Biochemistry and Pharmacology - Theses
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ItemUnderstanding the molecular mechanism of AAA+ ATPase p97 in complex with different cofactors( 2023-10)p97 has emerged as an attractive target for treatment of cancer, neurodegenerative and infectious diseases. This enzyme is a highly conserved and abundant AAA+ ATPase in all eukaryotic organisms. p97 is composed of two rings with a central pore and containing six identical subunits. Each subunit contains an N-terminal domain, two ATPase domains (D1 and D2), linkers connecting the domains (N-D1 linker and D1-D2 linker), and a disordered C-terminus. p97 is a key element of the ubiquitin proteasome system and, in concert with various cofactor proteins, extracts and unfold damaged or misfolded substrates through ATP hydrolysis. p97, in complex with cofactors, plays a crucial role in maintaining organelle and protein homeostasis through both ubiquitin dependent and independent pathways. These cofactors control the substrate selection, subcellular localization and regulate p97 enzymatic functions . Dysfunction of p97 has been associated with several diseases, including cancer and neurodegenerative disorders. Binding and assembly of cofactors to p97 are essential for its function. Therefore, understanding how the p97-cofactor form a complex and process substrates can provide valuable insights to develop inhibitors for specific pathways. In this project, we explored five structurally and functionally p97 cofactors including p37, SAKS1, Ufd1-Npl4, OTUD2 and UBXD7 to understand the mode of interaction of these cofactors with p97 and determine how these cofactors impact p97’s ATPase and unfoldase activities. We combined structural biology methods with structural dynamics techniques to investigate the structure of p97-cofactor complex and the conformational changes that occur upon interaction between proteins. We also performed in silico studies to determine the mode of interaction between p37 and p97 at the atomic level. According to the HAWKDOCK, HDOCK, Arpeggio and MM-GBSA binding free energy calculations, we found multiple hydrophobic interactions as well as two hydrogen bonds between the p37 UBX protein and the p97 N-D1 domain. In addition, we observed that the residues of the p37 UBX protein predicted to participate in interactions with the p97 N-D1 domain interface are remarkably conserved across UBX cofactors This finding indicates the significance of these interacting residues among UBA-UBX cofactors in interaction with p97. In addition, the in silico study provided the first structural insights into the p37-p97 complex through methods such as homology modeling, protein-protein docking, and molecular dynamics simulation. This approach allowed us to identify critical residues involved in the interaction between p37 and p97. We conducted circular dichroism (CD), Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS), and analytical ultra centrifugation (AUC) to determine the secondary structures, molar mass and oligomeric state of cofactors, respectively. Our results from surface plasmon resonance (SPR) assays indicated that all these cofactors interact with high binding affinity to p97. The cross-linking mass spectrometry (XL-MS) data showed that all cofactors bind to at least two domains or linkers of p97. The ATPase and Unfoldase assays revealed both SAKS1 and UBXD7 enhance p97’s ATPase activity and enable it to unfold polyubiquitilated substrate in vitro. We also employed advanced techniques such as cryo-electron microscopy (cryo-EM), XL-MS and Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) techniques to understand how the interaction between SAKS1 and the p97 N domain leads to the conformational change in SAKS1 that promotes substrate recruitment. In addition, for the first time, we resolved a cryo-EM map of the p97-SAKS1 complex obtained from a pull-down assay, which reveals the unique conformation of p97. We also resolved two novel structures of p97 in the presence of ADP-BeFx. The first structure is p97 hexamer in which all the N domains are in up conformation. The second structure is a dodecamer form of p97 in which two hexamers of p97 attached to each other from the C-terminus and all p97 N domains are in Up conformations.
