential for Characterization by NMR

et al. 2020

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Disulfide bridges establish a fundamental element in the molecular architecture of proteins and peptides which are involved e.g., in basic biological processes or acting as toxins. NMR spectroscopy is one method to characterize the structure of bioactive compounds including cystine-containing molecules. Although the disulfide bridge itself is invisible in NMR, constraints obtained via the neighboring NMR-active nuclei allow to define the underlying conformation and thereby to resolve their functional background. In this mini-review we present shortly the impact of cysteine and disulfide bonds in the proteasome from different domains of life and give a condensed overview of recent NMR applications for the characterization of disulfide-bond containing biomolecules including advantages and limitations of the different approaches.

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SecScan: a general approach for mapping disulfide bonds in synthetic and recombinant peptides and proteins

Abstract: Selenocysteine scanning (SecScan) is a novel technique to map disulfide networks in proteins independent of structure-based distance information and mass spectrometry.

Cited by 14 publications

References 23 publications

A knottin scaffold directs the CXC-chemokine–binding specificity of tick evasins

A knottin scaffold directs the CXC-chemokine–binding specificity of tick evasins

Tick saliva protein Evasin-3 modulates chemotaxis by disrupting CXCL8 interactions with glycosaminoglycans and CXCR2

Cysteines and Disulfide Bonds as Structure-Forming Units: Insights From Different Domains of Life and the Potential for Characterization by NMR

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