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

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.

Characterization of Protein-Membrane Interfaces through a Synergistic Computational-Experimental Approach

Alessio Prunotto

The characterization of biological interfaces is widely recognized as one of the main challenges for modern biology. In particular, biological membranes are nowadays known to be an active environment that allows membrane proteins to perform their work and modulates their function. Integral and peripheral membrane proteins constitute 1/3 of the human proteome, and account for about 50% of the targets of modern medicinal drugs. Despite their remarkable role, their interplay with the membrane is often poorly characterized, mainly because of the limits that the currently available experimental techniques encounter when treating hydrophobic environments. In particular, peripheral membrane proteins are often studied in their soluble version: this approach is highly limiting, as the interaction with the membrane is essential for the activity of these biomolecules. Here, we show the potential of a combined computational-experimental approach in order to overcome the aforementioned limits. In particular, we use molecular modeling to study two peripheral membrane proteins of interest, and successively design ad hoc wet lab experiments to verify the outcomes and predictions of the simulations. This approach allows to bypass the technical limits and high costs of the wet lab techniques, by guiding the experiments with the data of the computational simulations. We focused our attention on the following peripheral membrane proteins: New Delhi metallo-beta-lactamase (NDM-1). NDM-1 is a bacterial enzyme that causes antibiotic resistance. Within the class of metallo-beta-lactamases, it represents the most serious threat to global health. The larger resistance of NDM-1 with respect to other proteins of the same class, has been linked to its post-translational modification, which connects it to the outer bacterial membrane of Gram-negative bacteria: this event can significantly increase the chances of NDM-1 to spread through the infection through vesicles excretion. In the present work, we elucidated the mechanistic aspects of the NDM-1/bacterial membrane interaction, and identified the features that contribute to the efficiency of this mechanism. Golgi phosphorylated protein 3 (Golph3). Golph3 is a peripheral membrane protein present at the Golgi apparatus of most eukaryotic cells. Its normal function consists in binding glycosylating enzymes, and transport them through the Golgi cisternae. In humans, Golph3 has been found to be overexpressed in several forms of cancer: however, no Golph3 inhibitors are currently present in the pharmaceutical market. This is mainly due to the lack of structural information regarding the molecular mechanism of Golph3. Here, we clarify the features of Golph3 that allow it to bind to the Golgi, and elucidate the mechanism of membrane binding. We also propose a recognition mechanism between Golph3 and the glycoenzymes, based on events predicted by the computer simulations. Overall, in the present work we demonstrate the potential of computational-experimental approaches in structural biology, and in particular in the study of peripheral membrane proteins. We show that a combined approach constitutes the best way of overcoming the limits of each technique, and we discuss the repercussions on the study of systems of biological interest.

EPFL2020

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

Molecular Modeling of Membrane Embedded Proteins

Thomas Lemmin

Over the past years, molecular modeling and simulation techniques have had a major impact on experimental life sciences. They are capable of providing accurate insight into microscopic mechanisms, which are usually difficult to investigate experimentally. Moreover, the integration of experimental data with molecular modeling appears to be a promising strategy to better understand complex biological processes. In this thesis, molecular dynamics simulation has been used in combination with experimental data to investigate two transmembrane proteins: (i) the bacterial chemoreceptor PhoQ and (ii) the Amyloid Precursor Protein (APP). (i) Bacterial two-component system PhoQ and bacterial membranes. Two-component systems (TCSs) are signaling complexes essential for bacterial survival and virulence. PhoQ is the histidine kinase chemoreceptor of the PhoQ-PhoP tandem machine that detects the concentration of cationic species at the inner membrane of Gram-negative bacteria. A full understanding of the PhoQ signal transduction mechanism is currently hindered by the lack of a complete atomistic structure. In this thesis project, the first structural model of the transmembrane (TM) portion of PhoQ from Escherichia coli was assembled, by using molecular simulations integrated with cross-linking disulfide scanning data. Its structural and dynamic features induce a concerted displacement of the TM helices at the periplasmic side, which modulates a rotation at the cytoplasmic end. This supports the idea that signal transduction is promoted through a combination of scissoring and rotational movements of the TM helices. Knowledge of this complex mechanism is essential in order to understand how the chemical stimuli sensed by the periplasmic sensor domain trigger, via the relay of the HAMP domain, the histidine auto-phosphorylation and kinase/phosphatase activity at the cytoplasmic end. The PhoQ sensor domain lies in close proximity to the membrane. Interaction with anionic lipids, such as phosphophatidylglycerols (PG) and cardiolipins (CL), are thought to play a key role in PhoQ activity. Present in bacterial and mitochondrial membranes, cardiolipins have a unique dimeric structure, which carries up to two charges, i.e. one per phosphate group, and under physiological conditions, can be unprotonated or singly protonated. Exhaustive models and characterization of cardiolipins are to-date scarce; therefore an ab initio parameterization of cardiolipin species for molecular simulation consistent with commonly used force fields is proposed here. Molecular dynamics (MD) simulations based on these models indicate a protonation-dependent lipid packing. A noteworthy interaction with solvating mono- and divalent cations is also observed. The proposed models will contribute to the biophysical and biochemical characterizations of bacterial and mitochondrial membranes and membrane-embedded proteins. (ii) Structural and dynamic properties of the Amyloid Precursor Protein. The Amyloid Precursor Protein (APP) is a type I membrane glycoprotein present at the neuronal synapsis. The proteolytic cleavage of its C-terminal segment produces amyloid-β (Aβ) peptides of different lengths, the deposition of which is an early indicator of Alzheimer"s disease (AD). Recently, the backbone structure of the APP transmembrane (TM) domain in detergent micelles was determined by nuclear magnetic resonance (NMR, independently by two different experimental groups). The TM conformations of these two structures are however markedly different. One is characterized by a highly kinked α-helix, whereas the other is mainly straight. Molecular dynamics simulations showed that the APP TM region is highly flexible and its secondary structure is influenced by the surrounding lipid environment. The size of the embedding detergent micelles strongly affects the conformation of the APP α-helix, with solvation being the main driving force for the development of a helical curvature. Once embedded in a membrane bilayer, APP systematically prefers a straight helical conformation. This is also confirmed when analyzing in silico the atomistic APP population observed in double electron-electron resonance (DEER) spectroscopy. In summary, the APP transmembrane domain is highly flexible due to the presence of glycine residues and can readily respond to the lipid environment, a property that might be critical for proteolytic processing by γ-secretase enzymes. The presented thesis work clearly shows how molecular simulations and their interplay with available experimental input can help advance the understanding of the mechanism of complex biological systems and processes on a molecular scale. These results, in particular, go well beyond the current understanding of the functioning of two transmembrane proteins relevant for human health. Furthermore, the computational approaches and procedures developed in these projects will hopefully promote novel integrated strategies for investigating biological systems.

EPFL2013