A Closer Look into G Protein Coupled Receptor Activation: X-Ray Crystallography and Long-Scale Molecular Dynamics Simulations

G protein coupled receptors (GPCRs) are a large eukaryotic protein family of transmembrane receptors that react to a signal coming from the extracellular environment to generate an intracellular response through the activation of a signal transduction pathway mediated by a heterotrimeric G protein. Their diversity, dictated by the multiplicity of stimuli to which they respond and by the variety of intracellular signalling pathways they activate, make them one of the most prominent families of validated pharmacological targets in biomedicine. In recent years, major breakthroughs in structure determination of GPCRs have given new stimuli to the exploration of the biology of these proteins, providing a structural basis to understand the molecular origin of GPCR mechanisms of action. Based on the information coming from these structural studies, a number of recent in silico investigations used molecular dynamics (MD) simulations to contribute to our knowledge of GPCRs. In this review, we will focus on investigations that, taking advantage of the tremendous progress in both hardware and software, made testable hypotheses that have been validated by subsequent structural studies. These state-of-the-art molecular simulations highlight the potential of microsecond MD simulations as a valuable tool in GPCR structural biology and biophysics.

Computational Investigation of Intracellular Signalling Cascades

Siri Camee van Keulen

In order to adjust to changes in the environment, cells can communicate via signalling cascades. A common pathway for intercellular communication is the G-protein-coupled- receptor (GPCR) signal transduction cascade which is triggered by a (chemical or physical) extracellular signal, such as light or small molecules. This external interaction induces conformational changes that can lead either to GPCR activation or deactivation. In the active form, GPCRs can induce the activation of trimeric G proteins, which results in the dissociation of the Galpha from the beta and gamma subunits. Subsequently, active Galpha can interact with other cytosolic proteins, such as adenylyl cyclase (AC), which converts adenosine triphosphate to cyclic adenosine monophosphate (cAMP) upon stimulation. AC activation can be induced via stimulatory Galpha subunit (Galpha_s) interactions, while inhibition is related to inhibitory Galpha (Galpha_i) association. X-ray crystallography is crucial for the computational study of the GPCR signal transduction pathway. However, sometimes important molecules or moieties can become lost or cannot be incorporated during crystallisation procedures, which can have a significant effect on a protein’s conformation and function. In this thesis, three steps in the GPCR signal transduction cascade have been studied, rhodopsin activation (i), the conformation of active Galpha_i (ii) and the interaction between Galpha_i and AC (iii). In all three cases some groups or molecules that are absent in the X-ray structures play a vital role in the function of the proteins. In the first project, rhodopsin’s early intermediates after photon exposure are investigated together with a deprotonation reaction that takes place in the later stages of the activation pathway. The classical molecular dynamics (MD) and mixed quantum mechanics/molecular mechanics MD results suggest that photon exposure induces active site rearrangements which increase the stability of rhodopsin’s deprotonated Schiff base state. A bridging water molecule in the active site that is absent in the X-ray structure appears to play an important role during the deprotonation mechanism, which demonstrates the dependence of the protein’s function on its environment. The second study investigates the conformation of the active soluble form of Galpha_i, which has not yet been fully resolved via experimental studies due to crystallisation difficulties of the lipidated N-terminus. To investigate the effect of lipidation via classical MD simulations, a model of soluble lipid-bound Galpha_i was constructed. The simulations show that Galpha_i can form a hydrophobic pocket for the lipid on the protein surface. Other regions of the protein, important for protein-protein interaction, are adjusted as well. Hence, the post-translational modification appears to have a significant impact on Galpha_i’s active conformation and function, which was not captured via the unlipidated X-ray structures. The last project is devoted to a complex of active soluble Galpha_i and AC (Galpha_i :AC). While the interaction site for Galpha_s on AC is known, the interaction site for Galpha_i has not yet been identified. Classical MD simulations were performed on a docked Galpha_i:AC complex to understand the inhibition mechanisms as well as the differentiation between AC stimulation/inhibition. The results show that Galpha_i binding not only leads to AC inhibition via impeding ATP binding but also by preventing re-activation via Galpha_s through closure of the subunit’s interaction site on AC. Keywords: GPCR signal transduction pathway, G-protein-coupled recepter, rhodopsin, G protein, complex formation, cis-trans isomerisation, adenylyl cyclase.

EPFL2017

Studying membrane-protein interplay by modeling realistic conditions

Martina Audagnotto

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

EPFL2017

Early Steps of the Intramolecular Signal Transduction in Rhodopsin Explored by Molecular Dynamics Simulations

Leonardo Guidoni, Ursula Röthlisberger

We present mol. dynamics simulations of bovine rhodopsin in a membrane mimetic environment based on the recently refined x-ray structure of the pigment. The interactions between the protonated Schiff base and the protein moiety are explored both with the chromophore in the dark-adapted 11-cis and in the photoisomerized all-trans form. Comparison of simulations with Glu181 in different protonation states strongly suggests that this loop residue located close to the 11-cis bond bears a neg. charge. Restrained mol. dynamics simulations also provide evidence that the protein tightly confines the abs. conformation of the retinal around the C12-C13 bond to a pos. helicity. 11-cis to all-trans isomerization leads to an internally strained chromophore, which relaxes after a few nanoseconds by a switching of the ionone ring to an essentially planar all-trans conformation. This structural transition of the retinal induces in turn significant conformational changes of the protein backbone, esp. in helix VI. Our results suggest a possible mol. mechanism for the early steps of intramol. signal transduction in a prototypical G-protein-coupled receptor. [on SciFinder (R)]

2002

Computational Investigation of Intracellular Signalling Cascades

Siri Camee van Keulen

EPFL2017

Studying membrane-protein interplay by modeling realistic conditions

Martina Audagnotto

EPFL2017