New Strategies for Integrative Dynamic Modeling of Macromolecular Assembly

Data reporting on structure and dynamics of cellular constituents are growing with increasing pace enabling, as never before, the understanding of fine mechanistic aspects of biological systems and providing the possibility to affect them in controlled ways. Nonetheless, experimental techniques do not yet allow for an arbitrary level of resolution on cellular processes in situ. By consistently integrating a variety of diverse experimental data, molecular modeling is optimally poised to enhance to near-atomistic resolution our understanding of molecular recognition in large assemblies. Within this integrative modeling context, we briefly review in this chapter the recent progresses of molecular simulations at the atomistic and coarse-grained level of resolution to explore protein protein interactions. In particular, we discuss our recent contributions in this field, which aim at providing a robust bridge between novel optimization algorithms and multiscale molecular simulations for a consistent integration of experimental inputs. We expect that, with the ever-growing sampling ability of molecular simulations and the tireless progress of experimental methods, the impact of such dynamic-based approach could only be more effective with time, contributing to provide detailed description of cellular organization.

Molecular Modeling of Bacterial Nanomachineries

Matteo Thomas Degiacomi

Proteins have the ability to assemble in multimeric states to perform their specific biological function. Unfortunately, characterizing experimentally these structures at atomistic resolution is usually difficult. For this reason, in silico methodologies aiming at predicting how multiple protein copies arrange to forma multimeric complex would be desirable. We present Parallel OptimizationWorkbench (POW), a swarm intelligence based optimization framework able to deal, in principle, with any optimization problem. We show that POW can be applied to biologically relevant problems such as prediction of protein assemblies and the parameterization of a Coarse-Grained force field for proteins. By combining POW optimizations, Molecular Dynamics simulations, Poisson-Boltzmann calculations and a variety of experiments, we subsequently study two bacterial nanomachieries: Aeromonas hydrophila's pore-forming toxin aerolysin, and Yersinia enterocolitica injectisome. These structures are challenging both for their size, and for the timescales involved in their functioning. Aerolysin is a pore-forming toxin secreted as an hydrophilic monomer. By means of large conformational changes, the protein heptamerizes on the target cell's surface, and finally inserts β-barrel into its lipid bilayer, causing cell death. The main hurdle in the study of this structure is the complexity of the mode of action, which spans timescales currently unreachable by classical molecular dynamics. We show that aerolysin C-terminal region has the dual role of preventing premature oligomerization and helping the folding of tertiary structure, qualifying therefore as an intramolecular chaperone. We study the transmembrane β-barrel properties and compare them with those of the homologous protein α-hemolysin. We show that aerolysin's barrel is more rigid than α-hemolysin's, and should be anion selective. We present models for aerolysin heptamer both in prepore and, for the first time, in membrane-inserted conformation. Our results are validated experimentally, and are consistent with known biochemical and structural data. The injectisome is an example of a type III secretion system. Its most striking feature is probably its size: hundreds of proteins assemble in a unique structure spanning the Gram-negative bacterial double membrane, and protruding outside the cell as a needle for tenth of nanometers. Obtaining an atomistic representation of this massive structure, and therefore some insights about its mode of action, is one of the greatest challenges. We show that the final length of injectisome's needle is determined by the secondary structure content of a ruler protein located inside its cavity during assembly. Using POW, we also produce the first model for Yersinia injectisome's basal body, highlighting the flexibility of this region in adapting between the inner and outer membranes. As a whole, this work demonstrates that a synergy of dry and wet experiments can provide precious insights into macromolecular structure and function.

EPFL2012

Multiscale simulations of protein dynamics

Enrico Spiga

Characteristic timescales associated with the function of biomolecules, like proteins, range from femtoseconds up to minutes, whereas their corresponding spatial extent ranges from few ̊A to μm when associating in large macromolecular complexes. Moreover, biomolecules are functional in a large variety of different physico-chemical conditions strongly dependent on pH, ionic strength, crowding agents, etc. This huge complexity is hard to be studied with an arbitrary level of resolution embracing all these spatial and temporal scales. Molecular simulation is a well established approach to gain mechanistic insights into the function of biomolecular systems, producing atomistically detailed models of in vitro and/or in vivo conditions. I present in this thesis two projects that aim at improving on the current limitations of multiscale molecular simulations, namely (i) the sampling of large systems, and (ii) the detailed representation and description of realistic physiological conditions. Addressing the first issue, I propose a new coarse-grained model for proteins to be used in molecular dynamics simulations. This coarse-grained model is based on a more accurate description of protein electrostatics, which accounts for dipolar contributions. The parameterization of this force field is based on force-matching methods and on the use of a particle swarm optimization heuristic algorithm. The obtained results are encouraging being structural and electrostatic properties accurately reproduced with the coarse-grained model for a variety of protein folds. Moreover, the parameterization procedure can be straightforwardly applied to any protein, and can be extended to a larger dataset to generate a fully transferable coarse-grained force field, to be applied to any protein and any large macromolecular assemblies for which long all-atom simulations are still a challenge. While the development and use of coarse-grained models are important to tackle the limited sampling of large systems, it is still important to use all-atom molecular dynamics simulation to investigate with high accuracy in physiological conditions protein dynamics. For this reason, I present here the results of state-of-art molecular dynamics simulations applied to study the influence of crowding agents on the internal dynamics of the protein ubiquitin. Their analysis allows to describe how ubiquitin dynamics is slaved by crowding agents. This work demonstrates that the description of protein dynamics should take into account its intrinsic multiscale nature. The development and applications of coarse-grained models permit to simulate proteins at low computational cost, the use of atomistic simulations allows to accurately describe proteins in absence and presence of crowding agents, and both of them permit to highlight the essence of protein dynamics.

EPFL2013

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

Molecular Modeling of Bacterial Nanomachineries

Matteo Thomas Degiacomi

EPFL2012

Molecular Modeling of Membrane Embedded Proteins

Thomas Lemmin

EPFL2013

Multiscale simulations of protein dynamics

Enrico Spiga

EPFL2013