Computational Investigation of Intracellular Signalling Cascades

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.

Computational characterisation of the selectivity and binding mode of mono- and binuclear transition metal based anticancer compounds

Thibaud Edouard Alistair von Erlach

The rising resistance and the severe side effects associated with classical anticancer drugs such as cisplatin or mercaptopurines has triggered the development of novel antitumourous agents based on transition metal (e.g. ruthenium, osmium, and gold) organometallic complexes. In parallel, the tools of computational biochemistry have become more and more powerful and can nowadays provide important insights into the mechanistic origins of many systems of medicinal relevance creating a synergetic working environment between experimental and theoretical approaches. In this dissertation, a range of computational methods to study molecules from a quantum mechanical and/or classical mechanical point of view have been applied to characterise key properties of biological drug-target complexes. All of the computational research presented herein has been performed in tight collaboration with experimental groups, which determined X-ray structures of drug-target adducts and characterised the anticancer properties of the synthesised novel anticancer drugs studied here. In particular, the interactions of a small ruthenium-based complex acting as an DNA semi-intercalator,[(eta^6-5,8,9,10-tetrahydroanthracene)Ru(II)(ethylenediamine)X] with X representing a leaving group such as Cl-, were characterised exposing different binding modes of the compounds with free vs. nucleosomal DNA. Additionally, classical molecular dynamics simulations were used to complete a characterisation of the localisation of the linker of a novel binuclear antitumourous compound, that was designed in such a way as to take advantage of newly discovered allosteric pathways present in the nucleosome core particle with the aim of creating a synergistic effect in the uptake the two moieties. In addition to the localisation of the linker, the effects of the bound compound on the allostery of the nucleosome core particle has been explored to compare the mode of action of the newly synthesised binuclear compound with that of non-linked analogue with synergistic properties. Moreover, a similar study has been performed on a herpes virus peptide, (Met-Ala-Pro-Pro-Gly-Met-Arg-Leu-Arg-Ser-Gly-Arg-Ser-Thr-Gly-Ala-Pro-Leu-Thr-Arg- Gly-Ser), binding to the nucleosome core particle to observe whether or not it would be able to trigger allosteric pathways. Finally, some fundamental issues related to the selectivity of transition metal based anticancer compounds for specific binding sites on the nucleosome core particle were tackled by simulating the approach of three different ruthenium and osmium-based complexes, [(eta^6-cymene)Ru(II)(1,3,5-triaza-7-phosphoadamantane)X_2], [(eta^6-cymene)Ru(II)(N-phenylpyridine-2-carbothioamide)X] and [(eta^6-cymene)Os(II)( N-phenylpyridine-2-carbothioamide)X], from the bulk solvent to their bound state with the histone proteins with the help of classical and mixed quantum mechanical/molecular mechanical molecular dynamics simulations. A determination and analysis of the corresponding free energy profiles were used to provide some rationalisation of the observed preference of different compounds for specific binding sites. All the computational studies performed here and the insights gained can provide some guidance for the rational design of anticancer compounds with specific binding properties.

EPFL2020

Exploring the inhibition mechanism of adenylyl cyclase type 5 by n-terminal myristoylated Gαi1

Ursula Röthlisberger, Siri Camee van Keulen

Adenylyl cyclase (AC) is an important messenger involved in G-protein-coupled-receptor signal transduction pathways, which is a well-known target for drug development. AC is regulated by activated stimulatory (Gαs) and inhibitory (Gαi) G proteins in the cytosol. Although experimental studies have shown that these Gα subunits can stimulate or inhibit AC's function in a non-competitive way, it is not well understood what the difference is in their mode of action as both Gα subunits appear structurally very similar in a non-lipidated state. However, a significant difference between Gαs and Gαi is that while Gαs does not require any lipidation in order to stimulate AC, N-terminal myristoylation is crucial for Gαi's inhibitory function as AC is not inhibited by non-myristoylated Gαi. At present, only the conformation of the complex including Gαs and AC has been resolved via X-ray crystallography. Therefore, understanding the interaction between Gαi and AC is important as it will provide more insight into the unknown mechanism of AC regulation. This study demonstrates via classical molecular dynamics simulations that the myristoylated Gαi1 structure is able to interact with apo adenylyl cyclase type 5 in a way that causes inhibition of the catalytic function of the enzyme, suggesting that Gα lipidation could play a crucial role in AC regulation and in regulating G protein function by affecting Gαi's active conformation.

Public Library of Science2017

Computational Studies of the Proton-Coupled Metal Ion Transport in the SLC11/NRAMP Family of Transporters

Maria Letizia Merlini

In the last years, it has been demonstrated a link between the overload of metal ions inside nervous system cells and the onset of severe neurodegenerative diseases. This prompted the investigation of the structural and functional properties of transporters responsible for the uptake and regulation of metal ions inside cells. In particular, members of the Nramp/SLC11 ("natural resistance-associated macrophage proteins"/"solute carrier 11") family of transporters tightly regulate the influx of divalent transition-metal ions across cellular membranes contributing to cellular homeostasis. In these secondary active transporters, the translocation of divalent transition metal ions from the extracellular matrix to the cellular cytoplasm is coupled to proton transport (symport). The co-transport is realized using an alternate access mechanism in which the protein is alternately open towards the extracellular matrix, ("outward facing conformation", OFC) or towards the cellular cytoplasm ("inward facing conformation", IFC).Although a wide range of functional and structural studies provided fundamental insights on the proton-coupled transport, most mechanistic details are still unknown. As an example, the coordination sphere of the metal ion in the inward-facing conformation of the protein has only been partially resolved by crystallographic experiments and later attempts to provide comprehensive descriptions led to contradictory models. Furthermore, several experimental evidence proved that proton uniport in the absence of the metal ion, is also possible, suggesting an unusual symporter behaviour for Nramps, in which the two substrates are not tightly coupled. Despite the recent advances in the comprehension of Nramps proton transport, the underlying mechanistic details remain still ambiguous, as demonstrated by inconsistent mechanistic models proposed during the last years.In this thesis, computational methods have been used in order to provide new molecular level insights in the proton-coupled divalent transition metal ions transport performed by Nramps. In particular, a combination of classical molecular dynamics (MD) and QM/MM ab initio MD methods have been applied in order to refine the partially resolved coordination sphere of Mn2+ in the active site of Staphylococcus Capitis Divalent Metal-ion Transporter (ScaDMT), representing the only available crystal structure of a substrate-bound member of the family in an IFC. This QM/MM multiscale approach, using an accurate quantum treatment of the active site and a classical treatment of the surroundings, allowed for state-of-the-art calculations of the entire system that led to the unequivocal identification of the metal's coordination sphere. Furthermore, MD simulations have been performed for both the IFC ScaDMT and Eremococcus Coleocola DMT (EcoDMT), whose crystal structure has been obtained in OFC. Since experimental evidences proved that proton transport can occur even in the absence of the metal ion substrate, we performed simulations of both proteins in their apo forms. These simulations allowed us to characterize four amino acidic residues identified as likely key players in the proton transport process and to determine which role they assume in the process. Finally, we were able to identify the likely primary proton acceptor and proposed a potential route followed by protons in their influx toward the intracellular cytoplasm.