Thèses sur le sujet « NMR ligand-receptor interaction studies »

On a molecular level protein – ligand interactions are central to a number of biological processes, but their investigation is inherently difficult due to several problems, especially for membrane proteins. The study of this type of interactions poses a whole set of challenges, including the characterization of the dynamic behaviour and of the conformational properties of the ligands in complex with the target macromolecules. A variety of biophysical methods have been developed to study protein – ligand interactions and several NMR spectroscopic techniques have emerged as powerful methods to identify and characterize the binding of ligands with receptor proteins. Ligand-based methods do not require labeled protein, since only the ligand NMR signals are detected and only a small amount of protein is required. These techniques are particularly useful in the medium–low affinity range and, therefore they have been adopted to detect ligand interactions in various systems. Among the ligand-based NMR techniques, Saturation Transfer Difference (STD) and transferred-NOE focus on the NMR signals of the ligand and utilize NOE effects between protein and ligand. They are used for: i) the definition of the bioactive conformation of the ligand in the bound state (tr-NOESY), ii) the identification and characterization of the binding mode of the ligand to the receptor with epitope mapping of the ligand itself (STD). The use of the technique is limited to molecules that exhibit a dissociation constant Kd between 10-3 M and 10-7 M. During my PhD, I had the highly qualifying opportunity to grasp these new potent NMR methods, and to apply them for assessing the interactions of cell surface proteins with peptidomimetics. Since membrane proteins, such as integrins, change their conformation if extracted from their environment, it is clear the importance of working in the biophysical neighbourhood of the membrane itself and not in an isotropic extracellular medium. For this reason, when appropriate to the project, I have carried out NMR experiments using intact cells overexpressing the proteins of interest. Specifically, two main topics have been addressed: 1. The first and second year of my PhD have been mainly focused on the conformational study of peptidomimetic integrin ligands and on the investigation of their interaction with platelets and cancer cell overexpressing integrins on their membrane. This study has been developed within the framework of a PRIN project (MIUR-PRIN project 2010NRREPL “Synthesis and Biomedical Applications of Tumor-Targeting Peptidomimetics”) in collaboration with the research groups of Proff. Gennari and Piarulli (University of Insubria) as regards the synthetic activities and with the group of Dr. Belvisi as regards the computational and design studies. 2. The second part of my PhD was mainly focused on cadherins, a class of cell adhesion proteins that promote homophilic interactions. This work is at an early stage and has been developed within the framework of a FIRB project coordinated by Dr. Civera (MIUR-FIRB “Futuro in Ricerca” RBFR088ITV “Computer-aided design, synthesis and biological evaluation of peptidomimetics targeting N-cadherin as anticancer agents”). The NMR study has been aimed at obtaining a thorough understanding of the interaction of peptidomimetic molecules with isolated cadherin constructs containing relevant extracellular domains.

Because of their role in oncogenesis, inhibition of Ras proteins, particularly of their tumorigenic variants, represents today one of the principal strategies finalized to the obtainment of new antitumoral therapies. Among the different possible approaches, one of the most innovative and less explored is represented by the inhibition of this protein activation, key event for the explication of their biological activity, but also for the Ras-induced tumoral cell transformation. Objective of this thesis has been the development of new small molecules able to inhibit, at least partially (total inhibition in fact would result lethal for cell), Ras protein activation, in particular the GEFs-promoted GDP/GTP nucleotide exchange. Inhibitors able of inactivating Ras have been previously described by Schering-Plough. All these molecules contain a phenylhydroxylamino group that binds Ras in a region close to the nucleotide binding site and one aromatic group. Nevertheless, they present some negative characteristics that prevent their employment as potential drugs: (1) they are chemically unstable and (2) they are insoluble in water and in the most commonly used organic solvents. In order to obtain new more efficient inhibitors, we adopted the rational drug design strategy. Firstly, we studied the structure-activity relationship (SAR) of Schering-Plough compounds and of new molecules containing variants of their functional groups that we designed and synthesized. The data collected demonstrated that the phenylhydroxylamino group is an essential pharmacophore, while other positions are not so critical for the biological activity.Keeping in mind this, we prepared new compounds in which the phenylhydroxylamino moiety is supported on glycidic templates, in an attempt to try to take advantage of carbohydrate capability of orienting substituents in space, in this case in a suitable manner for the interaction with Ras proteins. In addition, the sugar portion can improve compound pharmacokinetic properties and decrease their toxicity. In this way, a new class of Ras inhibitors was obtained, their biological activity and the nature of their interaction with the molecular target were characterized.

