Photo-initiated crosslinking extends mapping of the protein–protein interface to membrane-embedded portions of cytochromes P450 2B4 and b5

Ječmen, Tomáš; Ptackova, Renata; Černá, Věra; Dračínská, Helena; Hodek, Petr; Stiborová, Marie; Hudeček, Jiřı́; Šulc, Miroslav

doi:10.1016/j.ymeth.2015.07.015

Cited by 9 publications

(8 citation statements)

References 39 publications

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“…This functionality has an advantage over site-specific cross-linking reagents, as non-specific photo-inducible chemistry permits cross-linking of residues in hydrophobic regions, extending the application of cross-linking to the structural determination of solvent-inaccessible interaction regions e.g. membrane-spanning protein complexes 135 . Photo-reactive functional groups (i.e.…”

Section: Developing New Cross-linking Chemistriesmentioning

confidence: 99%

“…Diazarine-incorporated amino acid analogs are unique from residue-specific cross-linkers in their ability to nonspecifically cross-link proximal residues. ,, This functionality permits cross-linking of residues in hydrophobic regions that often cannot be targeted by existing residue-specific reagents, extending the application of cross-linking to the structural determination of solvent-inaccessible interaction regions, e.g., membrane-spanning protein complexes . Photoreactive functional groups (i.e., diazarine, phenyl azide, benzophenone) have also been explored in conjunction with NHS esters to create short, heterobifunctional cross-linking reagents consisting of lysine-targeting and nonspecific ends. , However, while nonspecific chemistry is attractive for increasing surface coverage, it often results in highly complex cross-linked products, complicating database searching.…”

Section: Developing New Cross-linking Chemistriesmentioning

confidence: 99%

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Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology

2017

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Section: Developing New Cross-linking Chemistriesmentioning

confidence: 99%

Section: Developing New Cross-linking Chemistriesmentioning

confidence: 99%

Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology

2017

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“…Our model of interacting TM helices is consistent with recently published data, which were based on mapping of the protein−protein interface by photoinitiated cross-linking between membrane-embedded portions of rabbit cyt b 5 and P450 2B4. Using the diazirine analogue of methionin (in position 126 of cyt b5), Ječmen et al found an interaction of cyt b 5 TM helix with the TM helix of P450 2B4.…”

Section: Resultsmentioning

confidence: 99%

Membrane-Anchored Cytochrome P450 1A2–Cytochrome b₅ Complex Features an X-Shaped Contact between Antiparallel Transmembrane Helices

Jeřábek

Florián

Martínek

2016

Chem. Res. Toxicol.

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Eukaryotic cytochromes P450 (P450) are membrane-bound enzymes oxidizing a broad spectrum of hydrophobic substrates, including xenobiotics. Protein-protein interactions play a critical role in this process. In particular, the formation of transient complexes of P450 with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates catalytic activities of several P450s. To lay a structural foundation for the investigation of these effects, we constructed a model of the membrane-bound full-length human P450 1A2-cyt b5 complex. The model was assembled from several parts using a multiscale modeling approach covering all-atom and coarse-grained molecular dynamics (MD). For soluble P450 1A2-cyt b5 complexes, these simulations yielded three stable binding modes (sAI, sAII, and sB). The membrane-spanning transmembrane domains were reconstituted with the phospholipid bilayer using self-assembly MD. The predicted full-length membrane-bound complexes (mAI and mB) featured a spontaneously formed X-shaped contact between antiparallel transmembrane domains, whereas the mAII mode was found to be unstable in the membrane environment. The mutual position of soluble domains in binding mode mAI was analogous to the sAI complex. Featuring the largest contact area, the least structural flexibility, the shortest electron transfer distance, and the highest number of interprotein salt bridges, mode mAI is the best candidate for the catalytically relevant full-length complex.

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“…The protein band from SDS-PAGE was destained, cysteine residues were modified by iodoacetamide, and protein was digested by trypsin endoprotease (Promega Corp., Madison, USA). The resulting peptide mixture was extracted and the MS and MS/MS spectra of corresponding m / z signals for peptide identity verification were acquired and manually interpreted (54 % of protein sequence coverage) [ 51 ].…”

Section: Methodsmentioning

confidence: 99%

Heterologous expression of human cytochrome P450 2S1 in Escherichia coli and investigation of its role in metabolism of benzo[a]pyrene and ellipticine

et al. 2016

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Cytochrome P450 (CYP) 2S1 is ''orphan'' CYP that is overexpressed in several epithelial tissues and many human tumors. The pure enzyme is required for better understanding of its biological functions. Therefore, human CYP2S1 was considered to be prepared by the gene manipulations and heterologous expression in Escherichia coli. Here, the conditions suitable for efficient expression of human CYP2S1 protein from plasmid pCW containing the human CYP2S1 gene were optimized and the enzyme purified to homogeneity. The identity of CYP2S1 as the product of heterologous expression was confirmed by dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and mass spectrometry. To confirm the presence of the enzymatically active CYP2S1, the CO spectrum of purified CYP2S1 was recorded. Since CYP2S1 was shown to catalyze oxidation of compounds having polycyclic aromatic structures, the prepared enzyme has been tested to metabolize the compounds having this structural character; namely, the human carcinogen benzo[a]pyrene (BaP), its 7,8-dihydrodiol derivative, and an anticancer drug ellipticine. Reaction mixtures contained besides the test compounds the CYP2S1 enzyme reconstituted with NADPH:CYP reductase (POR) in liposomes, and/or this CYP in the presence of cumene hydroperoxide or hydrogen peroxide. High performance liquid chromatography was employed for separation of BaP, BaP-7,8-dihydrodiol, and ellipticine metabolites. The results found in this study demonstrate that CYP2S1 in the presence of cumene hydroperoxide or hydrogen peroxide catalyzes oxidation of two of the test xenobiotics, a metabolite of BaP, BaP-7,8-dihydrodiol, and ellipticine. Whereas BaP-7,8,9,10-tetrahydrotetrol was formed as a product of BaP-7,8-dihydrodiol oxidation, ellipticine was oxidized to 12-hydroxyellipticine, 13-hydroxyellipticine, and the ellipticine N 2 -oxide.

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Photo-initiated crosslinking extends mapping of the protein–protein interface to membrane-embedded portions of cytochromes P450 2B4 and b5

Cited by 9 publications

References 39 publications

Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology

Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology

Membrane-Anchored Cytochrome P450 1A2–Cytochrome b₅ Complex Features an X-Shaped Contact between Antiparallel Transmembrane Helices

Heterologous expression of human cytochrome P450 2S1 in Escherichia coli and investigation of its role in metabolism of benzo[a]pyrene and ellipticine

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Photo-initiated crosslinking extends mapping of the protein–protein interface to membrane-embedded portions of cytochromes P450 2B4 and b5

Cited by 9 publications

References 39 publications

Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology

Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology

Membrane-Anchored Cytochrome P450 1A2–Cytochrome b5 Complex Features an X-Shaped Contact between Antiparallel Transmembrane Helices

Heterologous expression of human cytochrome P450 2S1 in Escherichia coli and investigation of its role in metabolism of benzo[a]pyrene and ellipticine

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Membrane-Anchored Cytochrome P450 1A2–Cytochrome b₅ Complex Features an X-Shaped Contact between Antiparallel Transmembrane Helices