Studying membrane-protein interplay by modeling realistic conditions

Realistic models of cellular environments have long captured the imagination of biologists and physical scientists. More than 20 years ago, Goodshellâs inspiring rendering of cellular environments provided the foundation for the idea of capturing the full complexity of biological cells. Since then, increasingly realistic simulations of subcellular models have appeared, showing that we are rapidly moving towards comprehensive, as well as physically and biologically accurate cellular models. The major challenge in developing such models is the necessity to cover a wide range of scales. Indeed, atomistic details of molecular processes are on the length scale of 0.1 nm, while cellular dimensions range from 300 nm (for the smallest bacterial cells) up to 100 ÎŒm (for large eukaryotic cells). The biological range also encompasses the internal dynamics of individual molecules (from ns to ms) as well as entire biological processes (ms to hours). Although an atomistic representation of an entire cell is conceivable, the cost for achieving biologically significant timescales with most advanced computers is very high or nearly impossible currently. Therefore, the combination of in silico approaches with in vivo and in vitro experimental data, allows gaining further insights into the biophysics of the cell. In this thesis, a multi-scale approach was applied to investigate the interplay between proteins and their respective biological environments. In particular, two specific systems were the objects of my Ph.D. project: (i) the amyloid precursor protein (APP) and its interactions at the synaptic plasma membrane, and (ii) the human acyl-protein thioesterase I (hAPT1) and its role in protein depalmitoylation cycle across the Golgi and plasma membranes. Combining atomistic and coarse-grained molecular dynamics (MD) simulations, the stability of the dimeric conformation of APP in a realistic model of the synaptic plasma membrane was investigated. Within this approach, the stable dimeric conformation of the APP as a function of lipid membrane composition was identified, highlighting the important role of lipids unsaturation in protein stability and function. In detail, this indicated not only the preferential dimerization motif of the protein, but also the possible recruiting mode of cholesterol, which has previously been experimentally observed to correlate with Alzheimerâs disease. Furthermore, the role of the synaptic plasma membrane and its ability to form raft microdomains was explored for the interactions of APP with Î³-secretase, which specific catalytic cleavage is responsible for the production of physiological or toxic amyloid Î² peptides, responsible for the Alzheimerâs disease onset. This same multi-scale approach was applied to understand the role of S-acylation in the trafficking and function of hAPT1. In particular, the combination of experimental techniques (i.e., cell biology, biochemistry, microscopy and structural biology) with molecular modeling and simulation disclosed the substrate/enzyme interaction mode, the enzyme/membrane interface, and the role for hAPT1 for a key post-translational modification as S-palmitoylation. Based on our findings, we provided for the first time an atomistic representation of the hAPT1 enzyme mechanism, suggesting that hAPT1 is active in the monomeric form. In conclusion, these studies demonstrate once more the importance of modeling the molecular details of the biological environment at the most accu

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

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

Folding and Structure of the Pore Forming Toxin Aerolysin

Mircea Ioan Iacovache

The first obstacle encountered by a bacterial pathogen once inside the host is the plasma membrane surrounding the target cells. Throughout evolution bacteria has acquired and maintained genes that upon stimulation express proteins capable of damaging the membrane of other cells. Among these proteins pore forming toxins (PFTs) are a major class of bacterial effectors that are upregulated and secreted during bacterial infections. As their name suggests, pore forming toxins are proteins capable of inserting transmembrane pores in the membranes of the target cells which in turn leads to the lysis of the cell and release of nutrients. The mechanism by which the PFTs function during a bacterial attack has been the subject of extensive research over the years. In most cases PFTs are produced by the bacteria as soluble proteins that require the help of specialized secretion mechanisms to arrive as functional proteins in the external milieu. Once secreted by the producing bacteria these proteins diffuse towards the target cell and bind to the target membrane. Once bound to the plasma membrane of target cells they are capable of initiating a series of structural changes that will eventually lead to the conversion of the water-soluble PFT to a membrane inserted channel. The series of events and the characterization of the different structural changes required for a PFT to convert from a water-soluble protein to a membrane inserted channel is the subject of this thesis. Aerolysin, a PFT produced by Aeromonas hydrophilla, is one of the best candidates for a research into the details of the mode of action of bacterial PFTs. This particular PFT is produced by the bacterium as a soluble periplasmic protein and then secreted outside of the bacterium as a fully folded protein with the help of a type II secretion system. Binding to the target cell is achieved through two high affinity binding sites that recognize sugar modifications which are absent in A. hydrophila, a mechanism that insures that the producing cell is not damaged by its own PFT. Once bound to the target cell aerolysin requires proteolytic activation, a step which cleaves a C-terminal peptide (CTP). Activation is achieved using proteases present on the target cell and the removal of the CTP is thought to initiate the sequence of events leading to pore formation. Following activation aerolysin is able to oligomerize forming heptameric ring-like structures which spontaneously rearrange forming a transmembrane beta-barrel through the membrane. My thesis project, focused on the structural changes required in the mode of action of aerolysin, set off trying to identify the aminoacid sequence involved in the formation of the transmembrane beta-barrel. It was long thought that aerolysin would cross the membrane in a porin like fashin, forming a beta-barrel through the plasma membrane, primarily due to the lack of a hydrophobic patch of aminoacids in its sequence. An initial model proposed in the early '90s postulated that the only region that could form the transmembrane beta-barrel was the Domain 4 of the protein. In this model the removal of the CTP in the activation process would unravel the hydrophobic residues required for the beta-barrel formation and insertion. We and others were able to show however that the fourth domain of the protein is not involved directly in the formation of the pore and we identified a conserved loop in the third domain of the protein which is responsible for the formation of the beta-barrel. This loop presents an alternating pattern of hydrophobic and hydrophilic residues, a requirement for the formation of a transmembrane pore with a hydrophobic exterior and a hydrophilic cavity. Our research led us to propose a sequence of events upon insertion of the aerolysin pore in which a rearrangement of the DIII-loops of the seven monomers in the oligomer forms the initial beta-barrel and generates a hydrophobic tip which drives insertion of the structure through the membrane. Once the bilayer has been crossed the hydrophobic tips folds back on the membrane in a rivet like fashion, anchoring the pore. Following the identification of the DIII-loop as the region that forms the transmembrane pore my researched focused on the structural changes leading to the conversion of a water-soluble protein to a membrane inserted oligomer. While removal of the CTP is the key requirement for this conversion, the role of the CTP in aerolysin mode of action and the sequence of events triggered by its removal is not fully understood. Using a combination of in vivo, in vitro and in silico approaches we were able to show that the CTP plays a wider role in the aerolysin mode of action than previously thought. Indeed our research shows that the CTP is initially required for the correct folding of the soluble protein inside the bacterium, acting as a intramolecular chaperone during the folding of aerolysin. Following folding the CTP binds tightly to a hydrophobic pocket in the fourth domain of the protein locking the PFT in its soluble conformation, a role resembling C-terminal intramolecular chaperones previously described for tail spikes of bacteriophages or fiber forming collagen. This research will be continued with a study on the structural changes triggered by the removal of the CTP and their role in oligomerization and pore formation. The main focus of my thesis project has been however the determination of the structure of the oligomeric form of aerolysin. This part of the project is still ongoing and will be discussed in the final chapter of my thesis. Using 2D and 3D crystallography, AFM and modeling we hope to be able to improve our current understanding of the aerolysin heptameric form and the structural changes required in its formation.

EPFL2011