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ItemOrganellar translation and inhibition in Plasmodium falciparum( 2023-09)The malaria parasite Plasmodium falciparum has two prokaryote-derived organelles: the mitochondrion and a relic plastid known as the apicoplast. These contain their own distinct, reduced genomes which must be transcribed and translated to maintain parasite viability. The bacterial-like proteins and metabolic functions of these organelles make malaria parasites susceptible to many anti-bacterials. This study aims to investigate organellar translation in P. falciparum, including the expression of apicoplast-targeted translation enzymes, tracking the cellular consequences of apicoplast translation inhibition, and measuring active organellar protein synthesis. Aminoacyl tRNA synthetases are a family of essential enzymes required for protein translation in the cytosol, apicoplast and mitochondrion. Several of these enzymes are encoded by single genes, from which two protein isoforms are proposed to be generated by alternative translation initiation. One isoform contains an N-terminal apicoplast localisation sequence, while the other lacks this and is cytosolic. In this study we investigate the significance of the nucleotides surrounding canonical and proposed translation start sites and show that these are important for their recognition by translation machinery. Additionally, we verify one of these dual-localised enzymes - threonine aminoacyl tRNA synthetase - as the target of the potent anti-microbial agent borrelidin in P. falciparum. Most organelle translation inhibitors have a lethal, but slow phenotype, killing parasites in the cycle following their administration. This has been attributed to disruption of apicoplast translation, with parasite death due to the inability to continue synthesis of essential apicoplast-derived isoprenoid metabolites. The consequences of isoprenoid starvation has been partially characterised, implicating lipophilic prenyl and isoprene chains as important, however not all essential isoprenoid products have been identified. We therefore aimed to investigate other downstream consequences of apicoplast translation inhibitors in Plasmodium. We found that apicoplast isoprenoids are required for synthesis of the major parasite sugar anchor glycophosphatidylinositol. Following inhibition of apicoplast translation, proteins typically anchored via this glycoconjugate became untethered, resulting in parasite segmentation, egress, and invasion defects. Difficulty in detecting proteins derived from organellar genomes had made the verification of organellar translation inhibitors challenging. Here, we use a mass spectrometry approach to directly detect and measure organellar translation in P. falciparum. This has facilitated the confirmation of the anti-apicoplast mechanism of action for the clinically used anti-malarials doxycycline and clindamycin. In addition, doxycycline was determined to inhibit mitochondrial translation, which was found to affect the activity of the electron transport chain. Together, this work has confirmed both the direct mechanism of action and indirect cellular consequences of organellar translation inhibitors on P. falciparum. In verifying the essentiality of glycophosphatidylinositols for multiple processes during the asexual stages, we have highlighted the potential for designing therapies that directly target aspects of glycophosphatidylinositol maturation or their protein attachment. Furthermore, determining the secondary target of doxycycline to be the mitochondrion has important clinical implications and may influence which drugs can be safely recommended for combination treatments.
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ItemCellular and molecular defence mechanisms against Legionella infection( 2023-04)The two most prevalent causes of Legionnaires’ disease are L. pneumophila and L. longbeachae. The rising incidence of the Legionnaires’ disease over the past two decades together with the increasing prevalence of L. longbeachae in the Northern hemisphere highlights the necessity to gain a more detailed insight into the L. longbeachae-induced immune response. The aim of this thesis was to investigate the cellular and molecular mechanisms required for protection, focussing on immune cell responses and intracellular host-bacterial interactions. By combining a murine mouse model, a novel fluorescent reporter to track L. longbeachae, and cell depletion experiments, I uncovered the differential contribution of tissue-resident alveolar macrophages (AM) and infiltrating neutrophils to the defence against L. longbeachae. Early during infection, AM contained most of the bacteria. AM numbers sharply decreased during infection, which was accompanied by a large influx of neutrophils that also internalized bacteria. Comparative analysis of bacterial viability revealed that neutrophils were more efficient at killing and clearing of L. longbeachae than AM. In contrast, the results presented here indicated that lung-resident AM promoted infection, most likely by serving as a replicative niche. Defence against L. pneumophila is known to require IFN-gamma