NMR techniques allow to obtain structural information useful for the comprehension of biological processes. The aim of this work is to investigate interactions between biological macromolecules and binding partners, including other macromolecules, small ligands and therapeutically relevant compounds. These results will be exploited for the design and development of new potential drugs and medical devices.

Neurotransmission is the action of communication between neurons; the most common neurotransmitter in the mammalian CNS is glutamate. Excessive activation of glutamate receptors have been implicated in a number of neurodegenerative diseases; they are therefore of great interest for investigation as drug targets. The ionotropic glutamate receptor GluR2 has previously been expressed as a soluble construct representing the ligand binding domain known as S1S2J. GluR2 S1S2J contains two separate binding sites for different types of ligands; the glutamate binding site and the allosteric modulator binding site which is located the dimer interface formed between two GluR2 S1S2J monomers. Binding of ligands to the different sites has been implicated in two different mechanisms for receptor inactivation; both sites are targets for lead drug design. The GluR2 S1S2J construct has been studied in complex with its agonists and antagonists by X-ray crystallography however some questions remain about the exact structure of the ligand within the site and the allosteric modulator binding site has yet to be studied extensively.

Tritium NMR studies provide a convenient way of obtaining detailed information about conformational equilibria, dynamic processes and specific interactions in protein-ligand complexes provided that suitably 3H-labelled molecules are available. In this study [7,9-3H]- and [3',5',7-3H]folic acid, and [3',5',7-3H]methotrexate were synthesised and the NMR spectra of their complexes with Lactobacillus casei dihydrofolate reductase (DHFR) were assigned and analysed as a function of pH (DHFR-folate complexes) and temperature (DHFR-methotrexate complexes). From these data it was possible to obtain further evidence about the orientation of the pteridine ring in the complexes, and to monitor the dynamic processes in the bound ligands. In the 3H NMR spectra of the ternary complexes of the 3H-labelled folic acids with DHFR and NADP+, each labelled tritium gave rise to multiple signals, confirming previous findings that there are three interconverting, pH dependent, conformational forms of bound folate (forms I, IIa and IIb) in the ternary complex. The folate benzoyl ring could be shown to be in essentially the same environment in the different forms with the major differences being associated with the pterin ring. The appearance of a single resonance for the 3',5'-tritons showed that the benzoyl ring is flipping rapidly in all three forms. In contrast, the methotrexate binary complex and also the ternary complex with NADPH were shown to exist as a single conformational state with the benzoyl ring flipping rate being too slow to give a single averaged signal for the 3',5'-tritium nuclei over the temperature range 283 - 313 K. 3h{1h} Nuclear Overhauser enhancement experiments have been conducted on the small molecules, [3H]dimethyl sulphoxide, [3',5',7-3H]folic acid and [3',5',7-3H]methotrexate as a prelude to 3H-1H heteronuclear NOE experiments on binary and ternary complexes formed using Lactobacillus casei DHFR and the ligands [3',5',7-3H]methotrexate and [3',5',7-3H]folic acid.

