Computational microscopy of the supramolecular organization of the respiratory chain complexes

Dit proefschrift gaat over de associatie van eiwitcomplexen uit de ademhalingsketen in sterk georganiseerde structuren, zogenaamde supercomplexen (SC). Deze dissertatie gaat met name over de betrokkenheid van het membraan in dit associatieproces en de mogelijke rol van cardiolipine (CL), één van de hoofdcomponenten van het membraan. De belangrijkste hypothese is dat CL bijdraagt aan de stabiliteit van de ademhalingsketen door de complexen aan elkaar te ‘lijmen’.Met behulp van coarse grained moleculair dynamische (CGMD) simulatie technieken heb ik twee hoofdhypothesen onderzocht: 1) of CLs betrokken zijn in de stabilisatie van het associatieproces van de complexen tot supercomplexen; 2) aannemend dat er CL bindingsplaatsen zijn, kan men zich twee verschillende werkingsmechanismen indenken: CL kan zich als een slot of als een brug gedragen. In beide gevallen voorkomt CL ongunstige interacties door het afdekken van delen van het oppervlak van de complexen. De eerste hypothese wordt uitgewerkt in hoofdstukken III en IV van dit proefschrift, voor respectievelijk complex III (CIII, cytochrome bc1 complex) en complex IV (CIV, cytochrome c oxidase) van de ademhalingsketen. De tweede hypothese wordt beoordeeld door naar de zelf-associatie te kijken van de twee complexen en wordt gepresenteerd in hoofdstuk V. In een poging om de snelheid van de CGMD simulaties nog meer te verhogen heb ik een model ontwikkeld, dit wordt gepresenteerd in hoofdstuk VI. Het aantal vrijheidsgraden van het systeem wordt in dit model gereduceerd door het weghalen van de water fase, die meestal een aanzienlijk deel van de simulatie doos vormt.This thesis addresses the association of the protein complexes of the respiratory chain into highly organized structures called supercomplexes (SC). More specifically, the thesis focuses on the involvement of the membrane in this association process, and the potential roles of one of its main lipidic components, cardiolipin (CL). The main hypothesis being CL would contribute to the stability of the respiratory chain assembly by “gluing” the complexes together.Using the coarse grain molecular dynamics (CGMD) simulation technique, I investigated two main hypotheses: 1) if CLs are involved in stabilizing the association of the complexes into supercomplexes; 2) assuming the presence of CL binding sites, one can imagine two potential mechanisms of action: CL could act as locks or bridges. In both cases, unfavorable interactions between complexes are avoided by CL capping parts of the complex surfaces. The first hypothesis is developed in the Chapters III and IV of this thesis, for the respective complex III (CIII, cytochrome bc1 complex) and complex IV (CIV, cytochrome c oxidase) of the respiratory chain. The second hypothesis was then assessed by looking at the self-assembly of the two complexes and is presented in Chapter V. In an effort to further increase the speed of CGMD simulations, I developed a new model presented in Chapter VI. The number of degrees of freedom of the system was reduced by removing the aqueous phase, which usually represents a significant part of a simulation box

The flitting of electrons in complex I: A stochastic approach

AbstractA stochastic approach based on the Gillespie algorithm is particularly well adapted to describe the time course of the redox reactions that occur inside the respiratory chain complexes because they involve the motion of single electrons between the individual unique redox centres of a given complex. We use this approach to describe the molecular functioning of the peripheral arm of complex I based on its known crystallographic structure and the rate constants of electron tunnelling derived from the Moser and Dutton phenomenological equations. There are several possible electrons pathways but we show that most of them take the route defined by the successive sites and redox centres: NADH+ site – FMN – N3 – N1b – N4 – N5 – N6a – N6b – N2 – Q site. However, the electrons do not go directly from NADH towards the ubiquinone molecule. They frequently jump back and forth between neighbouring redox centres with the result that the net flux of electrons through complex I (i.e. net number of electrons reducing a ubiquinone) is far smaller than the number of redox reactions which actually occur. While most of the redox centres are reduced in our simulations the degree of reduction can vary according to the individual midpoint potentials. The high turnover number observed in our simulation seems to indicate that, in the whole complex I, one or several slower step(s) follow(s) the redox reactions involved in the peripheral arm. It also appears that the residence time of FMNH• and SQ• (possible producers of ROS) is low (around 4% and between 1.6% and 5% respectively according to the values of the midpoint potentials). We did not find any evidence for a role of N7 which remains mainly reduced in our simulations. The role of N1a is complex and depends upon its midpoint potential. In all cases its presence slightly decreases the life time of the flavosemiquinone species. These simulations demonstrate the interest of this type of model which links the molecular physico-chemistry of the individual redox reactions to the more global level of the reaction, as is observed experimentally

Molecular mechanism of cardiolipin-mediated assembly of respiratory chain supercomplexes

We reveal the molecular mechanism by which cardiolipin glues respiratory complexes into supercomplexes. This mechanism defines a new biophysico-chemical pathway of protein–lipid interplay, with broad general implications for the dynamic organization of crowded cell membranes.</p

High-Throughput Simulations Reveal Membrane-Mediated Effects of Alcohols on MscL Gating

