Crystallographic studies of macromolecular structures

National Institute Of Environmental Health Sciences

Investigators

Linked publications, trials & patents

Abstract

Our lab works on a variety of different biological systems including heparan/chondroitin sulfate biosynthesis as part of our independent work, representing about 25% of our effort and in other fields including DNA replication/repair, antibody/antigen interactions and viral infection in support of other labs within the DIR. Listed below are a few of our main projects form last year: 1. Work on glycosylaminoglycan (GAG) biosynthesis (in collaboration with Dr. Jian Liu at UNC) was centered around GAG biosynthesis enzymes that are utilized for the production of potential therapeutics dealing with coagulation, inflammation, and viral infection. By understanding how these enzymes interact with their substrates, we hope to modify them to improve their effectiveness in generating novel therapeutics with specific lengths, sequences, and sulfation patterns. There are seven isoforms of the 3-O-sulfotransferase (3OST). The historical dogma is isoform 1 generates anticoagulant heparan sulfate, isoform 3 generates the entry receptor for herpes simplex virus while isoform 5 can generate both. In this past year we have published structures of 3OST-5 bound to two substrates with different oligosaccharide sequences to better understand its specificity. These structures help us understand the preference for the enzyme to sulfate a glucosamine sugar flanked by glucuronic acids vs iduronic acids. In addition, work with our collaborators identified a novel oligosaccharide sequence produced by 3OST-5 that has a tighter IC50 for antithrombin than the currently approved anticoagulant fondaparinux. This oligosaccharide is hypothesized to have a longer half-life as well. We hope to apply this same approach to understand the specificity of other uncharacterized isoforms. 2. Double-strand breaks (DSBs) in DNA can result from exposure to DNA damaging agents, ionizing radiation, or reactive oxygen species generated during a cells normal metabolic processes. DSBs are extremely toxic and must be repair quickly and efficiently, as persistence of these lesions in the genome can lead to cell death and disease. In nonreplicating cells, or in cells that have not yet gone through DNA replication, the favored pathway of DSB repair is Nonhomologous End-Joining (NHEJ). In collaboration with Dr. Thomas Kunkel (NIEHS) and Dr. Dale Ramsden (UNC-CH), Andrea Kaminski from our group has used a hybrid approach combining molecular biology and X-ray crystallography to probe the structural determinants of DNA substrate selectivity for the X Family polymerases (Pols) and during NHEJ. We obtained crystal structures of these polymerases engaged in synapsis (end-bridging) of a variety of DSB ends, as snapshots before and throughout the gap-filling catalytic cycle. These structures reveal that specific structural motifs (Loop 1 and the thumb loop) in the polymerases can play a role in stabilizing different end configurations. Deletions or chimeric substitutions of these loops generated surprising and fortuitous alterations in substrate selectivity for DSB ends, allowing Pol to synapse and repair breaks entirely lacking sequence complementaritysubstrates that were previously determined to be Pol-specific substrates. 3. Over the past decade, our lab has worked to support the research of Dr. Geoff Mueller in understanding how the structure of allergens relates to the immune response. In 2020/2021 Dr. Jungki Min in my group worked to obtain crystal structure of Ara h 2 in complex with human IgG antibodies that were cloned from patients undergoing oral immunotherapy to peanut in a collaboration with Dr. Sarita Patil (Harvard). This work identified specific interactions of the antibody with three major bins that Dr. Patil identified to be important for antibody recognition. Using the crystal structures, Dr. Min designed three double mutants targeting each epitope bin based on the epitope-paratope analysis. We confirmed that the binding of these mutants to patient-derived IgGs is selective, affecting only the targeted bins. Preliminary ELISA showed that these mutants interacted less with IgEs in a peanut allergy patient serum, suggesting immunodominant roles of these conformational epitopes. We are currently testing a hexa-mutant to determine if it is a more potent hypoallergen. In addition to the peanut allergy project, Dr. Min solved crystal structures of a dog lipocalin allergen Can f 1 and its closely related (and cross-reactive) cat allergen Fel d 7. Structure comparison with reported dog lipocalins revealed that Can f 1 and Fel d 7 ligand binding pockets, called calyxes, are more open and positively charged. Dr. Min is currently utilizing NMR and MD approaches to investigate the substrate preferences of these enzymes.