Structural dynamics to therapeutic interactions : visualizing proline metabolic enzymes by kinetic crystallography and molecular dynamics, and antibody-antigen complexes from enterotoxigenic E. coli by cryo-EM
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Structural studies using molecular dynamics simulations, X-ray crystallography, negative stain and cryo-electron microscopy on a diverse set of disease-related contexts (i.e., cancer metastasis, proline metabolic disorders, and enterotoxin-induced diarrheal illness) are presented in this thesis. Insights from the dynamics and structural descriptions obtained from this work contribute essential knowledge for the development of treatments to these diseases. Chapter 1 describes a collaborative project initiated by Dr. Marie Migaud, at the University of South Alabama, an expert in synthetic organic chemistry and disease involving aberrant forms of metabolic dinucleotides. We show via molecular dynamics (MD) simulations that three ROS-generated oxidative states on the nicotinamide of NAD(H), termed ox-NADs, display distinct conformational preferences in solution, with comparisons drawn to the solution dynamics of the essential dinucleotides NAD+ and NADH. The mechanism of exactly how ox-NADs contribute to their observed role in cancer metastasis and age-related diseases is unknown. Current hypotheses are either (1) given their high similarity to NAD(H), they could behave as endogenous inhibitors to NAD(H)-binding proteins, or (2) they exacerbate disease progression via the depletion of available NAD(H) in the cell. Future work to test the first hypothesis could involve obtaining X-ray crystal structures of ox-NADs (primarily 4-ox-NAD, which behaved most similar to NAD(H) from our analyses) bound to NAD(H)-binding proteins, in addition to thorough binding assays. Chapter 2 details work centered around a medical case study through collaboration with medical researchers at the United Hospitals of Marche, Italy, in which a young child displayed symptoms of pyridoxine dependent epilepsy (PDE) linked to two missense mutations in their ALDH7A1 gene. When I joined the project, one of these point mutations in the ALDH7A1 protein, R134S, was well characterized structurally, and the biochemical reasoning for the reduced activity of the mutated ALDH7A1 that leads to PDE was clear (i.e., disruption of the tetrameric oligomer required for ALDH7A1 catalysis). The other mutation, R441C, displayed very similar structural characteristics to wildtype ALDH7A1, thus an explanation for the drastic 50-fold reduction in catalytic activity could not be drawn. I ran a series of MD simulations of the structure of the mutant R441C ALDH7A1 tetramer, along with simulations of the wildtype ALDH7A1 tetramer, and identified a very subtle, but apparent difference in the dynamics of the C-terminal “gate” that has been previously shown in ALDH7A1 to have a significant function in modulating substrate access to its active site. This work neatly combines X-ray crystallography, enzyme kinetics, sedimentation velocity analysis, and MD simulations to deduce the mechanisms of the two identified mutations in their contribution to PDE in the young child of this case study. Chapter 3 surrounds my most substantial, primary project under Dr. Tanner using X-ray crystallography and MD to study substrate channeling in the bifunctional proline catabolic enzyme, Sinorhizobium meliloti Proline Utilization A (SmPutA). The human forms of the individual, monofunctional enzymes PRODH and ALDH4A1 (a.k.a. GSALDH) that achieve this essential metabolic conversion of L-proline to L-glutamate are thought to engage in a protein-protein interaction and undergo substrate channeling similar to bacterial PutAs, and both enzymes have substantial implications in proline metabolic disorders, cancer, and atherosclerosis. Utilizing kinetic X-ray crystallography, or in crystallo enzymatic turnover of SmPutA, transient covalent intermediates and substrate/product complexes were captured, which provides a near complete structural understanding of how proline catabolism is achieved in PutA. We initiated MD simulations from one of the captured species, the PRODH product P5C-bound state, and observed product release events in the majority of simulations and sequestering of this intermediate within the large tunnel that connects the PRODH and ALDH4A1 active sites. This work provides the first ever MD simulations of any PutA, and could inform on how this process is achieved in humans (specifically in our description of the absolute requirement of the conserved aspartate-arginine ion gate in the PRODH active site opening prior to product release). Chapter 4 highlights my work after formal addition of Dr. Berndsen as my co-advisor, where we collaborated with Dr. James Fleckenstein at Washington University in St. Louis, a leader in the field of the molecular pathogenesis and prevention of enterotoxigenic E. coli (ETEC). ETEC is among the leading causes of diarrheal disease in young children in low-middle income countries, in regions lacking access to the fundamental need of clean drinking water. In this chapter, I contributed extensive negative stain electron microscopy data that outlined differential polyclonal antibody (pAb) targeting against a virulence factor and adhesin of ETEC, EtpA, from the sera of mice vaccinated with EtpA adjuvanted with double mutant heat-labile enterotoxin (dmLT; LT is also secreted by ETEC, one of the direct causative toxins of eventual diarrhea, and an activator of innate immunity in its double mutant form). We were able to obtain 2-dimensional class averages which detail a time-dependence during vaccination on the quality and quantity of pAbs targeting EtpA. EtpA is a ripe target in vaccine design due to its role in facilitating ETEC colonization, and this work aids in development of an ETEC vaccination strategy using EtpA. Chapter 5 describes my most substantial, primary project under Dr. Berndsen, and continued collaboration with the Fleckenstein lab, where we focus on another secreted virulence factor of ETEC, EatA (a serine protease autotransporter, or SPATE), using cryo-electron microscopy (cryo-EM). The role of EatA during ETEC infection is in its mucinase activity, specifically in cleaving the layer of complex glycoprotein MUC2 that lines intestinal epithelia. The Fleckenstein lab has identified human antibodies against EatA that neutralize MUC2 degradation through a combination of natural ETEC infection cases and controlled ETEC infection studies. We have obtained high resolution structures of four of the identified antibodies in their fragment antigen binding (Fab) form bound to EatA, and detail three unique EatA epitopes. We also show that one of the targeted epitopes is highly conserved in the related Shigella and enteroaggregative E. coli SPATE molecules, SepA and Pic, by obtaining cryo-EM Fab complexes with these molecules that display near identical binding modes to the Fab-EatA solved complex. Together, these results promote this identified structural motif as a prime candidate for design of a minimal immunogenic construct in the effort to create a broadly protective vaccine against ETEC and related enteric pathogens. Chapters 1-4 are adapted from published works, and chapter 5 is currently in the final stages of manuscript preparation for initial submission to bioRxiv.
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Ph. D.