UC San Diego
Evaluation of mechanisms of non-canonical G-protein signaling, membrane transporter dysregulation, and bacterial sialidase substrate preference using X-ray crystallography
- Author(s): Rees, Steven Douglas
- Advisor(s): Chang, Geoffrey A
- et al.
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.