Structural and functional basis for lipid synergy on the activity of the antibacterial peptide ABC transporter McjD

The lipid bilayer is a dynamic environment that consists of a mixture of lipids with different properties that regulate the function of membrane proteins; these lipids are either annular, masking the protein hydrophobic surface, or specific lipids, essential for protein function. In this study, using tandem mass spectrometry, we have identified specific lipids associated with the Escherichia coli ABC transporter McjD, which translocates the antibacterial peptide MccJ25. Using non-denaturing mass spectrometry, we show that McjD in complex with MccJ25 survives the gas-phase. Partial delipidation of McjD resulted in reduced ATPase activity and thermostability as shown by Circular Dichroism, both of which could be restored upon addition of defined E. coli lipids. We have resolved a phosphatidylglycerol lipid associated with McjD at 3.4 Å resolution, while molecular dynamic simulations carried out in different lipid environments assessed the binding of specific lipids to McjD. Combined, our data show a synergistic effect of zwitterionic and negatively charged lipids on the activity of McjD; the zwitterionic lipids provide structural stability to McjD whereas the negatively charged lipids are essential for its function

In silico analysis of membrane transport/permeability mechanisms

Lipid membranes are a fundamental component of living cells, mediating the physical separation of intracellular components from the external environment, as well as the different cellular organelles from cytoplasm. Transmembrane transport proteins confer permeability to lipid membranes, which is essential for nutrient translocation and energy metabolism. Crystallography of transmembrane proteins is a particularly challenging problem. Due to their natural localization and chemical properties only a limited number of structures are to date available at atomic resolution. In silico analysis can be successfully applied to address the structure and to propose testable models of transporters and pores and of their function. My PhD work focused on two main models: Pendrin (SLC26A4) and the Permeability Transition Pore (PTP). These two systems allowed me to investigate different membrane types and permeation mechanisms, i.e. the plasma membrane-specific anion exchange (SLC26A4) and the inner mitochondrial membrane (IMM) unselective PTP. Pendrin mutations are estimated to be the second most common genetic cause of human deafness, but a precise 3D structure of the protein is still missing. Aim of my work was to obviate the absence of structural information for pendrin transmembrane domain and to give a functional explanation for mutations collected in the MORL Deafness Variation Database. The human pendrin 3D model was inferred by homology with SLC26Dg and then validated analyzing the surface distribution of hydrophobic residues. The resulting high quality model was used to map 147 pathogenic human mutations. Three mutation clusters were found, while their localization suggested an innovative 14 transmembrane domain structure for pendrin. The nature of PTP has long remained a mystery. In 2013 Giorgio et. al. suggested dimers of F1FO (F)-ATP synthase to form the pore, however the exact PTP composition and how can a pore form from the energy-conserving enzyme is still matter of debate. PTP opening is triggered by an increased Ca2+ concentration in the mitochondrial matrix, and is favored by oxidative stress. To shed light on PTP function, I investigated the effect of Ca2+ binding to the Me2+ binding site of the F1 domain of F-ATP synthase through molecular dynamics (MD) simulations. A similar approach was also applied to the F-ATP synthase β subunit mutation T163S, which alters the relative affinity for Mg2+ and Ca2+. Experimental data show that Ca2+ binding stiffens the complex structure and that the T163S mutation induces resistance to PTP opening. Further, catalytic site rearrangement induced from different ion occupancy, as well as the mutation T163S, yields relevant variation of the interaction between F1 domain and OSCP subunit. I suggest that an unstructured loop between residues 82-131 of the β subunit transmits the structural rearrangement originated into catalytic site to the OSCP subunit and then to the inner membrane through the rigid lateral stalk. The critical role emerging for OSCP in the PTP regulation opens two parallel questions, i.e. (i) how the OSCP-mediated opening signal is transmitted to the trans-membrane region and (ii) what are the transmembrane PTP components. Variation in pore conductivity among species suggested that the putative pore-forming subunits may be different in different species. Sequence alignment was performed for all the subunits of F-ATP synthase, but we mainly focused on subunits e, g and b due to their localization in the complex and sequence conservation. Specific mutations affecting F-ATP synthase were collected and their functional effect is currently under analysis. In parallel, the presence and features of e, g and f subunits across eukaryotes was investigated by mean of phylogenetic analysis. Protein homologues of these specific subunits were found to be widespread in eukaryotes from yeast to plants while we found that Oomycetes lack subunits e and g and green algae subunit e. This observation suggest an ancient evolution for the F-ATP synthase dimerization subunits and possibly for the PTP. Further analysis and experimental validation are planned to clarify this aspect

