scholarly journals Characterization of the structure and interactions of P450 BM3 using hybrid mass spectrometry approaches

2020 ◽  
Vol 295 (22) ◽  
pp. 7595-7607 ◽  
Author(s):  
Laura N. Jeffreys ◽  
Kamila J. Pacholarz ◽  
Linus O. Johannissen ◽  
Hazel M. Girvan ◽  
Perdita E. Barran ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Cytochrome P450 ◽  
Bound States ◽  
Catalytic Mechanism ◽  
Catalytic Domain ◽  
Full Length ◽  
P450 Bm3 ◽  
Interaction Sites ◽  
Domain Interactions ◽  
Hdx Ms

The cytochrome P450 monooxygenase P450 BM3 (BM3) is a biotechnologically important and versatile enzyme capable of producing important compounds such as the medical drugs pravastatin and artemether, and the steroid hormone testosterone. BM3 is a natural fusion enzyme comprising two major domains: a cytochrome P450 (heme-binding) catalytic domain and a NADPH-cytochrome P450 reductase (CPR) domain containing FAD and FMN cofactors in distinct domains of the CPR. A crystal structure of full-length BM3 enzyme is not available in its monomeric or catalytically active dimeric state. In this study, we provide detailed insights into the protein-protein interactions that occur between domains in the BM3 enzyme and characterize molecular interactions within the BM3 dimer by using several hybrid mass spectrometry (MS) techniques, namely native ion mobility MS (IM-MS), collision-induced unfolding (CIU), and hydrogen-deuterium exchange MS (HDX-MS). These methods enable us to probe the structure, stoichiometry, and domain interactions in the ∼240 kDa BM3 dimeric complex. We obtained high-sequence coverage (88–99%) in the HDX-MS experiments for full-length BM3 and its component domains in both the ligand-free and ligand-bound states. We identified important protein interaction sites, in addition to sites corresponding to heme-CPR domain interactions at the dimeric interface. These findings bring us closer to understanding the structure and catalytic mechanism of P450 BM3.

The catalytic mechanism of cytochrome P450 BM3 involves a 6 Å movement of the bound substrate on reduction

1996 ◽  
Vol 3 (5) ◽  
pp. 414-417 ◽  
Author(s):  
Sandeep Modi ◽  
Michael J. Sutcliffe ◽  
William U. Primrose ◽  
Lu-Yun Lian ◽  
Gordon C.K. Roberts
Keyword(s):  
Cytochrome P450 ◽  
Catalytic Mechanism ◽  
P450 Bm3 ◽  
Cytochrome P450 Bm3

scholarly journals Combining small-molecule bioconjugation and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to expose allostery: the case of human cytochrome P450 3A4

2020 ◽  
Author(s):  
Julie Ducharme ◽  
Christopher J. Thibodeaux ◽  
Karine Auclair
Keyword(s):  
Mass Spectrometry ◽  
Cytochrome P450 ◽  
Cytochrome P450 3A4 ◽  
Hydrogen Deuterium Exchange ◽  
Allosteric Site ◽  
Exchange Mass Spectrometry ◽  
Human Cytochrome ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

AbstractWe report herein a novel approach to study allostery which combines the use of carefully selected bioconjugates and hydrogen-deuterium exchange mass spectrometry (HDX-MS). The utility of our method is demonstrated using human cytochrome P450 3A4 (CYP3A4). CYP3A4 is arguably the most important drug-metabolizing enzyme, and as such, is involved in numerous drug interactions. Diverse allosteric ligand effects have been reported for this enzyme, yet the structural mechanism of these phenomena remain poorly understood. We have described different CYP3A4-effector bioconjugates, some of which mimic the allosteric effect of positive effectors on CYP3A4, while others show activity enhancement even though the label does not occupy the allosteric pocket (agonistic), or do not show activation while still blocking the allosteric site (antagonistic). These bioonjugates were studied here by HDX-MS, which enabled us to better define the position of the allosteric site, and to identify important regions involved in CYP3A4 activation.

scholarly journals Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

2021 ◽  
Author(s):  
Valentine V. Courouble ◽  
Sanjay Kumar Dey ◽  
Ruchi Yadav ◽  
Jennifer Timm ◽  
Jerry Joe E. K. Harrison ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mass Spectrometry ◽  
Conformational Dynamics ◽  
Three Dimensional ◽  
Full Length ◽  
Structural Proteomics ◽  
Exchange Mass Spectrometry ◽  
Dimeric Unit ◽  
Hydrogen Deuterium ◽  
Hdx Ms

