Since DHODH in vivo is permanently membrane-associated, we speculated that mitochondrial lipids may be of importance for its biological function. We performed DHODH activity assays either in detergent alone or supplemented with any of the three major human mitochondrial lipids phosphatidyl ethanolamine (PE), phosphatidyl choline (PC), and cardiolipin (CDL). Monitoring the reduction of the soluble electron acceptor benzoquinone showed that all three lipids increased the reaction rate (V) by 30%–60% relative to the detergent-solubilized protein. Since all three lipids caused a similar increase in activity, the effect is unlikely to be mediated by specific lipid recognition in DHODH ( Figure S2 ).

Hanson et al., 2003 Hanson C.L.

Ilag L.L.

Malo J.

Hatters D.M.

Howlett G.J.

Robinson C.V. Phospholipid complexation and association with apolipoprotein C-II: insights from mass spectrometry.

Laganowsky et al., 2014 Laganowsky A.

Reading E.

Allison T.M.

Ulmschneider M.B.

Degiacomi M.T.

Baldwin A.J.

Robinson C.V. Membrane proteins bind lipids selectively to modulate their structure and function.

Landreh et al., 2017 Landreh M.

Costeira-Paulo J.

Gault J.

Marklund E.G.

Robinson C.V. The effects of detergent micelles on lipid binding to proteins in electrospray ionization mass spectrometry.

Figure 2 DHODH Exhibits Limited Binding of Mitochondrial Phospholipids Show full caption (A) Structures of the detergent LDAO and the three major phospholipid species in the human mitochondrial membrane. (B) nESI-MS shows that binding of PC, PE, and CDL, can be preserved in the gas phase. CDL exhibits the most stable association with DHODH, while PC interactions are not well retained. (C) Mass spectra of DHODH in the presence of increasing amounts of PE reveal a maximum number of lipid adducts for both apo- and holo-DHODH. Above a 20-fold excess (180 μM) of PE over DHODH, a maximum of three lipids are bound per protein. (D) The DHODH-lipid complexes are less sensitive to DMSO-induced dissociation than the DHODH-FMN complexes (indicated by blue arrows). Spectra recorded without and with DMSO are normalized to the 14+ charge state of holo- and apo-DHODH, respectively. See also Figure S2

Having established that we can detect intact DHODH complexes in the gas phase, we used MS to investigate the interactions between DHODH and phospholipids in detail ( Figure 2 A). nESI-MS spectra recorded in the presence of any of the three lipids show additional peaks indicating the formation of protein-lipid complexes. For CDL and PE, these were readily detected at a protein to lipid ratio of 1:4. Interactions with PC, on the other hand, resulted in fewer lipid adducts that could only be detected at higher lipid concentrations ( Figure 2 B). Since the lipids chosen for analysis have identical acyl chains, the different extent to which their association is preserved after desolvation can be attributed to differences in head group interactions. For PC, for which only minor adducts are detected, hindrance from the tertiary amine may negatively affect favorable electrostatic head group interactions. CDL, for which strong adduct peaks are observed, contains two negatively charged phosphates that may easily form hydrogen-bonding interactions with DHODH. The observed preference for anionic head groups suggests that the protein-lipid complexes are held together by charge interactions in the gas phase. To further characterize the interaction, we recorded mass spectra of the protein in the presence of increasing concentrations of PE, the most abundant of the three mitochondrial lipids. Interestingly, a maximum of three lipid adducts could be observed even at a 20-fold excess of PE over DHODH, and the protein-lipid complex peaks remained at lower intensities than the peaks corresponding to the lipid-free protein ( Figure 2 C). The limited lipid binding indicates that the protein makes little contact with the detergent-solubilized lipids. As a result, only a few lipid molecules remain attached to the protein after detergent release, in contrast to integral membrane proteins, which bind and retain a large number of lipid adducts (). We conclude that DHODH associates only superficially with membrane lipids, consistent with binding to the membrane-associated region. To test this hypothesis, we measured the effect of lipids on the thermal stability of detergent-solubilized DHODH. All three lipids induced only a very minor shift in melting temperature ( Figure S2 ). The fact that lipids do not provide stabilization against global unfolding of the protein is compatible with a peripheral association.