but not IL-18, a strong IFN-gamma inducer. However, Il18r1–/– mice were less able to clear L. longbeachae and had significantly impaired IFN-gamma levels in the lung. Furthermore, IL-18R signalling was critical for the efficient bacterial killing by neutrophils via production of reactive oxygen species. Previous bone marrow chimera experiments in our laboratory suggested that IL-18R expression in epithelial cells was necessary and sufficient for protection against L. longbeachae. However, genetic in vivo experiments using the Cre-lox-system revealed that IL-18R+ NK cells and T cells were central players in the IL-18-dependent anti-L. longbeachae defence via IFN-gamma secretion, whereas IL-18R expression by ciliated bronchiolar epithelial cells did not confer protection. Finally, our results highlighted key mechanisms by which Legionella subverts host macrophages to form an intracellular endoplasmic reticulum (ER)-like vacuole as its intracellular replicative niche. Establishment of the Legionella-containing vacuole (LCV) by recruitment of ER-derived vesicles induces ER stress. Although the relevance of ER stress in this process is unclear, effector proteins secreted by L. pneumophila are known to inhibit onset of the ER stress response. Using the pharmacological ER stress inducer thapsigargin, we showed that ER stress induces a protective host response promoting the secretion of pro-inflammatory cytokines, limiting intracellular L. pneumophila replication, and improving host survival. Mechanistically, ER stress induced a novel non-canonical activation of the transcription factor STAT1 via the IRE1 kinase driving transcription of the IFN-gamma-induced chemokine CXCL10. These results highlighted a potential role of the host ER stress response in the initiation of a protective cellular immune response towards L. pneumophila.
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ItemHyper-nutritional cell culture medium distorts the expression of anti-cancer drug targets( 2023-07)In vitro cell cultures are instrumental in deciphering the mechanisms that underlie cell processes in vivo including differentiation, migration, growth and mechanics, all of which are impacted by both biochemical and biomechanical microenvironments. Evolving technologies provide continuous improvement to cell culture towards more physiologically relevant systems that better emulate in vivo conditions. However, the cell culture media as one of the fundamental elements for in vitro cell culture have been overlooked with incremental rather than the transformational improvements that are required. Nutrient availability in the tumour microenvironment is essential for metabolism, cellular processes and may be a determinant of anti-cancer drug responses. Nevertheless, the overwhelming majority of current in vitro biomedical research is conducted in unphysiological conditions, using conventional cell culture medium developed generations ago with hyper-physiological level of nutrients. In addition, most of the cell culture were in static conditions, lacking the flow/renewal of the in vivo situation to provide appropriate biochemical and mechanical cues and prevent accumulation of metabolic by-products. We therefore developed an improved physiological medium, “Melbourne Medium” (MM) with human plasma-like composition for continuous supply to multi-well cell culture environments using the custom-built RPM2 multiplexed superfusion system. Our focus here is on the imperative to understand the influence of physiological media on cellular behaviours by contrast with conventional hyper-nutritional media. Culture of non-small cell lung cancer (NSCLC) A549 cells in MM slowed proliferation, promoted an epithelial-mesenchymal transition (EMT)-like phenotype with an increased motility and induced paclitaxel resistance. Global proteomic analyses revealed expected differential expression of metabolism-related gene ontologies, but also multiple biological pathways of critical importance to the growth and spread of NSCLC. Notably, significant distortion was identified in the expression of key anti-cancer drug targets including EGFR, STAT3, TGFBI and Smad3, as well as tubulins, specifically betaII and betaIV tubulin isoforms implicated in paclitaxel resistance. The most up- and down- regulated proteins in MM, CNBP and 15-hydroxy prostaglandin dehydrogenase (15-PGDH), respectively, are each associated with lung cancer survival, the former association being discovered in this thesis study. The cell metabolism processes critical to tumour progression were perturbed by hyper-nutritional medium, as indicated by both proteomic and metabolomic analyses. For the first time, by applying the RPM2 multiplexed superfusion of media, we established that the physiological nature of MM could be maintained, while minimising accumulation of both bioactive metabolic by-products and previously unappreciated metabotoxin formation. Together, these studies have demonstrated that hyper-nutritional (conventional) media profoundly distort tumour cell functions, whereas superfusion of MM facilitated a more physiological metabolic environment, associated with improved drug target validation and drug screening.
