Neurodegenerative diseases are defined as diseases wherein nerve cells are damaged or destroyed and are a major health concern. Millions around the world have Alzheimer's and Parkinson's diseases, causing untold suffering and costing billions of dollars annually. Understanding the pathogenesis of these diseases on a molecular level is crucial to developing cures.

Parkinson's disease destroys dopaminergic neurons in the substantia nigra pars compacta area of the brain, causing many movement related disorders and ultimately death. A hallmark of Parkinson's disease is the cytosolic, filimental inclusion known as the Lewy Body, which consists mostly of the protein α-synuclein. α-Synuclein is a 140-residue unstructured neural peptide that has been linked to the pathogenesis of Parkinson's disease through genetics and animal models. Genetic mutations arising in Parkinson's are rare, and the disease appears to be instigated more commonly by external influences such as pesticides and heavy metals, in particular copper and aluminum. In vitro studies have indirectly demonstrated interaction between α-synuclein and copper ions. However, previous attempts to characterize the monomeric, unstructured α-synuclein-Cu2+ interaction more directly have lead to some confusion and conflicting results regarding the stoichiometry, chelation structures and binding affinity.

Alzheimer's disease is characterized as a dementia and is the most common neurological disorder, primarily affecting the cognitive abilities leading to extreme confusion, aggressive behavior and difficulty speaking. The disease ultimately leads to death within seven years after diagnosis. A hallmark of Alzheimer's disease is extracellular deposits, termed senile plaques, consisting mostly of the aggregated 40-42 residue protein Aβ. These plaques are shown to have elevated levels of copper relative to the rest of the brain and Aβ binds copper strongly in its monomeric form. Indirect evidence has suggested that aggregated Aβ chelates copper with higher affinity than the monomeric form. Direct evidence of this phenomenon, though, is lacking.

In this dissertation we use electron paramagnetic resonance (EPR) to directly investigate how the proteins α-synuclein and aggregated Aβ interact with Cu2+ ions. EPR is a technique that is uniquely suited to probe the interaction of natively unstructured proteins with paramagnetic species such as Cu2+. EPR combined with site-directed mutagenesis is a powerful tool for directly determining the protein-paramagnetic species stoichiometry and protein residues involved in copper chelation. Dissociation constant based EPR competition studies directly measure the affinity of the species of interest for the paramagnetic ions. Using these techniques we have determined the stoichiometry, chelation structures and affinities for both unstructured (chapter 2) and membrane bound (chapter 3) α-synuclein. Furthermore, in chapter 4, EPR competition studies between aggregated Aβ and human serum albumin demonstrate that the aggregated form of Aβ is capable of outcompeting albumin for Cu2+ whereas monomeric Aβ is not. This information helps to shed light on both of these proteins disease pathogenesis and perhaps their natural function as well. This knowledge is critical for the development of new therapies for these diseases.

