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Open Access Publications from the University of California

Scholarly Works (8 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2018)

X-ray crystallography remains a popular technique for achieving atomic resolution of macromolecular interactions. Protein crystal structures offer a snapshot of how these complex molecules fold in three-dimensional space, often too difficult to simulate de novo with high accuracy, as well as where substrates, activators, inhibitors, regulators, and other regulatory partners bind them. While the physics of crystallography can be a steep learning curve, the methodology offers a powerful tool for analyzing protein crystals.

This dissertation is a summation of three major projects I undertook in Dr. Geoffrey Chang’s lab at UCSD. These projects differ highly in their focus areas, moving from environmental pollution to sugar-derived microbiome changes, and still further to cancer-linked non-canonical G protein signaling. All three of these projects were linked not by topic, but by their problems: all required a means of seeing a protein-ligand interaction at high resolution, one that had not yet been previously achieved. Each required its own solutions to difficult crystallographic situations, including low resolution, poor ligand occupancy, abnormal crystal packing, and subtle binding pocket changes, yet all benefitted from applying the crystallographic method to solve them.

This dissertation began by exploring the hypothesis that persistent organic pollutants (POPs) dysregulate efflux transporters at the barriers between main circulation and the body’s major organ systems. We identified a high number of POPs in yellowfin tuna, one of the most consumed fish species in the US, many of which inhibited xenobiotic efflux by P-glycoprotein, a well-characterized efflux transporter. We utilized five asymmetrically placed Br atoms around PBDE-100, a common POP, to place this ligand in the density of a P-gp co-crystal structure, the first known structure of an efflux transporter with a POP. This structure confirmed occupancy of this ligand in an area known to be bound by inhibitors, which corroborated biochemical characterization of this interaction.

My work in crystallography continued with a collaboration with the Karsten Zengler lab at UCSD. In this work, metagenomics studies identified changes in the gut microbiome of mice in response to diets enriched with Neu5Ac or Neu5Gc, the two common mammalian sialidases with disparate presence in red meat. In particular, the first microbial sialidases with specificity for Neu5Gc were identified. I led the protein purification and structure determination efforts in this project. The structures for this project, including the first structure of any protein with a Neu5Gc-like ligand, indicated which residues likely play a role in Neu5Gc preference for these sialidases, paving the way for future work into engineering Neu5Gc-specific enzymes, which could play a role in reducing gut inflammation due to red meat-containing diets.

Another sought-after collaboration with the Pradipta Ghosh lab at UCSD explored non-canonical signaling of G protein systems by Guanine nucleotide exchange modulators (GEMs). Previous work has shown that G proteins can be activated in signaling cascades independent of canonical GPCR signaling, but little was known about how G proteins interacted with GEMs. We serendipitously discovered a GDI-like motif that aided in crystallization of a complex between the alpha G protein subunit Gi with the C-terminus of GIV/Girdin, a well-characterized GEM. This structure represents the first of a Gi protein with a natural GEM.

These projects, while rooted in highly diverse scientific fields, found commonality in their unique crystallographic challenges. A crystallographer, or protein chemist, could not have asked for a better means of not only learning the intricacies of this complex field, but also how to apply them in new ways to solve old and new structural problems.