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ItemDesign, Synthesis, and Analysis of BDNF Mimetic Peptides( 2023-06)Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family essential for development of both CNS and PNS. It is widely thought to be involved in, and possess clinical potential for, the treatment of a variety of diseases. It mediates its cellular effects through the TrkB receptor and the p75 neurotrophin receptor (p75NTR). Actions of BDNF vary vastly depending on the cell types and receptor abundance. In chemical biology research, we need molecules that are capable of selectively targeting a single receptor type. When studying proteins with multiple receptor targets, like BDNF, the protein itself can be a useful control, but compounds capable of targeting each receptor selectively are vital to elucidate and study protein-receptor activity in a disease context. To meet that need, our lab specialises in developing peptides that mimic specific loops of large proteins. Cyclo-pAKKR and TDP6 are the two lead peptidomimetics developed from BDNF by my original lab leader, Professor Tony Hughes, over two decades ago, which selectively targeting the p75NTR and TrkB receptors respectively. Although these compounds have generated promising and valuable biological data, further and broader research using these mimetic peptides, particularly TDP6 (a complicated tricyclic dimeric peptide), was hindered by the challenging chemical synthesis and low synthesis yields, despite many approaches attempted over the years. A next-generation TrkB-targeting BDNF mimetic peptide with improved chemical synthesis was urgently needed for further functional, biological, and other assays. In contrast to the limited chemistries available when TDP6 was first designed, in recent years numerous bio-orthogonal chemistries have been explored and published, providing new synthetic chemistry opportunities in the development of TDP6 analogues. These new analogues would need to maintain the activity of TDP6, but be higher yielding, less time-consuming, more economical, and less labour-intensive. This project involved designing, chemically synthesising, and evaluating peptide mimetics of BDNF that selectively target either TrkB or p75NTR. Particular attention was paid to the design and chemical synthesis of new TrkB-targeting BDNF mimetic peptides, as these steps are far from trivial. As exemplar, the step-by-step development of a new TrkB-targeting BDNF mimetic peptide, TDP7, is described in this thesis. As a next-generation analogue of TDP6, prepared utilising alternative ligation chemistries, TDP7 can be produced more efficiently, and with 22-fold higher yield than TDP6, which enabled a wider range of analyses, and broadened its utility as a chemical biology tool. In summary, development of peptides mimicking specific loop regions of BDNF is an effective approach to study the receptor-specific actions of BDNF, to ultimately then expand knowledge of BDNF biology. Production of BDNF mimetic peptides, especially the polycyclic peptides, in sufficient quantity for characterisation and subsequent analysis, has been the challenge met in this project. The exploration of ligation chemistries, including orthogonal chemistries for polycyclic peptides, can serve as a guide beyond this project for production of more chemically complex peptides.
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ItemNo Preview AvailableUsing Artificial Intelligence to Improve the Diagnosis and Treatment of Cancer( 2022-12)Cancer is a complex and heterogeneous disease driven by the accumulation of mutations at the genetic and epigenetic levels—making it particularly challenging to study and treat. Despite Whole-genome sequencing approaches identifying thousands of variations in cancer cells and their perturbations, fundamental gaps persist in understanding cancer causes and pathogenesis. Towards this, my PhD focused on developing computational approaches by leveraging genomic and experimental data to provide fundamental insights into cancer biology, improve patient diagnosis, and guide therapeutic development. The increased mutational burden in most cancers can make it challenging to identify mutations essential for tumorigenesis (drivers) and those that are just background accumulation (passenger), impacting the success of targeted treatments. To overcome this, I focused on using insights about the mutations at the protein sequence and 3D structure level to understand the genotype-phenotype relationship to tumorigenesis. I