The cellular prion protein (PrPC) is comprised of two domains – a globular C-terminal domain and an unstructured N-terminal domain. Copper drives tertiary contact in PrPC, inducing a neuroprotective cis interaction that structurally links the protein’s two domains. The location of this interaction on the C-terminus overlaps with the sites of human pathogenic mutations and toxic antibody docking. Combined with recent evidence that the N-terminus is a toxic effector regulated by the C-terminus, there is an emerging consensus that this cis interaction serves a protective role, and that the disruption of this interaction by misfolded PrP oligomers may be a cause of toxicity in prion disease. We demonstrate here that two highly conserved histidines in the C-terminal domain of PrPC are essential for the protein’s cis interaction, which helps to protect against neurotoxicity carried out by its N-terminus. We show that simultaneous mutation of these histidines drastically weakens the cis interaction and enhances spontaneous cationic currents in cultured cells - the first C-terminal mutant to do so. Whereas previous studies suggested that Cu2+ coordination was localized solely to the protein’s N-terminal domain, we find that both domains contribute equatorially coordinated histidine residue side chains, resulting in a novel bridging interaction. We also find that extra N-terminal histidines in pathological familial mutations involving octarepeat expansions inhibit this interaction by sequestering copper from the C-terminus. Our findings further establish a structural basis for PrPC’s C-terminal regulation of its otherwise toxic N-terminus. The glycans of the prion protein are located in close proximity to the C-terminal histidines that we have shown are necessary for the neuroprotective copper tether. The recombinant protein used for the previously described experiments lacked glycans, and we wondered if they would have any effect on the observed interaction. Since the glycans are fairly large and located on the surface of the protein, we thought that they may sterically hinder the cis interaction, falsifying or requiring modification of the interpretation of the previously described results. We set out to create glycosylated protein in order to directly test the impact of these moieties on the protective cis interaction. Bacterially expressed proteins used in NMR studies lack glycans, and proteins from other organisms are neither 15N labelled, nor glycosylated homogenously. We developed a method to add two artificial glycans to uniformly 15N labelled prion protein, using a buffer system that evolves over a pH range in order to accommodate the conflicting pH requirements of the substrate and enzymes, without the need to fine tune buffer conditions. NMR and CD spectroscopy of the protein indicates that the glycans do not influence its fold. Using our glycosylated, 15N labelled protein, we tested the effects of the glycans on the protein’s previously observed cis interaction. In opposition to our hypothesis that the glycans would prevent the interaction through steric hindrance, we observed that the interaction was refined by the glycans; the glycans narrow down the region of the C-terminus with which copper interacts. We then found that the presence of glycans partially restores the lost cis interaction observed when C-terminal histidines are mutated away. Together, the work in this dissertation shows that both N and C-terminal histidines of the prion protein bind copper ions, driving a neuroprotective self-regulatory interaction, and that the glycans of the protein refine and strengthen this interaction.

The melanocortin 4-receptor (MC4R) is a G-protein coupled receptor (GPCR) that is expressed in the hypothalamus and is essential for energy homeostasis. Mutations in MC4R are the most common monogenic cause of obesity. MC4R signaling is regulated by its agonist ???? -MSH and by its endogenous antagonist the agouti-related peptide (AgRP). AgRP’s orexigenic effects are extraordinarily long-lasting. Additionally, several accessory proteins have been implicated in the regulation of MC4R. This dissertation focuses on two accessory proteins, syndecan-3 and the melanocortin receptor accessory protein 2 (MRAP2). Syndecan-3 is a cell-surface heparan sulfate proteoglycan that is hypothesized to increase the local concentration of AgRP at MC4R neurons. AgRP’s prolonged orexigenic effects are dependent on positively charged residues outside of its inhibitor cystine knot (ICK) core. These positively charged residues are not required for MC4R binding or antagonism but are crucial for electrostatic interactions with heparan sulfate. Using AgRP peptides with varying positive charge in its non-ICK segments along with glycan arrays and feeding studies with AgRP-treated mice, we find that AgRP increases food intake and reduces energy expenditure. These actions are dependent on AgRP’s positive charges and the AgRP variant that has the most protracted feeding response also has the greatest affinity for heparan sulfate. These findings further support the role of syndecan-3 in facilitating AgRP signaling. MRAP2 increases ????-MSH-induced MC4R signaling and also modulates several other hypothalamic GPCRs that regulate metabolism. MRAP2 is a single-pass membrane protein that forms highly unusual anti-parallel dimers. Despite MRAP2’s important role in energy balance, very little is known regarding the molecular details that drive its unique structure and how this relates to its ability to modulate MC4R. Using a panel of MRAP2 variants, biochemical experiments reveal that MRAP2 dimerizes through its transmembrane domain. We also show that MRAP2 can form parallel dimers using a luciferase-based molecular complementation assay. Elucidating the functions and structures of MC4R’s accessory proteins will not only further our understanding of GPCR signaling but also aid in the development of therapeutics aimed at modulating this complex signaling system.