Six novel platinum(II) intercalators of the form [Pt(AL)(IL)]Cl2, where AL = ethylenediamine (en), 1R,2R-diaminocyclohexane (R,R-dach), or 1S,2S-diaminocyclohexane (S,S-dach) and IL = 4,7-dihydroxy-1,10-phenanthroline (4,7-dhp) or 4,7-dicarboxy-1,10-phenanthroline (4,7-dcp), were synthesised. All complexes were prepared by the addition of the intercalating ligand followed by the addition of the diamine ancillary ligand. The complexes with 4,7-dhp were soluble in DMSO and were characterised by 1H, 13C, and 195Pt NMR, elemental analysis, UV-vis, ESI-MS, and CD. The complexes with 4,7-dcp were only soluble in a highly acidic solution and, therefore, were characterised only by 1H NMR and elemental analysis. The cytotoxicity of the 4,7-dhp complexes was tested in the L1210 murine leukaemia cell line. [Pt(S,S-dach)(4,7-dhp)]Cl2 showed an IC50 value of > 80 μM. The antitumour and antibacterial activities of all six complexes were tested in vitro using the Kirby-Bauer disc diffusion method with Staphylococcus aureus and Agrobacterium tumefaciens. The 4,7-dhp complexes showed no activity to these bacteria strains. The activities of the 4,7-dcp complexes were not able to be tested due to their solubility only in acidic solutions, which itself inhibits cell growth. The diffusion coefficients of the Pt(II) intercalators of the form [Pt(AL)(IL)]Cl2, where AL = en, R,R-dach, or S,S-dach and IL = phen, 4-mp, 4,7-dmp, 4,7-dhp, 4,7-dcp or 3,4,7,8-tmp and various starting materials used during the synthesis of these complexes were measured using pulsed gradient spin-echo (PGSE) NMR. The diffusion coefficients of both 4,7-dcp and [Pt(4,7-dcp)Cl2] were observed to be lower than other compounds with similar molecular weights indicating dimerisation of the compounds. The binding studies of the systems, [Pt(en)(phen)]Cl2 to (i) BSA, (ii) delipidated BSA, and (iii) d(GTCGAC)2 were studied using a simple two-site binding model with diffusion NMR. The binding of [Pt(en)(phen)]Cl2 – BSA was well described by the model giving the values Kd = 0.0021 ± 0.0002 M and n = 5.85 ± 0.31. On the contrary, the binding of [Pt(en)(phen)]Cl2 – delipidated BSA showed a poor fit to the model. From the poor fit of the data, it was speculated that the transverse relaxation of BSA largely affected the system. The binding of [Pt(en)(phen)]Cl2 – d(GTCGAC)2 showed results where the diffusion coefficient decreases as the concentration of the drug increases but an opposite effect was observed from the point where the drug reached equimolar concentrations to d(GTCGAC)2. It was speculated that the drug undergoes allosteric binding to the biomolecule or that a conformational change occurred as the drug concentration increases in the system. A further study of [Pt(en)(phen)]Cl2 and K2PtCl4 using 195Pt diffusion NMR was conducted giving a diffusion coefficient of 3.08 ± 0.04 × 10-10 m2 s-1 for K2PtCl4. The diffusion coefficient of [Pt(en)(phen)]Cl2, however, were unobtainable due to the short transverse relaxation of the Pt complex.

Thesis (M.Sc.) (Hons)--University of Western Sydney, 2008.
A thesis presented to the University of Western Sydney, College of Health and Science, School of Biomedical and Health Sciences, in fulfilment of the requirements for the degree of Master of Science (Honours). Includes bibliographies.

The members of the mammalian neuropeptide Y family, i.e. the peptides neuropeptide Y (NPY), peptide YY (PYY) and pancreatic polypeptide (PP), are all involved in regulation of food intake. In human and most other mammals they act via receptors Y1, Y2, Y4 and Y5. NPY is released in the hypothalamus and is one of the strongest appetite-stimulating neurotransmitters whereas PP and PYY are secreted from gut endocrine cells after meals and function as appetite-reducing hormones. This thesis describes studies of the NPY system at both the molecular and the physiological level. The first part describes two investigations of receptor-ligand interactions with the human Y1 and Y2 receptors. The results clarify the importance of several amino-acid residues of the human Y1 receptor. Three amino acids previously suggested by others to form a binding pocket for the carboxy-terminus of the peptide were confirmed to be crucial for interaction with peptide ligands. However, they were found to be too distantly located from each other to be able to form a binding pocket. Further investigation of the three corresponding positions in the human Y2 receptor showed that only one of the positions was important for interaction with full-length peptides. The results indicate overlapping but, surprisingly, non-identical binding of the different peptides to human Y1 and Y2 receptors, despite the fact that the two receptors share a common ancestor. The second part of the thesis describes an investigation of the effect of PP on food intake in six beagle dogs and a test for personality characteristics in dogs (TFPC). Treatment with physiological doses of PP decreased both the appetitive and the consummatory drive but had no effect on the amount food consumed. The TFPC protocol was used to map individual behavioral differences in a population of sixteen beagle dogs. The test, which included several situations that may appear in an experimental study, revealed considerable inter-individual differences in behavioral responses despite the fact that the dogs were born and housed in the same animal facility in constant controlled conditions. These results demonstrate that PP can influence food intake in distantly related mammals and emphasize the importance of considering differences in personality in experimental animals.