    Cover page: Evaluation of mechanisms of non-canonical G-protein signaling, membrane transporter dysregulation, and bacterial sialidase substrate preference using X-ray crystallography
    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2021)
    Marine pollutants bioaccumulate at high trophic levels of marine food webs and are transferred to humans through consumption of apex species. Yellowfin tuna (Thunnus albacares) are marine predators, and one of largest commercial fisheries in the world. Previous studies have shown that yellowfin tuna can accumulate high levels of persistent organic pollutants, including Transporter Interfering Chemicals (TICs), which are chemicals shown to bind to mammalian xenobiotic transporters and interfere with their function. Here, we examined the extent to which these same compounds might interfere with the activity of the yellowfin tuna (Thunnus albacares) ortholog of this transporter. To accomplish this goal we identified, expressed, and functionally assayed tuna ABCB1. The results demonstrated a common mode of vertebrate ABCB1 interaction with TICs that predicts effects across these species, based on high conservation of specific interacting residues. Importantly several TICs showed potent inhibition of Ta-ABCB1, such as the organochlorine pesticides Endrin (EC50 = 1.2 ± 0.2 μM) and Mirex (EC50 = 2.3 ± 0.9 μM). However, unlike the effects observed on mouse ABCB1, low concentrations of the organochlorine pesticide TICs p,p'-DDT and its metabolite p,p'-DDD co-stimulated verapamil-induced Ta-ABCB1 ATPase activity possibly suggesting a low transport activity for these ligands in tuna. These results provide a mechanistic basis for understanding the potential vulnerability of tuna to these ubquitous pollutants.
      Cover page: Transporter-interfering chemicals inhibit P-glycoprotein of yellowfin tuna (Thunnus albacares).
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2016)
      The world's oceans are a global reservoir of persistent organic pollutants to which humans and other animals are exposed. Although it is well known that these pollutants are potentially hazardous to human and environmental health, their impacts remain incompletely understood. We examined how persistent organic pollutants interact with the drug efflux transporter P-glycoprotein (P-gp), an evolutionarily conserved defense protein that is essential for protection against environmental toxicants. We identified specific congeners of organochlorine pesticides, polychlorinated biphenyls, and polybrominated diphenyl ethers that inhibit mouse and human P-gp, and determined their environmental levels in yellowfin tuna from the Gulf of Mexico. In addition, we solved the cocrystal structure of P-gp bound to one of these inhibitory pollutants, PBDE (polybrominated diphenyl ether)-100, providing the first view of pollutant binding to a drug transporter. The results demonstrate the potential for specific binding and inhibition of mammalian P-gp by ubiquitous congeners of persistent organic pollutants present in fish and other foods, and argue for further consideration of transporter inhibition in the assessment of the risk of exposure to these chemicals.
        Cover page: Global marine pollutants inhibit P-glycoprotein: Environmental levels, inhibitory effects, and cocrystal structure
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2019)
        Heterotrimeric G proteins are key molecular switches that control cell behavior. The canonical activation of G proteins by agonist-occupied G protein-coupled receptors (GPCRs) has recently been elucidated from the structural perspective. In contrast, the structural basis for GPCR-independent G protein activation by a novel family of guanine-nucleotide exchange modulators (GEMs) remains unknown. Here, we present a 2.0-Å crystal structure of Gαi in complex with the GEM motif of GIV/Girdin. Nucleotide exchange assays, molecular dynamics simulations, and hydrogen-deuterium exchange experiments demonstrate that GEM binding to the conformational switch II causes structural changes that allosterically propagate to the hydrophobic core of the Gαi GTPase domain. Rearrangement of the hydrophobic core appears to be a common mechanism by which GPCRs and GEMs activate G proteins, although with different efficiency. Atomic-level insights presented here will aid structure-based efforts to selectively target the noncanonical G protein activation.
          Cover page: Structural basis for GPCR-independent activation of heterotrimeric Gi proteins
          • Article
          • Peer Reviewed
          UC Santa Barbara Previously Published Works (2019)
          Dietary habits have been associated with alterations of the human gut resident microorganisms contributing to obesity, diabetes and cancer1. In Western diets, red meat is a frequently eaten food2, but long-term consumption has been associated with increased risk of disease3,4. Red meat is enriched in N-glycolylneuraminic acid (Neu5Gc) that cannot be synthesized by humans5. However, consumption can cause Neu5Gc incorporation into cell surface glycans6, especially in carcinomas4,7. As a consequence, an inflammatory response is triggered when Neu5Gc-containing glycans encounter circulating anti-Neu5Gc antibodies8,9. Although bacteria can use free sialic acids as a nutrient source10-12, it is currently unknown if gut microorganisms contribute to releasing Neu5Gc from food. We found that a Neu5Gc-rich diet induces changes in the gut microbiota, with Bacteroidales and Clostridiales responding the most. Genome assembling of mouse and human shotgun metagenomic sequencing identified bacterial sialidases with previously unobserved substrate preference for Neu5Gc-containing glycans. X-ray crystallography revealed key amino acids potentially contributing to substrate preference. Additionally, we verified that mouse and human sialidases were able to release Neu5Gc from red meat. The release of Neu5Gc from red meat using bacterial sialidases could reduce the risk of inflammatory diseases associated with red meat consumption, including colorectal cancer4 and atherosclerosis13.