have looked at proteins that participate in two DNA repair processes: primarily non homologous end joining (NHEJ) along with eukaryotic homologous recombination (HR), where missense mutations have been linked to many diverse cancers. The molecular consequences of these mutations on protein dynamics, stability, and binding affinities to other interacting partners were evaluated using in silico biophysical tools. This highlighted that cancer-causing mutations were associated with structure destabilization and altered protein conformation and network topology, thus impacting cell signalling and function. Interestingly, my work on NHEJ DNA repair machinery highlighted diverse driving forces for carcinogenesis among core components like Ku70/80 and DNA-PKcs. Cancer-causing mutations in anchor proteins (Ku70/80) impacted crucial protein-protein interactions, while those in catalytic components (DNA-PKcs) were likely to occur in regions undergoing iii purifying selection. This insight led to a consensus predictor for identifying driving mutations in NHEJ. While when assessing the functional consequences of BRCA1 and BRCA2 genes of HR DNA repair at the protein sequence level, this methodology underlined that cancer-causing mutations typically clustered in well-established structural domains. Using this insight, I developed a more accurate predictor for classifying pathogenic mutations in HR repair compared to existing approaches. This broad heterogeneity of cancers complicates potential treatment opportunities. I, therefore, next explored the properties of compounds potentially active against one or various types of cancer, including screens against 74 distinct cancer cell lines originating from 9 tumour types. Overall, the identified active molecules were shown to be enriched in benzene rings, aligning with Lipinski's rule of five, although this might reflect screening library biases. These insights enabled the development of a predictive platform for anticancer activity, thereby optimizing screening libraries with potentially active anticancer molecules. Similarly, I used compounds' structural and molecular properties to accurately predict those compounds with increased teratogenicity early in the drug development process and prioritize drug combinations to augment combinatorial screening libraries, potentially alleviating acquired drug resistance. The outcomes of this doctoral work highlight the potential benefits of using computational approaches in unravelling the underlying mechanisms of carcinogenesis and guiding drug discovery for designing more effective therapies. Ultimately, the predictions generated by these tools would improve our understanding of the genotype-phenotype association, enabling promising patient diagnosis and treatment.
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ItemMolecular dynamics simulation-based image analysis methods for structural studies of biological macromolecular complexes( 2023-07)Cryo-Electron Microscopy (cryo-EM) is an imaging technique that allows observing nanoscale molecular machines such as proteins and DNA. These molecular machines are flexible and constantly undergo internal motions that change their conformations in order to achieve their biological function. Identifying conformational changes of macromolecules is of great biological importance and is used in the development of new drugs. Deciphering continuous conformational transitions of macromolecules, through the main cryo-EM processing techniques, Single Particle Analysis (SPA) and cryo-Electron Tomography (cryo-ET), is challenging partly due to the low signal-to-noise ratio and is currently an active field of research. During my PhD thesis, I investigated new image processing methods based on Molecular Dynamics (MD) simulations that allow extracting continuous conformational variability from SPA and cryo-ET data. My work resulted in the development of MDSPACE and MDTOMO, the two first methods using MD simulations, empowered by Normal Mode Analysis (NMA), to extract the continuous conformational landscape from SPA and cryo-ET datasets, respectively. The developed methods were employed to investigate the conformational behavior of diverse systems such as 80S ribosome and AAA ATPase p97 (MDSPACE) and SARS-CoV2 spike protein in situ (MDTOMO). The methods are competitive as they allow an atomic-scale determination of the conformational spaces of various macromolecules from heterogeneous cryo-EM data, which can be advantageous in structure-based drug development.