The work presented in this thesis concerns the opioid peptide dynorphin A (DynA). DynA functions primarily as a neurotransmitter and belongs to the family of typical opioid peptides. These peptides are a part of the opioid system, together with the opioid receptors, a family of GPCR membrane proteins. The opioid system system is involved or implicated in several physiological processes such as analgesia, addiction, depression and other types of neurological disorders. In this thesis, two biologically relevant aspects of DynA have been investigated with biophysical methods. First, interactions between DynA and an opioid receptor, and second, the direct membrane interactions of DynA. The DynA–receptor studies were focused on the selectivity-modulating second extracellular loop (EL2) of the kappa-opioid receptor (KOR). A protein engineering approach was used in which the EL2 was grafted onto a soluble protein scaffold. The results show that DynA binds with low affinity but high specificity to EL2 in the construct protein environment. The strength of the interaction is in the micromolar range, and we argue that this interaction is part of the receptor recognition event. With bicelles as a mimetic, membrane interactions were probed for wild-type DynA and for two DynA peptide variants linked to a neurological disorder. R6W–DynA and L5S–DynA were shown to be very different in terms of bicelle association, penetration and structure induction. In these experiments, as well as in investigations of DynA dynamics in bicelles, the lipid environment was shown to have much larger effects on peptide dynamics than on structure; and both these properties depend on lipid charge. Additionally, in a methodological project, DHPC/DMPC bicelle morphology as a function of total PC concentration was characterised by diffusion NMR in combination with two-way decomposition. The results may contribute to providing guidelines for the appropriate use of bicelles as a membrane mimetic.

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 5: Manuscript.

Current genomic and proteomic research is generating a considerable number of potential new therapeutic targets. To tackle all these pharmacological targets, the development of rapid and reliable screening methods, which give information on protein-ligand interactions, are of great importance. New advances in these technologies are often emerging and more will surely follow.

La voie de signalisation du récepteur des androgènes (AR) est impliquée dans la progression du cancer de la prostate, et il a été montré que des mutations dans ce domaine étaient responsables de l'activation constitutive des gènes placés sous le contrôle des hormones androgènes. Une de ces mutations transforme un résidu thréonine du DBD en alanine (T575A). Des expériences permettant de mesurer l'activité de transcription ont permis à l'équipe du Dr. Ceraline à l'IRCAD de montrer que la mutation T575A induit un changement de spécificité du récepteur. Alors que l'activité de promoteurs placés sous le contrôle d'éléments de réponse spécifique de AR diminue, celle des promoteurs placés sous le contrôle d'éléments non spécifique augmente. Ce changement de spécificité est corrélé à une modification de l'affinité du récepteur pour les éléments de réponse spécifiques et non spécifiques. Afin de comprendre le mécanisme de cette "reprogrammation" à l'échelle moléculaire, l'étude structurale des domaines DBD des récepteurs sauvage et muté a été entreprise par RMN. La comparaison des deux structures en solution a montré que la mutation n'altère pas le repliement du domaine et donc que la différence de reconnaissance des éléments de réponse n'est pas liée directement à la structure tridimensionnelle du domaine. Nous avons ensuite cherché à déterminer si l'altération de la fonction n'était pas due à une différence de dynamique de la chaîne peptidique. Afin d'étudier les mouvements moléculaires le long de la chaîne, des mesures de relaxation hétéronucléaire ont été effectuées et ont montré également une grande similarité dans le comportement dynamique des deux domaines, à l'exception d'une région située dans le premier doigt de zinc à proximité d'une histidine (H570), qui est conservée dans l'ensemble de la famille des domaines DBD des récepteurs nucléaires. Cette différence nous a conduit à mesurer, par RMN, le pKa de cette histidine pour les deux protéines. Nous avons ainsi montré que la mutation T575A induit une diminution de 0,5 unité de pH par rapport à la même histidine dans le domaine sauvage. L'analyse de la structure a permis de montrer que cette différence de pKa est liée à la perte d'une interaction entre le groupe hydroxyle de la thréonine 575 et le cycle imidazole de l'histidine. L'effet de la mutation sur le mécanisme de reconnaissance s'explique donc par un effet indirect dans lequel un acide aminé situé à distance de la région d'interaction modifie la surface électrostatique du domaine DBD. L'effet de la charge positive en position 570 sur la spécificité de reconnaissance de l'élément de réponse a ensuite été étudiée en construisant plusieurs mutants portant ou non une charge à cette position (mutants H570R et H570A). Ces études ont permis de confirmer l'importance de cette charge et l'ensemble de nos travaux fournissent un éclairage inédit sur les mécanismes de reconnaissance de l'ADN par les récepteurs nucléaires. .
The androgen receptor (AR) is a ligand-activated transcriptional factor and a member of the nuclear receptor super family. AR shares a common structural and functional architecture with other members of nuclear receptors. The DNA binding domain of AR (ARDBD) binds to specific response elements as a homodimer. In the clinic, certain mutations in AR are associated with the progression of prostate cancer and have consequences for the treatment of patients with advanced prostate cancer. Previous studies showed that the mutation T575A, locating in the DNA binding domain, enhances the transcriptional activity regulated by full-length AR on promoters containing the non-specific response element compared to the wild type domain does not. These differences prompted us to study the molecular mechanism of ARDBD wild type and the T575A mutant. Structures of ARDBD wild type and T575A mutant revealed high similarity. However, dynamic behavior showed distinct differences between wild type and T575A mutant domains. The protonation state of H570 in ARDBD was found to be differed by the mutation. This loss of charge of H570 results in changes in transcriptional activity of AR. .