ABSTRACTCoronavirus (CoV) non-structural proteins (nsps) assemble to form the replication-transcription complex (RTC) responsible for viral RNA synthesis. nsp7 and nsp8 are important cofactors of the RTC, as they interact and regulate the activity of RNA-dependent RNA polymerase (RdRp) and other nsps. To date, no structure of full-length SARS-CoV-2 nsp7:nsp8 complex has been published. Current understanding of this complex is based on structures from truncated constructs or with missing electron densities and complexes from related CoV species with which SARS-CoV-2 nsp7 and nsp8 share upwards of 90% sequence identity. Despite available structures being solved using crystallography and cryo-EM representing detailed snapshots of the nsp7:nsp8 complex, it is evident that the complex has a high degree of structural plasticity. However, relatively little is known about the conformational dynamics of the complex and how it assembles to interact with other nsps. Here, the solution-based structural proteomic techniques, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and crosslinking mass spectrometry (XL-MS), illuminate the structural dynamics of the SARS-CoV-2 full-length nsp7:nsp8 complex. The results presented from the two techniques are complementary and validate the interaction surfaces identified from the published three-dimensional heterotetrameric crystal structure of SARS-CoV-2 truncated nsp7:nsp8 complex. Furthermore, mapping of XL-MS data onto higher order complexes suggests that SARS-CoV-2 nsp7 and nsp8 do not assemble into a hexadecameric structure as implied by the SARS-CoV full-length nsp7:nsp8 crystal structure. Instead our results suggest that the nsp7:nsp8 heterotetramer can dissociate into a stable dimeric unit that might bind to nsp12 in the RTC without altering nsp7-nsp8 interactions.

The Role of Protein Plasticity in Computational Rationalization Studies on Regioselectivity in Testosterone Hydroxylation by Cytochrome P450 BM3 Mutants

2012 ◽  
Vol 13 (2) ◽  
pp. 155-166 ◽  
Author(s):  
Stephanie B.A. de Beer ◽  
Laura A.H. van Bergen ◽  
Karlijn Keijzer ◽  
Vanina Rea ◽  
Harini Venkataraman ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
P450 Bm3 ◽  
Cytochrome P450 Bm3 ◽  
Testosterone Hydroxylation

scholarly journals Engineering the biomimetic cofactors of NMNH for cytochrome P450 BM3 based on binding conformation refinement

RSC Advances ◽  
2021 ◽  
Vol 11 (20) ◽  
pp. 12036-12042
Author(s):  
Yao Liu ◽  
Yalong Cong ◽  
Chuanxi Zhang ◽  
Bohuan Fang ◽  
Yue Pan ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Rational Design ◽  
Design Strategy ◽  
Efficient Utilization ◽  
P450 Bm3 ◽  
Binding Conformation ◽  
Cytochrome P450 Bm3

A rational design strategy was proposed to improve the efficient utilization of alternative biomimetic cofactor by P450 BM3 enzyme.

scholarly journals An electron transfer competent structural ensemble of membrane-bound cytochrome P450 1A1 and cytochrome P450 oxidoreductase

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Goutam Mukherjee ◽  
Prajwal P. Nandekar ◽  
Rebecca C. Wade
Keyword(s):  
Electron Transfer ◽  
Cytochrome P450 ◽  
Drug Targets ◽  
Catalytic Domain ◽  
Cytochrome P450 1A1 ◽  
P450 Oxidoreductase ◽  
Distal Side ◽  
Cytochrome P450 Oxidoreductase ◽  
Membrane Bound ◽  
Transient Interactions

AbstractCytochrome P450 (CYP) heme monooxygenases require two electrons for their catalytic cycle. For mammalian microsomal CYPs, key enzymes for xenobiotic metabolism and steroidogenesis and important drug targets and biocatalysts, the electrons are transferred by NADPH-cytochrome P450 oxidoreductase (CPR). No structure of a mammalian CYP–CPR complex has been solved experimentally, hindering understanding of the determinants of electron transfer (ET), which is often rate-limiting for CYP reactions. Here, we investigated the interactions between membrane-bound CYP 1A1, an antitumor drug target, and CPR by a multiresolution computational approach. We find that upon binding to CPR, the CYP 1A1 catalytic domain becomes less embedded in the membrane and reorients, indicating that CPR may affect ligand passage to the CYP active site. Despite the constraints imposed by membrane binding, we identify several arrangements of CPR around CYP 1A1 that are compatible with ET. In the complexes, the interactions of the CPR FMN domain with the proximal side of CYP 1A1 are supplemented by more transient interactions of the CPR NADP domain with the distal side of CYP 1A1. Computed ET rates and pathways agree well with available experimental data and suggest why the CYP–CPR ET rates are low compared to those of soluble bacterial CYPs.

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