An Isoprene Lipid-Binding Protein Promotes Eukaryotic Coenzyme Q Biosynthesis

The biosynthesis of coenzyme Q presents a paradigm for how cells surmount hydrophobic barriers in lipid biology. In eukaryotes, CoQ precursors-among nature's most hydrophobic molecules-must somehow be presented to a series of enzymes peripherally associated with the mitochondrial inner membrane. Here, we reveal that this process relies on custom lipid-binding properties of COQ9. We show that COQ9 repurposes the bacterial TetR fold to bind aromatic isoprenes with high specificity, including CoQ intermediates that likely reside entirely within the bilayer. We reveal a process by which COQ9 associates with cardiolipin-rich membranes and warps the membrane surface to access this cargo. Finally, we identify a molecular interface between COQ9 and the hydroxylase COQ7, motivating a model whereby COQ9 presents intermediates directly to CoQ enzymes. Overall, our results provide a mechanism for how a lipid-binding protein might access, select, and deliver specific cargo from a membrane to promote biosynthesis.

Characterization of Coenzyme Q Biosynthesis Proteins through Integrative Modeling at the Protein-Membrane Interface

Deniz Aydin

Integral and peripheral membrane proteins account for one-third of the human proteome, and they are estimated to represent the target for over 50% of modern medicinal drugs. Despite their central role in medicine, the complex, heterogeneous and dynamic nature of biological membranes complicates the investigation of their mechanism of action by both experimental and computational techniques. Among the different membrane bound compartments in eukaryotic cells, mitochondria are highly complex in form and function, and they harbor a unique proteome that remains largely unexplored. A growing number of inherited metabolic diseases are associated with mitochondrial dysfunction, which necessitates the structural and functional elucidation of mitochondrial proteins. In this thesis, we combine experimental and computational methods to explore the activity of COQ8 and COQ9, two functionally elusive proteins of the biosynthetic complex that produces coenzyme Q, a redox-active lipid component of the mitochondrial electron transport chain. (i) Conserved Lipid Modulation of Ancient Kinase-Like UbiB Family Member COQ8. We demonstrate that COQ8 has an ATPase function that is activated when it specifically associates with cardiolipin-containing membranes. We identify its interaction surface with the inner mitochondrial membrane, which gives hints about the possible interaction surfaces with other members of the coenzyme Q synthesis machinery and has implications on how it mediates functional interactions with lipids. Collectively, this work reveals how the positioning of COQ8 on the inner mitochondrial membrane is key to its activation, and therefore advances our understanding of the COQ8 function. (ii) Membrane, Lipid, and Protein Interactions of Coenzyme Q Biosynthesis Protein COQ9. We explore the lipid binding activity of COQ9, and we reveal that COQ9 repurposes an ancient bacterial fold to selectively bind aromatic isoprenes, including CoQ intermediates that reside within the bilayer. We elucidate the mechanistic details of its membrane binding process, by which COQ9 warps the membrane surface and creates a tightly sealed hydrophobic region to access its lipid cargo. Finally, we establish a potential molecular interface between COQ9 and COQ7, the enzyme that catalyzes the penultimate step in CoQ biosynthesis, suggesting a model whereby COQ9 presents intermediates to CoQ enzymes to overcome the hydrophobic barrier of the membrane. Collectively, our results provide a mechanism for how a lipid binding protein might access, select, and extract specific cargo from a membrane and present it to a peripheral membrane enzyme. In conclusion, our work is a good illustration of the interplay between experiment and modeling in protein research and specifically in understanding how proteins perform their action in direct synergy with membrane environments. We anticipate our integrative methodologies and mechanistic findings will prove relevant to other membrane proteins, whose fine functional modulation at the membrane-water interface has been historically challenging to characterize.

EPFL2019

Kinetic reconstruction and analysis of sphingolipid metabolism

Vassily Hatzimanikatis, Howard Riezman, Georgios Savoglidis

Lipids are major constituents of the cell. They are responsible for major properties of the cellular membranes: hydrophobicity, selective permeability and being the scaffold of signaling proteins. Many diseases are associated with alterations in the lipid distribution in the cell and the composition of membrane domains. Metabolic syndrome, obesity, atherosclerosis, as well as Alzheimer’s, Huntington’s diseases and cancer, have an impact in the levels of lipids, with observed alterations in their concentrations compared to the healthy state. Applying computational techniques along with systematic modeling of lipid metabolism can provide insights that can guide biomedical research and develop potential strategies for prevention and cure. In the present study we developed a comprehensive model of the sphingolipid biosynthesis in the yeast Saccharomyces cerevisiae. Sphingolipids are one of the four major lipid categories, along with (glycero)phospholipids, sterols and fatty acids synthesized in the yeast S. cerevisiae. The importance of sphingolipids present in any higher eukaryote has been demonstrated in many recent studies. For this study, S. cerevisiae has been chosen as a model organism due to its high homology of cellular processes with mammalian cells. The developed model will be an essential part towards the construction of a detailed kinetic model of the whole lipidome of the cell. We first constructed a stoichiometric model that contains all the currently known reactions for the biosynthesis of ceramides and complex sphingolipids in the yeast. Additionally, we have accounted for all five reported hydroxylation states, along with the reactions synthesizing the necessary precursor metabolites from other lipid pathways (i.e. palmitate-CoA from fatty acid synthesis and phosphatidylinositol from the phospholipids metabolism). We next developed a kinetic model and we used a large number of lipidomic measurements of wild type yeast to consistently calibrate our model. Curation of the kinetic information of the model came from the comprehensive mining of references for operation of the enzymes as well as ranges of kinetic parameters from online databases. These datasets created the pool for a sampling technique that accounts for the uncertainty in the parameters of the model. This lead to a robust dynamic model, containing mass balances for all the components of the biochemical network as well as terms that accounted for the dillution of these molecules in the cell due to growth. We performed a thorough kinetic analysis of the system by examining the impact of different assumptions in enzyme operation on the levels of ceramides and complex sphingolipids (e.g. substrate competition, (un)competitive inhibition). By applying the principles of Metabolic Control Analysis (MCA) we were able to quantify the effect of enzyme activities on the lipid profiles and we identified enzymes of the biochemical reaction network as efficient targets for metabolic engineering towards a desired state. We also applied a reverse engineering method and we were able to identify enzyme perturbations responsible for an observed altered state compared to the wild type cellular lipidomic profile. Such an approach could lead to the identification of genetic mutations by imploring the information containd within metabolomic measurements. Although we demonstrate the application of the method in models of lipid metabolism it is possible to be applied to biochemical systems of various parts of metabolism and sizes creating a modular platform for kinetic analysis of cellular operations.