Topographical constraint is the most powerful approach for the design of bioactive peptides to explore the bioactive conformation of crucial side-chain pharmacophores of amino acid residues in peptide-receptor recognition and signal transduction. Novel topographically constrained amino acids β-isopropylphenylalanine and 2',6'-dimethyl-2,3-methanophenylalanine have been designed and synthesized. Incorporation of the four optically pure β-isopropylphenylalanine stereoisomers into deltorphin I produced four peptide analogues of [β-iPrPhe]Deltorphin I with differentiated bioactivities. The most potent and selective analogue, [(2S,3R)-β-iPrPhe]Deltorphin I showed an IC₅₀ nM binding affinity, and a 29000 fold selectivity for the δ-opioid receptor over the μ opioid receptor. Combined molecular modeling and NMR studies indicated that the (2S,3R)-β-iPrPhe³ residue in the analogue favors the trans rotamer, and can induce the linear peptide to form a low-energy folded conformation which was proposed as the bioactive conformation for the δ-opioid receptor. Coupling four optically pure, conformationally constrained β-methyl-2',6'-dimethyltyrosine (TMT) with L-Tic formed four dipeptide analogues of TMT-L-Tic. The most potent and selective analogue, (2S,3R)-TMT-L-Tic showed 9 nM binding affinity and 4000 fold selectivity to the δ vs μ opioid receptor. The lowest-energy conformation of (2S,3R)-TMT-L-Tic was suggested to be the bioactive one in which TMT side chain is trans and Tic side chain is in a gauche (+) conformation. Bicyclic oxytocin antagonist [dPen¹, cyclo(Glu⁴ Lys⁸)]OT (BC-OT) (pA₂ = 8.10) is an excellent template to examine further topographical ideas. Substitution of Tyr² with the topographically constrained para-methoxy-β-methyl-2',6'-dimethyltyrosine (p-MeOTMT) amino acids produced two very potent antagonists [(2S,3S)-p-MeOTMT²]BC-OT (pA₂ = 8.26) and [(2R,3R)-p-MeOTMT²]BC-OT(pA₂ = 7.80), and two inactive analogues [(2S,3R)-p-MeOTMT²]BC-OT and [(2R,3S)-p-MeOTMT²]BC-OT. These interesting results can be attributed to the biased side-chain conformation, gauche(+) and gauche(-) in (2S,3S)-p-MeOTMT and (2R,3R)-p-MeOTMT respectively, and trans in both (2S,3R)-p-MeOTMT and (2R,3S)-p-MeOTMT residues. Rational design of non-peptide mimetics from peptide leads is still elusive. Based on the δ-opioid selective lead [(2S,3R)-TMT¹]DPDPE and SAR of δ-opioid selective ligands, the first generation of non-peptide mimetics have been designed and synthesized. The new lead SL-3111 showed binding affinity IC₅₀ = 8 nM, and over 2000 fold selectivity for the δ-opioid receptor over the μ receptor.