            Cover page: Gut bacteria responding to dietary change encode sialidases that exhibit preference for red meat-associated carbohydrates
            • Article
            UC Davis Previously Published Works (2021)
            CASP (Critical Assessment of Structure prediction) conducts community experiments to determine the state of the art in computing protein structure from amino acid sequence. The process relies on the experimental community providing information about not yet public or about to be solved structures, for use as targets. For some targets, the experimental structure is not solved in time for use in CASP. Calculated structure accuracy improved dramatically in this round, implying that models should now be much more useful for resolving many sorts of experimental difficulty. To test this, selected models for seven unsolved targets were provided to the experimental groups. These models were from the AlphaFold2 group, who overall submitted the most accurate predictions in CASP14. Four targets were solved with the aid of the models, and, additionally, the structure of an already solved target was improved. An a-posteriori analysis showed that in some cases models from other groups would also be effective. This paper provides accounts of the successful application of models to structure determination, including molecular replacement for X-ray crystallography, backbone tracing and sequence positioning in a Cryo-EM structure, and correction of local features. The results suggest that in future there will be greatly increased synergy between computational and experimental approaches to structure determination.
              Cover page: Computational models in the service of X-ray and cryo-EM structure determinationCreative Commons 'BY' version 4.0 license
              • Article
              • Peer Reviewed
              UC Davis Previously Published Works (2021)
              Critical assessment of structure prediction (CASP) conducts community experiments to determine the state of the art in computing protein structure from amino acid sequence. The process relies on the experimental community providing information about not yet public or about to be solved structures, for use as targets. For some targets, the experimental structure is not solved in time for use in CASP. Calculated structure accuracy improved dramatically in this round, implying that models should now be much more useful for resolving many sorts of experimental difficulties. To test this, selected models for seven unsolved targets were provided to the experimental groups. These models were from the AlphaFold2 group, who overall submitted the most accurate predictions in CASP14. Four targets were solved with the aid of the models, and, additionally, the structure of an already solved target was improved. An a posteriori analysis showed that, in some cases, models from other groups would also be effective. This paper provides accounts of the successful application of models to structure determination, including molecular replacement for X-ray crystallography, backbone tracing and sequence positioning in a cryo-electron microscopy structure, and correction of local features. The results suggest that, in future, there will be greatly increased synergy between computational and experimental approaches to structure determination.
                Cover page: Computational models in the service of X‐ray and cryo‐electron microscopy structure determination
                • Article
                • Peer Reviewed
                UC San Diego Previously Published Works (2019)
                Sensing and responding to environmental water deficiency and osmotic stresses are essential for the growth, development, and survival of plants. Recently, an osmolality-sensing ion channel called OSCA1 was discovered that functions in sensing hyperosmolality in Arabidopsis Here, we report the cryo-electron microscopy (cryo-EM) structure and function of an OSCA1 homolog from rice (Oryza sativa; OsOSCA1.2), leading to a model of how it could mediate hyperosmolality sensing and transport pathway gating. The structure reveals a dimer; the molecular architecture of each subunit consists of 11 transmembrane (TM) helices and a cytosolic soluble domain that has homology to RNA recognition proteins. The TM domain is structurally related to the TMEM16 family of calcium-dependent ion channels and lipid scramblases. The cytosolic soluble domain possesses a distinct structural feature in the form of extended intracellular helical arms that are parallel to the plasma membrane. These helical arms are well positioned to potentially sense lateral tension on the inner leaflet of the lipid bilayer caused by changes in turgor pressure. Computational dynamic analysis suggests how this domain couples to the TM portion of the molecule to open a transport pathway. Hydrogen/deuterium exchange mass spectrometry (HDXMS) experimentally confirms the conformational dynamics of these coupled domains. These studies provide a framework to understand the structural basis of proposed hyperosmolality sensing in a staple crop plant, extend our knowledge of the anoctamin superfamily important for plants and fungi, and provide a structural mechanism for potentially translating membrane stress to transport regulation.
                  Cover page: Cryo-EM structure of OSCA1.2 from Oryza sativa elucidates the mechanical basis of potential membrane hyperosmolality gating
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