The mechanosensitive channels of large conductance (MscL) are bacterial membrane proteins that serve as last resort emergency release valves in case of severe osmotic downshock. Sensing bilayer tension, MscL channels are sensitive to changes in the bilayer environment and are, therefore, an ideal test case for exploring membrane protein coupling. Here, we use high-throughput coarse-grained molecular dynamics simulations to characterize MscL gating kinetics in different bilayer environments under the influence of alcohols. We performed over five hundred simulations to obtain sufficient statistics to reveal the subtle effects of changes in the membrane environment on MscL gating. MscL opening times were found to increase with the addition of the straight-chain alcohols ethanol, octanol, and to some extent dodecanol but not with hexadecanol. Increasing concentration of octanol increased the impeding effect, but only up to 10–20 mol %. Our in silico predictions were experimentally confirmed using reconstituted MscL in a liposomal fluorescent efflux assay. Our combined data reveal that the effect of alcohols on MscL gating arises not through specific binding sites but through a combination of the alcohol-induced changes to a number of bilayer properties and their alteration of the MscL–bilayer interface. Our work provides a key example of how extensive molecular simulations can be used to predict the functional modification of membrane proteins by subtle changes in their bilayer environment

The Integrin Receptor in Biologically Relevant Bilayers: Insights from Molecular Dynamics Simulations

Integrins are heterodimeric (αβ) cell surface receptors that are potential therapeutic targets for a number of diseases. Despite the existence of structural data for all parts of integrins, the structure of the complete integrin receptor is still not available. We have used available structural data to construct a model of the complete integrin receptor in complex with talin F2–F3 domain. It has been shown that the interactions of integrins with their lipid environment are crucial for their function but details of the integrin/lipid interactions remain elusive. In this study an integrin/talin complex was inserted in biologically relevant bilayers that resemble the cell plasma membrane containing zwitterionic and charged phospholipids, cholesterol and sphingolipids to study the dynamics of the integrin receptor and its effect on bilayer structure and dynamics. The results of this study demonstrate the dynamic nature of the integrin receptor and suggest that the presence of the integrin receptor alters the lipid organization between the two leaflets of the bilayer. In particular, our results suggest elevated density of cholesterol and of phosphatidylserine lipids around the integrin/talin complex and a slowing down of lipids in an annulus of ~30 Å around the protein due to interactions between the lipids and the integrin/talin F2–F3 complex. This may in part regulate the interactions of integrins with other related proteins or integrin clustering thus facilitating signal transduction across cell membranes

Parameterization of a coarse-grained model of cholesterol with point-dipole electrostatics

© 2018, Springer Nature Switzerland AG. We present a new coarse-grained (CG) model of cholesterol (CHOL) for the electrostatic-based ELBA force field. A distinguishing feature of our CHOL model is that the electrostatics is modeled by an explicit point dipole which interacts through an ideal vacuum permittivity. The CHOL model parameters were optimized in a systematic fashion, reproducing the electrostatic and nonpolar partitioning free energies of CHOL in lipid/water mixtures predicted by full-detailed atomistic molecular dynamics simulations. The CHOL model has been validated by comparison to structural, dynamic and thermodynamic properties with experimental and atomistic simulation reference data. The simulation of binary DPPC/cholesterol mixtures covering the relevant biological content of CHOL in mammalian membranes is shown to correctly predict the main lipid behavior as observed experimentally

Mechanism of allosteric regulation of β2-adrenergic receptor by cholesterol

There is evidence that lipids can be allosteric regulators of membrane protein structure and activation. However, there are no data showing how exactly the regulation emerges from specific lipid-protein interactions. Here we show in atomistic detail how the human b2- adrenergic receptor (b2AR) – a prototypical G protein-coupled receptor – is modulated by cholesterol in an allosteric fashion. Extensive atomistic simulations show that cholesterol regulates b2AR by limiting its conformational variability. The mechanism of action is based on the binding of cholesterol at specific high-affinity sites located near the transmembrane helices 5–7 of the receptor. The alternative mechanism, where the b2AR conformation would be modulated by membrane-mediated interactions, plays only a minor role. Cholesterol analogues also bind to cholesterol binding sites and impede the structural flexibility of b2AR, however cholesterol generates the strongest effect. The results highlight the capacity of lipids to regulate the conformation of membrane receptors through specific interactions.There is evidence that lipids can be allosteric regulators of membrane protein structure and activation. However, there are no data showing how exactly the regulation emerges from specific lipid-protein interactions. Here we show in atomistic detail how the human b2- adrenergic receptor (b2AR) – a prototypical G protein-coupled receptor – is modulated by cholesterol in an allosteric fashion. Extensive atomistic simulations show that cholesterol regulates b2AR by limiting its conformational variability. The mechanism of action is based on the binding of cholesterol at specific high-affinity sites located near the transmembrane helices 5–7 of the receptor. The alternative mechanism, where the b2AR conformation would be modulated by membrane-mediated interactions, plays only a minor role. Cholesterol analogues also bind to cholesterol binding sites and impede the structural flexibility of b2AR, however cholesterol generates the strongest effect. The results highlight the capacity of lipids to regulate the conformation of membrane receptors through specific interactions.Peer reviewe