Computational Modeling of Realistic Cell Membranes

Cell membranes contain a large variety of lipid types and are crowded with proteins, endowing them with the plasticity needed to fulfill their key roles in cell functioning. The compositional complexity of cellular membranes gives rise to a heterogeneous lateral organization, which is still poorly understood. Computational models, in particular molecular dynamics simulations and related techniques, have provided important insight into the organizational principles of cell membranes over the past decades. Now, we are witnessing a transition from simulations of simpler membrane models to multicomponent systems, culminating in realistic models of an increasing variety of cell types and organelles. Here, we review the state of the art in the field of realistic membrane simulations and discuss the current limitations and challenges ahead

Computational Approaches to Simulation and Analysis of Large Conformational Transitions in Proteins

abstract: In a typical living cell, millions to billions of proteins—nanomachines that fluctuate and cycle among many conformational states—convert available free energy into mechanochemical work. A fundamental goal of biophysics is to ascertain how 3D protein structures encode specific functions, such as catalyzing chemical reactions or transporting nutrients into a cell. Protein dynamics span femtosecond timescales (i.e., covalent bond oscillations) to large conformational transition timescales in, and beyond, the millisecond regime (e.g., glucose transport across a phospholipid bilayer). Actual transition events are fast but rare, occurring orders of magnitude faster than typical metastable equilibrium waiting times. Equilibrium molecular dynamics (EqMD) can capture atomistic detail and solute-solvent interactions, but even microseconds of sampling attainable nowadays still falls orders of magnitude short of transition timescales, especially for large systems, rendering observations of such "rare events" difficult or effectively impossible. Advanced path-sampling methods exploit reduced physical models or biasing to produce plausible transitions while balancing accuracy and efficiency, but quantifying their accuracy relative to other numerical and experimental data has been challenging. Indeed, new horizons in elucidating protein function necessitate that present methodologies be revised to more seamlessly and quantitatively integrate a spectrum of methods, both numerical and experimental. In this dissertation, experimental and computational methods are put into perspective using the enzyme adenylate kinase (AdK) as an illustrative example. We introduce Path Similarity Analysis (PSA)—an integrative computational framework developed to quantify transition path similarity. PSA not only reliably distinguished AdK transitions by the originating method, but also traced pathway differences between two methods back to charge-charge interactions (neglected by the stereochemical model, but not the all-atom force field) in several conserved salt bridges. Cryo-electron microscopy maps of the transporter Bor1p are directly incorporated into EqMD simulations using MD flexible fitting to produce viable structural models and infer a plausible transport mechanism. Conforming to the theme of integration, a short compendium of an exploratory project—developing a hybrid atomistic-continuum method—is presented, including initial results and a novel fluctuating hydrodynamics model and corresponding numerical code.Dissertation/ThesisDoctoral Dissertation Physics 201

Uncovering the molecular mechanisms of cardiac ion channels’ regulation by lipids and pore formation in membranes using computer simulations

Membranes are complex cellular structures consisting of many different lipid types, a variety of bound proteins, and other molecules. Growing evidence suggests that membranes and lipids play significant bioactive roles in modulating protein function across several cellular processes. Molecular dynamic (MD) simulations have proven to be a valuable method to study lipid organization and membrane protein activity. In this thesis, I used MD simulations to study how lipids regulate two types of membrane proteins: ion channels and pore-forming proteins. Previous simulations and experimental studies showed that polyunsaturated fatty acids (PUFAs) activate KCNQ1 channels while blocking hERG channels. However, some questions regarding how the channel state or PUFA structural properties influence their molecular mechanisms remained unclear. In part of my work, I built a cardiomyocyte membrane model to study the molecular mechanism underlying the interactions between PUFAs and two voltage-gated potassium channels involved in the cardiac action potential: KCNQ1 and hERG. My results revealed that when KCNQ1 voltage sensor domain (VSD) was in the resting state or ‘down’ conformation, the PUFAs established short-lasting interactions that were different from the long-lasting interactions previously observed in the KCNQ1 intermediate state, where the VSD is in the ‘up’ conformation. Additionally, my studies showed that the number of double bonds in the PUFA acyl tail and the size of the polar head regulates their affinity for KCNQ1. Moreover, MD simulations of the hERG channel in the cardiomyocyte membrane unveiled the PUFA interacting site on hERG at the interface between the VSD and the PD in the open and closed states. I anticipate that this detailed molecular understanding of how PUFAs interact with KCNQ1 and hERG will aid in developing future drugs that utilize these mechanisms. As part of this work, I also studied the pore-forming mechanism of the N-terminal peptide StII1-30, derived from the actinoporin StII. My results revealed that this peptide followed a toroidal pore formation mechanism. Additionally, I unveiled the role of curved lipids as cofactors in the formation of toroidal pores. This work has the potential to lead to strategies for the rational use of these peptides as immunotoxins for immunotherapy in cancer tumors. The overall work in this thesis enhances our understanding of lipid-protein interactions in voltage-gated ion channels and the mechanism underlying pore formation by lytic peptides