Six novel platinum(II) intercalators of the form [Pt(AL)(IL)]Cl2, where AL = ethylenediamine (en), 1R,2R-diaminocyclohexane (R,R-dach), or 1S,2S-diaminocyclohexane (S,S-dach) and IL = 4,7-dihydroxy-1,10-phenanthroline (4,7-dhp) or 4,7-dicarboxy-1,10-phenanthroline (4,7-dcp), were synthesised. All complexes were prepared by the addition of the intercalating ligand followed by the addition of the diamine ancillary ligand. The complexes with 4,7-dhp were soluble in DMSO and were characterised by 1H, 13C, and 195Pt NMR, elemental analysis, UV-vis, ESI-MS, and CD. The complexes with 4,7-dcp were only soluble in a highly acidic solution and, therefore, were characterised only by 1H NMR and elemental analysis. The cytotoxicity of the 4,7-dhp complexes was tested in the L1210 murine leukaemia cell line. [Pt(S,S-dach)(4,7-dhp)]Cl2 showed an IC50 value of > 80 μM. The antitumour and antibacterial activities of all six complexes were tested in vitro using the Kirby-Bauer disc diffusion method with Staphylococcus aureus and Agrobacterium tumefaciens. The 4,7-dhp complexes showed no activity to these bacteria strains. The activities of the 4,7-dcp complexes were not able to be tested due to their solubility only in acidic solutions, which itself inhibits cell growth. The diffusion coefficients of the Pt(II) intercalators of the form [Pt(AL)(IL)]Cl2, where AL = en, R,R-dach, or S,S-dach and IL = phen, 4-mp, 4,7-dmp, 4,7-dhp, 4,7-dcp or 3,4,7,8-tmp and various starting materials used during the synthesis of these complexes were measured using pulsed gradient spin-echo (PGSE) NMR. The diffusion coefficients of both 4,7-dcp and [Pt(4,7-dcp)Cl2] were observed to be lower than other compounds with similar molecular weights indicating dimerisation of the compounds. The binding studies of the systems, [Pt(en)(phen)]Cl2 to (i) BSA, (ii) delipidated BSA, and (iii) d(GTCGAC)2 were studied using a simple two-site binding model with diffusion NMR. The binding of [Pt(en)(phen)]Cl2 – BSA was well described by the model giving the values Kd = 0.0021 ± 0.0002 M and n = 5.85 ± 0.31. On the contrary, the binding of [Pt(en)(phen)]Cl2 – delipidated BSA showed a poor fit to the model. From the poor fit of the data, it was speculated that the transverse relaxation of BSA largely affected the system. The binding of [Pt(en)(phen)]Cl2 – d(GTCGAC)2 showed results where the diffusion coefficient decreases as the concentration of the drug increases but an opposite effect was observed from the point where the drug reached equimolar concentrations to d(GTCGAC)2. It was speculated that the drug undergoes allosteric binding to the biomolecule or that a conformational change occurred as the drug concentration increases in the system. A further study of [Pt(en)(phen)]Cl2 and K2PtCl4 using 195Pt diffusion NMR was conducted giving a diffusion coefficient of 3.08 ± 0.04 × 10-10 m2 s-1 for K2PtCl4. The diffusion coefficient of [Pt(en)(phen)]Cl2, however, were unobtainable due to the short transverse relaxation of the Pt complex.
Master of Science (M.Sc.) (Hons)

Proteins modularity enhances the multi-functionality and versatility of proteins by providing such properties as multiple and various ligand-binding sites, increased ligand affinity through the avidity effect, and the juxtaposition of ligand-binding modules near catalytic domains. An NMR-based "dissect-and-build" approach to studying modular protein structure and function has proven very successful, whereby modules are initially characterized individually and then correlated with the overall function of a protein. We have used the dissect-and-build approach and NMR to study two modular protein systems. Chapter 2 details the NMR solution structure of the weak-lysine-binding kringle IV type 8 (KIV8) module from the apolipoprotein(a) (apo(a)) component of lipoprotein(a) was determined and its ligand-binding properties assessed. In vitro studies have demonstrated the importance of the apo(a) KIV7 and KIV8 modules in mediating specific lysine-dependent interactions with the apolipoproteinB-100 (apoB-100) component of LDL in the initial non-covalent step of lipoprotein assembly. Notable differences identified in the lysine binding site (LBS) of the KIV8 were deemed responsible for the differential modes of apoB-100 recognition by KIV7 and KIV8. In addition, the KIV8 structure has brought to light the importance of an RGD sequence at the N-terminus of the apo(a) KIV8 module, which may mediate important apo(a)-integrin interactions. In Chapters 3-6, structure-function studies of the CpGH84C X82 and the CpGH84A dockerin-containing modular pair were conducted to understand how the varying modularity unique to the C-terminal regions of the secreted multi-modular family 84 glycoside hydrolases influences the spreading of Clostridium perfringens. Identification of a CpGH84C cohesin module (X82), and the structural characterization of a dockerin-containing modular pair provides the first evidence for multi-enzyme complex formation mediated by non-cellulosomal cohesin-dockerin interactions. The formation of large hydrolytic enzyme complexes introduces a novel mechanism by which C. perfringens may enhance its role in pathogenesis.
Thesis (Ph.D, Biochemistry) -- Queen's University, 2007-09-27 11:46:38.753

