Dynamics and Location of the Allosteric Midazolam Site in Cytochrome P4503A4 in Lipid Nanodiscs

Redhair, Michelle; Hackett, John C.; Pelletier, Robert; Atkins, William M.

doi:10.1021/acs.biochem.9b01001

Cited by 32 publications

(63 citation statements)

References 54 publications

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“…Alterations in conformational dynamics upon ligand binding were described in several earlier reports [11,12,14,17,19,[21][22][23]25,126,130]; in selected studies, an allosteric effect could also be identified. A great example of perturbed conformational dynamics was published by Zhou et al in 2017 [25], where HDX-MS analysis shed light on the role of conformational dynamics, which was considerably altered upon ligand binding, in the mechanism of action of the DXPS enzyme; the closed conformation of DXPS proved to be critical for stabilization of the transition state, whereas the open conformation is apparently required for releasing the lactyl-thiamin diphosphate final product.…”

Section: Analysis Of Protein Interactionsmentioning

confidence: 90%

“…Last year, a group of eminent researchers in the field formulated clear recommendations for performing and interpreting HDX-MS experiments [ 4 ] and hence paved the way towards truly standardized experiments. The applications of HDX-MS are very versatile: they include studies of protein complexes [ 5 , 6 , 7 , 8 , 9 , 10 ], ligand binding [ 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 ], dynamic properties like conformational changes and folding/unfolding/refolding [ 26 ], conformational changes that arise from allosteric effects [ 27 , 28 , 29 ], structure and stability of biopharmaceuticals [ 17 , 30 , 31 ] and epitopes [ 15 , 32 , 33 ], etc. Unlike several other biophysical techniques, HDX-MS possesses the advantages of no or very high size limit [ 8 ] and is useful for studying individual proteins as well as large complexes [ 34 , 35 ].…”

Section: Introductionmentioning

confidence: 99%

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Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

Ozohanics

Ambrus

2020

Life

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Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) is a rapidly evolving technique for analyzing structural features and dynamic properties of proteins. It may stand alone or serve as a complementary method to cryo-electron-microscopy (EM) or other structural biology approaches. HDX-MS is capable of providing information on individual proteins as well as large protein complexes. Owing to recent methodological advancements and improving availability of instrumentation, HDX-MS is becoming a routine technique for some applications. When dealing with samples of low to medium complexity and sizes of less than 150 kDa, conformation and ligand interaction analyses by HDX-MS are already almost routine applications. This is also well supported by the rapid evolution of the computational (software) background that facilitates the analysis of the obtained experimental data. HDX-MS can cope at times with analytes that are difficult to tackle by any other approach. Large complexes like viral capsids as well as disordered proteins can also be analyzed by this method. HDX-MS has recently become an established tool in the drug discovery process and biopharmaceutical development, as it is now also capable of dissecting post-translational modifications and membrane proteins. This mini review provides the reader with an introduction to the technique and a brief overview of the most common applications. Furthermore, the most challenging likely applications, the analyses of glycosylated and membrane proteins, are also highlighted.

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Section: Analysis Of Protein Interactionsmentioning

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

Ozohanics

Ambrus

2020

Life

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“…25 Although it has been speculated that this peripheral binding site may stem from a crystallographic artifact, many biochemical and biophysical studies support the hypothesis that the F'-helix is involved in effector binding and allosteric activation. 14,22,23,26,[29][30][31][32][33][34][35] How a binding event in this region may lead to CYP3A4 catalytic enhancement remains unknown. Structural changes leading to allosteric activation are often dynamic, and can be transient, making them difficult to fully capture with X-ray crystallography.…”

Section: Discussionmentioning

confidence: 99%

“…42 To our knowledge, only two HDX-MS studies have looked specifically at CYP3A4, one investigating membrane association and inhibitor binding, 38 and the other examining the binding of the substrate and the positive effector, midazolam. 43,44 Herein, using CYP3A4 as a model system, we demonstrate the utility of combining bioconjugation and HDX-MS to characterize allosteric mechanisms. The results presented also advance our mechanistic understanding of CYP3A4 in several ways: i) they confirm the existence of functionally relevant flexible regions in wild type CYP3A4; ii) confirm that the F'-helix of CYP3A4 is part of the allosteric site; iii) identify coupled structural elements involved in CYP3A4 allosteric activation; and iv) demonstrate the importance of F-helix flexibility in CYP3A4 allosteric activation.…”

Section: Discussionmentioning

confidence: 99%

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

Ducharme

Thibodeaux

Auclair

2020

Preprint

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We 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.

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“…Cumulatively, these data build on the growing body of evidence for the functional relevance of conformational dynamics in CYP3A4. [28][29][30]…”

Section: Introductionmentioning

confidence: 99%

Structural dynamics of cytochrome P450 3A4 in the presence of substrates and cytochrome P450 reductase

Ducharme

Sevrioukova

Thibodeaux

et al. 2021

Preprint

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Cytochrome P450 3A4 (CYP3A4) is the most important drug-metabolizing enzyme in humans and has been associated with harmful drug interactions. The activity of CYP3A4 is known to be modulated by several compounds, as well as by the electron transfer partner, cytochrome P450 reductase (CPR). The underlying mechanism of these effects however is poorly understood. We have used hydrogen-deuterium exchange mass spectroscopy (HDX-MS) to investigate the impact of CPR and three different substrates (7-benzyloxy-4-trifluoromethyl-coumarin, testosterone and progesterone) on the conformational dynamics of CYP3A4. Here, we report that interaction of CYP3A4 with substrates or with the oxidized or reduced form of CPR leads to a global rigidification of the CYP3A4 structure. This was evident from a suppression of deuterium exchange in several regions of CYP3A4, including those known to be involved in protein-protein interactions (C-helix) as well as substrate binding and specificity (B′-, E-helices and K/β1-loop). Furthermore, the bimodal isotopic distributions observed for some CYP3A4-derived peptides were drastically impacted by CPR and/or substrates, suggesting the existence of stable CYP3A4 conformational populations that are perturbed by ligand/CPR binding. The results have implications for understanding the mechanisms of allostery, ligand binding, and catalysis in CYP enzymes.

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Dynamics and Location of the Allosteric Midazolam Site in Cytochrome P4503A4 in Lipid Nanodiscs

Cited by 32 publications

References 54 publications

Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

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

Structural dynamics of cytochrome P450 3A4 in the presence of substrates and cytochrome P450 reductase

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