2014

Structural characterisation and membrane insertion of M13 procoat, M13 coat and Pf3 coat proteins

Membrane proteins fulfill many central functions in the biological membrane. The insertion process of these proteins and their structure, which are intimately linked to their function, are not yet well understood. As a model we studied three proteins reconstituted into lipid bilayers (which serve as a model for the biological membrane): the major coat proteins of phages Pf3 and M13 and the precursor of the latter, M13 procoat protein, which spans the inner membrane two times and serves as a model for a multi-spanning membrane protein. Whereas Pf3 coat protein can insert into the membrane in its mature form, M13 coat protein needs the signal sequence present in the precursor to insert. A new, simple and efficient purification method has been developed for M13 procoat, M13 and Pf3 coat proteins. Homogenous preparations were obtained in isopropanol/0.l% TFA, where M13 coat protein is found to be dissolved in a monomeric form, and the two other proteins as dimers. The conformations of these particular proteins in different environments have been determined by circular dichroism and infrared spectroscopy. In organic solvents, the proteins adopt a conformation with an average helix content of 90%. In lipid bilayers composed of phospatidylcholine and phosphatidylglycerol lipids, the average helix content is 50% for M13 procoat protein, 60% for M13 coat protein and 75% for Pf3 coat protein. In order to characterize the orientational distribution the orientational order parameter Sα of the protein helices in planar lipid bilayers have been determined by polarized infrared measurements in the amide I spectral range. The helices of the three proteins are oriented preferentially parallel to the membrane normal, with Sα = 0.63 for M13 procoat protein, Sα = 0.58 for Pf3 coat protein and a distinctly higher value of Sα = 0.81 for M13 coat protein. The topology of M13 and Pf3 coat proteins was investigated using limited proteolysis in lipid vesicles with proteinase K combined with mass spectroscopy to analyze the lytic fragments. The C-terminal part of both proteins was found to be inaccessible to proteinase K, only 14 (Pf3) or 15 (M13) residues on the N-terminal were protease accessible from the outside of the vesicles. Furthermore, the standard free energy change, ΔGo, of a membrane-inserting protein with a leader sequence has been determined experimentally for the first time, using M13 procoat protein as an example. The partition coefficient for the distribution of this protein between the aqueous phase and the membrane phase of preformed lipid vesicles yielded a value of Γ = 6.5×105 M-1, corresponding to a ΔGo of -10.4 kcal/mol, based on measurements of the fluorescence energy transfer between the intrinsic tryptophan of the protein and a suitably labeled lipid membrane of POPC. For comparison, the partition coefficient of the M13 coat protein between the aqueous and the POPC lipid bilayer phase was determined to be distinctly lower: Γ = 1×105 M-1 (ΔGo = -9.3 kcal/mol). Proteinase K digestion experiments have been performed, showing that 20% of the procoat protein bound to lipid vesicles spontaneously integrate in a transbilayer form, whereas 80% remain inserted in the interfacial membrane region. By taking together these results, an upper limit for the free energy change of the transmembrane insertion of procoat protein was estimated to be -14.8 kcal/mol. In order to distinguish further the contribution arising from insertion of the procoat protein into the membrane interfacial region from that due to transmembrane insertion, the partition coefficient of a mutant procoat protein OM30R (which contains a positively charged amino acid in its mature hydrophobic segment (exchange of a Val to an Arg residue at position 30)) was determined, yielding Γ = 0.3×105 M-1 (ΔGo = -8.6 kcal/mol). Previously reported in vivo experiments have shown that the OM30R mutant protein is not translocated across Escherichia coli inner membrane, but only binds to the inner surface. The results presented here indicate that although the insertion of the procoat protein into the interfacial region of the lipid bilayer contributes the major part to ΔGo, it is the final energy gain of the interaction of the hydrophobic portions of the folded pre-protein with the lipid chains which drives the transmembrane insertion of the M13 procoat protein. Neither the leader sequence nor the mature coat protein alone yields this free energy gain. For the different proteins investigated here, spontaneous membrane insertion occurs only for fluid lipid bilayers, but not for membranes in the crystalline lipid phase. Furthermore, by using lipid bilayers with negative membrane surface charges, it was shown that both procoat and coat proteins are electrostatically attracted to the surface of the lipid membrane, though only to a small extent, with apparent partition coefficients of the same order of magnitude as for the phosphatidylcholine lipid membrane. This work has yielded new information about the structure and the insertion of the phage coat proteins. The new methods developed in this work might be of general applicability to study the structural dynamics of membrane protein assembly.

EPFL1996