Acetohydroxyacid synthase is a multisubunit enzyme that catalyses the first committed step in the biosynthesis of the branched chain amino acids viz., valine, leucine and isoleucine. In order to understand the structural basis for the observed allosteric feedback inhibition in AHAS, the regulatory subunit of AHAS isozymes I from E. coli was cloned, expressed, purified and the conditions were optimized for solution NMR spectroscopy. IlvN was found to exist as a dimer both in the presence and absence of the feedback inhibitor. Using high-resolution multidimensional, multinuclear NMR experiments, the structure of the dimeric valine-bound 22 kDa IlvN was determined. The ensemble of twenty low energy structures shows a backbone root mean square deviation of 0.73 ± 0.13 Å and a root mean square deviation of 1.16 ± 0.13 Å for all heavy atoms. Furthermore, greater than 98% of the backbone φ, ψ dihedral angles occupy the allowed and additionally allowed regions of the Ramachandran map. Each protomer exhibits a βαββαβα topology that is a characteristic feature of the ACT domain fold that is observed in regulatory domains of metabolic enzymes. In the free form, IlvN exists as a mixture of conformational states that are in intermediate exchange on the NMR timescale. Important structural properties of the unliganded state were probed by H-D exchange studies by NMR, alkylation studies by mass spectrometry and other biophysical methods. It was observed that the dynamic unliganded IlvN underwent a coil-to-helix transition upon binding the effector molecule and this inherent conformational flexibility was important for activation and valine-binding. A mechanism for allosteric regulation in the AHAS holoenzyme was proposed. Study of the structural and conformational properties of IlvN enabled a better understanding of the mechanism of regulation of branched chain amino acid biosynthesis. Solution structural studies of 32 kDa Protease-VPg (PVPg) from Sesbania mosaic virus (SeMV) Polyprotein processing is a commonly found mechanism in animal and plant viruses, by which more than one functional protein is produced from the same polypeptide chain. In Sesbania Mosaic Virus (SeMV), two polyproteins are expressed that are catalytically cleaved by a serine protease. The VPg protein that is expressed as a part of the polyprotein is an intrinsically disordered protein (by recombinant expression) that binds to various partners to perform several vital functions. The viral protease (Pro), though possessing the necessary catalytic residues and the substrate binding pocket is unable to catalyse the cleavage reactions without the VPg domain fused at the C-terminus. In order to determine the structural basis for the aforementioned activation of protease by VPg I undertook the structural studies of the 32 kDa PVPg domains of SeMV by solution NMR spectroscopy. NMR studies on this protein were a challenge due to the large size and spectral overlap. Using a combination of methods such as deuteration, TROSY-enhanced NMR experiments and selective ‘reverse-labelling’, the sequence specific assignments were completed for ~80% of the backbone and 13C nuclei. NMR studies on mutants such as the C-terminal deletion mutant, I/L/V to A mutants in VPg domain were conducted in order to identify the residues important for aliphatic-aromatic interactions observed in PVPg. Attempts were made to obtain NOE restraints between Pro and VPg domains through ILV labelled samples; however these proved unsuccessful. It was observed that ‘natively unfolded’ VPg possessed both secondary and tertiary structure in PVPg. However, 30 residues at the C-terminus were found to be flexible. Even though atomic-resolution structure could not be determined, the region of interaction between the domains was determined by comparing NMR spectra of Pro and PVPg. The conditions for reconstitution of the Protease-VPg complex by recombinantly expressed Pro and VPg proteins were standardised. These studies lay the foundation for future structural investigations into the Protease-VPg complex.