Emerging Diversity in Lipid-Protein Interactions

Membrane lipids interact with proteins in a variety of ways, ranging from providing a stable membrane environment for proteins to being embedded in to detailed roles in complicated and well-regulated protein functions. Experimental and computational advances are converging in a rapidly expanding research area of lipid-protein interactions. Experimentally, the database of high-resolution membrane protein structures is growing, as are capabilities to identify the complex lipid composition of different membranes, to probe the challenging time and length scales of lipid-protein interactions, and to link lipid-protein interactions to protein function in a variety of proteins. Computationally, more accurate membrane models and more powerful computers now enable a detailed look at lipid-protein interactions and increasing overlap with experimental observations for validation and joint interpretation of simulation and experiment. Here we review papers that use computational approaches to study detailed lipid-protein interactions, together with brief experimental and physiological contexts, aiming at comprehensive coverage of simulation papers in the last five years. Overall, a complex picture of lipid-protein interactions emerges, through a range of mechanisms including modulation of the physical properties of the lipid environment, detailed chemical interactions between lipids and proteins, and key functional roles of very specific lipids binding to well-defined binding sites on proteins. Computationally, despite important limitations, molecular dynamics simulations with current computer power and theoretical models are now in an excellent position to answer detailed questions about lipid-protein interactions

An investigation of 5-fluorouracil resistance in Leishmania and Trypanosoma species