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ItemApplication of activity-based probes to interrogate the contribution of cathepsin X to dendritic cells( 2023-08)Cathepsin X/Z/P (Cat X) is a lysosomal cysteine protease that exhibits mono-carboxypeptidase activity. Increased cathepsin X expression is associated with cancer and inflammation; however, its roles in normal physiology are poorly understood. Cathepsin X is highly expressed by antigen-presenting cells such as dendritic cells (DCs). DCs undergo functional changes upon agonism of pattern recognition receptors (e.g., Toll-like receptors) by pathogen-associated molecular patterns (e.g., bacterial DNA – CpG). Mature DCs can secrete cytokines and present antigens to activate adaptive immunity. We hypothesised that cathepsin X contributes to DC function. Using activity-based probes, immunoblotting, and immunofluorescence imaging, we measured active and total levels of cathepsin X in naive immortalised DCs (DC1940) or those stimulated with TLR-1/2, 2/6, 3, 4, 7/8, and 9 agonists (Pam3, FSL-1, Poly I:C, LPS, R-848, and CpG, respectively). Lysosomal and secreted cathepsin X levels were significantly elevated in response to TLR-9 agonist CpG treatment and to a much lesser extent with the other agonists. We further investigated this mechanism and found that IL-6 secreted upon TLR-9 agonism promotes cathepsin X upregulation. To examine the impact of cathepsin X on DC function, we generated cathepsin X-deficient DC1940 cells using CRISPR-Cas9. DC maturation, antigen uptake and antigen presentation were not affected by cathepsin X deficiency. We also investigated cytokine secretion and found that IL-6, IL-10, IL-12, TNFalpha, and MCP-11 were not affected by cathepsin X deficiency, while secretion of IL-1beta was impaired. We further conducted shotgun proteomics to broadly investigate the impact of cathepsin X deficiency on DC immune function. We discovered that the expression of TMEM176B was significantly elevated in cathepsin X-deficient cells. TMEM176B has been reported to be a negative regulator of inflammasome activation. We hypothesise that the impaired secretion of IL-1beta was due to the upregulation of TMEM176B in cathepsin X-deficient cells. We next investigated the impact of cathepsin X deficiency on the overall lysosomal proteolytic environment. Cathepsin X-deficient cells exhibited altered processing of cathepsin L, while the processing and activities of other lysosomal proteases and their inhibitors were unaffected. We further found that cathepsin X deficient cells exhibited lower levels of nuclear cathepsin L. Whether or not the altered processing of cathepsin L was related to nuclear localisation is still under investigation. Furthermore, lower nuclear cathepsin L in the absence of cathepsin X resulted in reduced processing of its nuclear substrate, lamin B1. Collectively, our data indicate that cathepsin X was strongly upregulated by TLR-9 agonism in DCs. While it may not be essential for DC maturation, antigen uptake, and antigen presentation, cathepsin X may regulate IL-1beta secretion through TMEM176B. In addition, the processing of cathepsin L was altered in cathepsin X-deficient cells, and the level of nuclear cathepsin L was reduced. Future studies will focus on the mechanistic interaction between cathepsin X and TMEM176B and investigate the impact of reduced nuclear cathepsin L on nuclear proteolysis to better understand the role of cathepsin X in pathophysiology.
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ItemInvestigating the structure and function of novel Coxiella burnetii effector proteins( 2023-04)Coxiella burnetii is an obligate intracellular bacterial pathogen and the causative agent of the zoonotic disease Q Fever. The danger that emerging C. burnetii strains present and the lack of effective treatments for Q Fever has prompted research into the pathogenesis of C. burnetii in recent years. Essential to C. burnetii pathogenesis and replication in infected host cells is the establishment of an intracellular niche termed the Coxiella-Containing Vacuole (CCV). CCV formation is dependent on the C. burnetii type IV secretion system (T4SS), which translocates C. burnetii type IV effector (T4E) proteins into the host cytoplasm to modulate host cell pathways thereby creating an intracellular environment that is permissive for C. burnetii replication. Many C. burnetii T4Es have been implicated in CCV biogenesis, however, most remain almost entirely uncharacterised. This thesis focuses on three T4Es implicated in CCV biogenesis; Cig57, Cbu1752, and Cbu1754. Cig57, Cbu1752, and Cbu1754 share no significant sequence similarity with other known proteins, making predication of protein structure and function unreliable. Thus, experimental investigation of C. burnetii T4E structure-function relationships is required. Prior to our work, structural techniques had not been applied to novel C. burnetii T4Es, in part due to lack of established techniques for their purification. The