Agouti Signaling Peptide (ASIP) is a small disulfide-rich peptide, which acts as an inverse agonist at melanocortin receptor 1 (MC1R) and reduces the production of melanin. ASIP has recently been shown to increase melanoma treatment sensitivity. Here we develop strategies to improve ASIP potency by increasing its affinity and specificity to MC1R. We find no increase in binding for the constructs we synthesized. We then increase the stability of ASIP via head-to-tail cyclization using hydrazide intermediates to facilitate native chemical ligation. We find the imposed chemical restraint does not alter peptide structure or function as a melanocortin antagonist. We compared the stability of linear and cyclic ASIP in human serum, finding that cyclic ASIP has a slight increase in resistance to proteolysis. These studies have the potential to revolutionize the treatment of melanoma through the use of ASIP as a peptide therapeutic.

The cellular prion protein (PrPC) is a membrane-anchored glycoprotein consisting of two domains: a flexible N-terminal domain that participates in metal binding, and a mainly helical C-terminal domain that converts to β-sheet structure in the course of prion disease. These two domains have traditionally been thought of as non-interacting; however, recent cellular and biophysical evidence has forced a reconsideration of this view. Here we employ nuclear magnetic resonance spectroscopy (NMR), electron paramagnetic resonance spectroscopy (EPR), and fluorescence resonance energy transfer (FRET) to investigate inter-domain structure in PrPC in response to metal binding. We develop a new strategy for probing domain-domain interactions in PrP that employs the genetic incorporation and labeling of the unnatural amino acid pAcPhe. Through 1H-15N HSQC NMR experiments on full-length and truncated PrP constructs, we demonstrate a novel tertiary fold in which the Zn2+-bound octarepeat domain contacts the exposed surfaces of helices 2 and 3. These results are supported by inter-domain distance measurements from EPR and FRET. The apparent stability of this interaction is diminished in several mutant PrPs that result in familial prion disease, suggesting a potential role for inter-domain structure in disease progression. We then examine global structure in Cu2+-bound PrPC using site-directed spin labeling and EPR spectroscopy. Distance measurements between Cu2+, bound with high affinity to the octarepeat domain, and spin labeled residues of the globular C-terminus reveal that the copper-bound octarepeats interacts with a negatively charged surface defined by helices 2 and 3. This interaction surface is qualitatively equivalent to the tertiary contact site identified in the Zn2+-bound protein. Our results are supported by molecular dynamics simulations and indicate that this cis interaction is stabilized by electrostatics. Our findings suggest that metal-induced tertiary structure may be a general property of PrPC, and that disruption of this interaction may be a contributing factor in prion disease pathology.

The Melanocortin system of G-Protein coupled receptors and ligands plays a vital role in cell signaling of many important physiological processes, including pigmentation, steroidogenesis, and metabolic control. These receptors are all activated by ligands derived from the proopiomelanocortin (POMC). This prohormone is processed by tissue specific expression of various proteases. Three receptors within this system have a secondary level of regulation. Melanocortin receptors (McRs) 1, 3 and 4 also respond to endogenously expressed antagonist proteins, the agouti-signaling protein (ASIP) and agouti-related protein (AgRP). ASIP and AgRP compete with POMC ligands for receptor binding and signal for reduction in cAMP. Both antagonists are sequentially and structurally similar; yet show differing tissue expression and receptor binding specificities. AgRP is expressed in the hypothalamus and signals for an increase in appetite and lower energy expenditure through Mc3R and Mc4R. ASIP is primarily expressed in the skin and affects pigmentation through Mc1R, but can interact with Mc3R and Mc4R through some genetic mutations.

