Lipids Shape the Electron Acceptor-Binding Site of the Peripheral Membrane Protein Dihydroorotate Dehydrogenase

Abstract

SummaryThe interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among co-factor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins.

Scratching the surface: native mass spectrometry of peripheral membrane protein complexes

“…To dissect the relationship between DHODH folding, inhibitor binding, and membrane interactions, Costeira-Paulo et al [44] performed nMS analysis of the protein embedded in detergent micelles that were subsequently stripped away by collisional activation to obtain well-resolved spectra of DHODH with bound FMN cofactor and ligands (Figure 2A). The energies required to release DHODH were lower than those normally used for integral membrane proteins, in line with a more labile protein-micelle interaction.…”

“…• We outline here that most of the nMS strategies tailored for integral membrane proteins can be applied with no, or only minor, modifications to the study of peripheral membrane proteins. As a result, mass spectrometric analysis of intact peripheral membrane protein complexes is now possible, capturing their interactions with detergent micelles [44], lipid nanodiscs [64], lipid vesicles [74], and even integral membrane protein partners [80]. • Based on these developments, we predict that nMS will enable new insights into elusive lipidprotein interactions.…”

“…(B) nESI-MS of DHODH in the presence of detergent and Brequinar shows a series of peaks corresponding to the protein with its FMN cofactor in complex with one Brequinar molecule and zero, one, two, or three detergent molecules. The deconvoluted spectrum is shown above the corresponding mass spectrum.Adapted from reference[44] with permission. Copyright 2018 Elsevier.…”

Cerpegin-derived furo[3,4-c]pyridine-3,4(1H,5H)-diones enhance cellular response to interferons by de novo pyrimidine biosynthesis inhibition

“…The vast majority of these inhibitors were found to bind a hydrophobic funnel localized at the contact interface of DHODH with the mitochondrial membrane. This lipophilic pocket was considered as the binding site for ubiquinone allowing a direct contact with FMN at its extremity as recently modelized by Costeira-Paulo J. et al [42]. As shown in Fig.…”

“…Ubiquinone is usually located in the mitochondrial membrane. Close contact between DHODH and the membrane allows ubiquinone to shuttle into a hydrophobic channel of this enzyme in order to contact FMN as recently modelled [42]. A majority of DHODH inhibitors that have been described in the literature compete with ubiquinone for binding to this channel, and interact with a limited number of well-characterized residues [1][2][3].…”