work of this thesis has generated efficient protocols for the purification of Cig57, Cbu1752, and Cbu1754, facilitating interaction and structural studies. These protocols also serve as important groundwork for the biochemical investigation of T4Es more broadly. Cig57 was previously shown to interact with the host nucleator of clathrin transport, FCHO2. In this thesis, the Cig57-FCHO2 interaction is characterised as a direct binding interaction mediated by the Cig57 C-terminal domain. Using small-angle X-ray scattering (SAXS) we validated a crystal structure of the Cig57 central domain that we previously solved and combining our experimental data with computational models of full-length Cig57, we resolve structural, dynamic, and functional properties of Cig57. We demonstrate a novel interaction between Cbu1752 and the clathrin heavy chain N-terminal domain (CHC NTD). Cbu1752 was found to have a dynamic N-terminal domain that directly mediates interactions with CHC NTD, and a C-terminal globular structured domain, which we hypothesise mediates Cbu1752 localisation. A computational model for the Cbu1752 C-terminal domain structure was supported by SAXS, providing a useful tool for further investigation of Cbu1752 structure. Unlike Cbu1752, computational models for Cbu1754 were found to be inaccurate indicating that caution is required when interpreting computational models of T4Es, and associated confidence statistics. Thus, we highlight the importance of experimental validation of computational models of novel T4Es and show that SAXS is a valuable tool for this purpose. Our structural and biochemical analysis of Cig57, Cbu1752, and Cbu1754 has led to an updated model of CCV biogenesis. In our model, we propose that clathrin transport is directly targeted by C. burnetii to inhibit recycling of lysosomes from the CCV. Broadly, the work presented in this thesis provides a better understanding of C. burnetii effector protein structure and function, which will to aid the development of future strategies to study and combat C. burnetii infection.
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ItemThe mechanisms of coupled folding and binding in the interaction of neurotensin with the neurotensin receptor 1( 2023-05)Neurotensin, NT, is a 13 residue, pELYENKPRRPYIL, linear disordered peptide that activates two different G-protein coupled receptors, GPCR, in the human body, neurotensin receptor 1, NTS1 and its homologue NTS2. NTS1 is regarded as a potential drug target for treatment of schizophrenia and cancer. Whereas hypothermic effects following activation of NTS1 hampers the therapeutic benefits of NTS2 activation as an opioid-independent analgesic target. Development of subtype specific drugs against this subclass of GPCRs is challenging in part due to the unknown mechanisms of ligand recognition and activation of these receptors. Here we combined a wide range of biophysical and biochemical techniques to investigate the mechanism underlying protein recognition in interaction of NT with the thermostabilized variant of NTS1, en2NTS1. Methods include solution NMR spectroscopy, kinetic analysis by stopped-flow fluorescence, hydrogen-deuterium exchange mass spectrometry (HDX-MS) with cell-based assays and molecular biology techniques, including site-specific incorporation of unnatural amino acids using amber codon suppression. The project was divided into two parts. In the first part, we focused on the mechanisms of receptor recognition and binding of NT to NTS1. Our data indicated that the presence of Pro7 and Pro10 in NT reduces the conformational space of NT to form transient structures that are important for formation of encounter complexes between the N-terminal segment of NT and the extracellular loop 2, ECL2, of the receptor, which steers the peptide to the binding pocket. Differences in the surface electrostatic potential in NTS1 and NTS2, especially between ECL2, propose the role of N-terminal region of NT in receptor subtype selectivity of NT. The second part of this project adopted ligand-observed and receptor-observed 19F NMR approaches combined with HDX-MS and numerical analysis of the kinetics of NT binding to NTS1 to investigate the mechanisms of NT recognition by NTS1. Our results showed that the conformational dynamics at the extracellular surface of the receptor are distinctly modified upon binding of ligands with different potencies. HDX-MS confirmed NMR data by indicating that an interaction between the N-terminal region and ECL2 of the receptor, promoted upon NT binding, as the origin of conformational change and probably responsible for the contraction of the orthosteric binding pocket of NTS1 upon binding to NT. Kinetic analysis of NT binding to NTS1 proposes that this conformational change is promoted by NT binding by an induced fit mechanism via formation of encounter complexes. Our data provides new insight into the kinetic regulation of ligand recognition by GPCRs. Finally, we propose a model for NT recognition and activation of NTS1.