The ligand expression and binding specificity at McRs 1, 3 and 4 provide a unique set of tools for understanding structural determinants required for regulation of pigmentation and energy balance and homeostasis. Here study a novel third antagonist sequence, AgRP2, discovered in teleost fish and expressed in the light sensitive Pineal gland of the brain. This protein shares the same cysteine spacing observed for ASIP and AgRP, and pharmacological studies revealed AgRP2 is selective for the zebrafish Mc1R. Unlike the human homologue, the zebrafish Mc1R is highly expressed in the brain. Knockdown studies reveal that the AgRP2 protein signals for pigment granule aggregation and is a vital molecule in the background adaptive process. Next, NMR analysis of a β-MSH mutant sequence, identified a tyrosine residue, not involved in receptor binding contacts, that participates in forming a turn motif. Mutation of this residue to cysteine significantly reduces this turn structure and dramatically reduces the agonist binding and activity at Mc4R. This mutation predisposes carriers to early-onset obesity. Finally, we study the non-ICK regions of the AgRP protein. Isothermal titration calorimetry experiments implicate the C-loop in proteoglycan binding and motivated rat feeding studies on C-terminal constructs. Through truncation of this domain, a correlation between peptide charge and appetite stimulation was characterized and led to the design of so-called "Super-AgRP" proteins that stimulate feeding in rodents by more than twice that of wild-type AgRP. These studies have the potential to revolutionize the treatment of cachexia and other negative energy balance metabolic disorders.

The Retinoblastoma Protein (Rb) is a sentinel of the cell division process. When phosphorylated, Rb is inactivated and physically releases its protein-binding partner, E2F; a potent transcription factor that up-regulates many of the genes required for DNA synthesis and cell division. Aberrant deregulation of Rb, either through the loss of functional Rb or the persistence of hyper-phosphorylated Rb, is a common event in the development and progression of cancer. This study uses protein X-ray crystallography, NMR, ITC and SAXS to characterize three distinct structural changes to Rb that are caused by certain phosphorylation events. Specifically, we find phosphorylation of S608 induces the formation of a helix within the pocket linker region of Rb, which competitively inhibits E2F-binding. T373 phosphorylation promotes a docking interaction between Rb's two structured domains; this causes an allosteric change to a critical E2F-binding surface of Rb and dramatically weakens the Rb-E2F interaction. Lastly, phosphorylation of Rb's C-terminus at S788 and S795 also promotes an intramolecular interaction that destabilizes the Rb-E2F complex. These three distinct phosphorylation-induced mechanisms provide supporting evidence for structure-based theories of how Rb phosphorylation promotes the release and activation of E2Fs. A knowledge of these mechanisms is the first step in understanding how Rb may be therapeutically targeted to regain its function in cancer cells.

The misfolding of the cellular prion protein (PrPC) into the aggregate prone conformer (PrPSc) is at the heart of a class of neurodegenerative diseases called transmissible spongiform encephalopathies (TSEs). These diseases affect humans, bovine, sheep, and other mammals. PrPC is a well conserved mammalian protein and is highly expressed in the brain. PrPC is composed of two main domains consisting of the unstructured N-terminus and the structured C-terminus. The two main domains are connected by a short linker called the central region (CR). Previous studies have shown internal amino-proximal deletions spanning the CR generate a profound neurotoxicity in mice. The toxicity generated by these deletions have been shown to parallel bona fide prion diseases, and thus making them a good model system for studying prion diseases. The neurotoxicity elicited by CR deletion mutants is thought to originate from a misregulation of the extreme N-terminus of PrPC. However, these deletion mutants make two major changes that could potentially lead to the misregulation of the N-terminus. These include: 1) weakening of a protective metal-driven cis-interaction, and 2) deletion of the locus important in a regulatory cleavage event. This study used designed mutations to allow for one of these changes at a time while not altering the other. We find that neither blocking the metal-driven cis-interaction nor cleavage generates toxicity. Conversely, we show that the WT PrPC CR sequence is necessary for regulating the toxicity. Moreover, we find that the WT PrPC CR sequence is necessary for PrPC homodimerization on the cell surface, which could be a protective mechanism from the deleterious effects of a misregulated extreme N-terminus. Additionally, we found that pathological mutations in the CR causing a certain set of prion diseases in humans do not affect the metal-driven cis-interaction. Furthermore, these result suggests a difference in the origin of neurotoxicity between CR pathological mutations and C-terminal pathological mutations.