Leishmaniasis is a parasitic vector-borne disease caused by the Leishmania parasite, which resides in female sandflies. African sleeping sickness or African trypanosomiasis is also a parasitic disease but it is spread by the tsetse fly (Glossina species). Chagas disease or American trypanosomiasis is a tropical disease caused by Trypanosoma cruzi and spread by insects called kissing bugs, Triatominae. Drug resistance has been one of the most important obstacles to the treatment of leishmaniasis and trypanosomiasis. For example, there has been evidence of resistance to melarsoprol and pentamidine for gHAT, and eflornithine for late stage HAT particularly in the T. b. rhodesiense, and pentavalent antimonials for leishmaniasis. This has limited the treatment options for these diseases. This has limited the treatment options for these diseases. Recent evidence has shown that pyrimidine metabolism is an excellent anti-protozoan drug development target, with multiple enzymes that are genetically essential. Pyrimidine nucleobase and nucleoside analogues have shown promising activity against Leishmania and Trypanosoma spp. Drugs like 5-fluorouracil and 5-fluoro-2’deoxyuridine are rapidly metabolized by the parasites into metabolic intermediates such as 5F-UDP-glucose, 5F-2’dUMP, 5F-UDP-galactose and 5F-UDP-N-acetylglucosamine, and incorporated into RNA. Pyrimidine analogue 5-FU was found to be a good inhibitor of high-affinity uracil transporters in T. b. brucei (TbU1 and TbU3) and Leishmania (LmajU1 and LmexU1). Although the transporters for therapeutically active nucleobase allopurinol and antiparasitic nucleoside analogues have been identified, the transporter for 5-FU is still unknown. However, following the exclusion method, it is concluded that the 5-FU transporter is not an ENT transporter in Trypanosoma and Leishmania spps as their ENT transporters have all been cloned and characterised. Hence, our main interest is identifying the transporter gene (family) of kinetoplastids for pyrimidine nucleobases, using the antimetabolite 5-FU as a probe. It is expected that the 5-FU transporter is not of a gene family that has been previously associated with that activity. Resistance to 5-FU was generated in both T. b. brucei s427-wild type BSF and L. mexicana promastigotes, producing clonal lines Tbb-5FURes and Lmex-5FURes, respectively. RNA-seq and RIT-seq analyses of 5-FU resistant cell lines have identified candidate genes for pyrimidine transporters, including genes annotated as cation transporters (Tbb-CAT1-4), fatty acid desaturase (Tbb-FAD and Lmex-FAD) and glucose transporters. Apart from some of the glucose transporters, none of these potential transport genes have been previously characterised in protozoa and as such they are of interest in their own right as well. Using the Alamar blue assay, the sensitivity to 5-FU in a single knockout of Tbb-CAT1-4 genes in T. b. brucei s427 WT cells was determined, and found to have no significant difference. Also, the results showed that [3H]-uracil uptake in T. b. brucei s427 WT + Tbb-CAT1-4+/- was almost the same as in wild type cells. Further, according to our results, the overexpression of Lmex-FAD gene in Lmex-5FURes and Tbb-FAD gene in Tbb-5FURes did not cause increased sensitivity to 5-FU in vitro, and similarly, did not change the rate of transport of [3H]-uracil. Following a full knockout of glucose transporter genes, their sensitivity to 5-FU was determined, revealing a significantly reduced sensitivity of the LmexGT1-3 double knockout genes in L. mexicana to 5-FU, in comparison to the wild type cell lines. Our results also revealed that the Lmex-GT1-3 KO cells do not accumulate 5-FU and uracil. In the re-expression of single LmexGT in Lmex-GT1-3 KO cells, the sensitivity to 5-FU increased significantly, but not quite back to the level of wild-type cells. After the introduction of the glucose transporter genes, all the three genes did appear to have a very similar ability of functioning with regards to the (regulation of) uptake of 5-FU and uracil, restoring uptake to ~50% of 5-FU and ~30% of uracil uptake of wild type, respectively. It was also discovered that 5-FU and uracil did not have any measurable effects on the transport of glucose by LmexGT1, LmexGT2 and LmexGT3, an indication that none of them inhibits the transport of 0.1 μM of [3H]-2-deoxy-D-glucose up to 2.5 mM and therefore, the GTs are not themselves transporting uracil. We successfully expressed and characterized the FurD transporter in the 5-FU resistant cell lines (Tbb-5FURes and Lmex-5FURes) in order to investigate whether the sensitivity to 5-FU in vitro resistant strains could be restored by the introduction of a confirmed uracil/5-FU transporter. This would allow a functional screening of potential transporter genes. However, we found that the EC50 values of 5-FU of the 5-FU resistant cell lines and the FurD-expressing cell lines were not significantly different, although the expression of FurD in Lmex-5FURes induced a very high level of [3H]-uracil/5-FU uptake, even much above the wild type activity. We also characterised the transport activity of FurD in Lmex-5FURes promastigotes, and found FurD to be a highly selective and high-affinity transporter for uracil with Km of 0.97 ± 0.17 μM. Interestingly, our results confirmed that the anticancer drug 5-FU was as good a substrate as uracil for FurD in Lmex-5FURes, with a Ki of 0.76 ± 0.25 μM. Using a targeted CRISPR-Cas9 gene knockout strategy, we show that deletion of the LmexNT1 locus in L. mexicana-Cas9 promastigotes completely abolished adenosine and thymidine uptake. Moreover, it became highly resistant to tubercidin (and its analogues) and to 5-fluoro-2’-deoxyuridine. We also tested the possibility of using L. mexicana-Cas9ΔNT1 promastigotes as a surrogate system for the expression of TcrNT2 and TcrNB2 transporters of T. cruzi. We found TcrNT2 to be a high-affinity thymidine transporter with a Km 0.156 ± 0.017 μM, while TcrNB2 could not be characterised despite our efforts. To conclude, the results obtained in this study provide significant contributions to the identification of the pyrimidine transporter genes. The results also increase our understanding of the cytotoxic activity of 5-FU in kinetoplastid parasites, gaining insight into the complex pyrimidine metabolism that occurs in these parasites. In addition, the study shows that pyrimidine transport mechanisms could potentially be exploited as drug carriers against kinetoplastid parasites