Thibodeaux, C. | bioRxiv Science

bioRxiv2021

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

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/{beta}1-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.

biochemistry↗

bioRxiv2020

Exploring the conformational landscape of a lanthipeptide synthetase using native mass spectrometry

Lanthipeptides are ribosomally-synthesized and post-translationally modified peptide (RiPP) natural products that are biosynthesized in a multistep maturation process by enzymes (lanthipeptide synthetases) that possess relaxed substrate specificity. Recent evidence has suggested that some lanthipeptide synthetases are structurally dynamic enzymes that are allosterically activated by precursor peptide binding, and that conformational sampling of the enzyme-peptide complex may play an important role in defining the efficiency and sequence of biosynthetic events. These "biophysical" processes, while critical for defining the activity and function of the synthetase, remain very challenging to study with existing methodologies. Herein, we show that native nanoelectrospray ionization coupled to ion mobility mass spectrometry (nanoESI-IM-MS) provides a powerful and sensitive means for investigating the conformational landscapes and intermolecular interactions of lanthipeptide synthetases. Namely, we demonstrate that the class II lanthipeptide synthetase (HalM2) and its non-covalent complex with the cognate HalA2 precursor peptide can be delivered into the gas phase in a manner that preserves native structures and intermolecular enzyme-peptide contacts. Moreover, gas phase ion mobility studies of the natively-folded ions demonstrate that peptide binding and mutations to dynamic structural elements of HalM2 alter the conformational landscape of the enzyme, and that the precursor peptide itself exhibits higher order structure in the mass spectrometer. Cumulatively, these data support previous claims that lanthipeptide synthetases are structurally dynamic enzymes that undergo functionally relevant conformational changes in response to precursor peptide binding. This work establishes nanoESI-IM-MS as a versatile approach for unraveling the relationships between protein structure and biochemical function in RiPP biosynthetic systems.

biochemistry↗