Crystallographic studies of macromolecular structures

National Institute Of Environmental Health Sciences

Investigators

Linked publications & trials

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. 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 3-OST-3 with different substrates as well as generated structures of 3-OST-5 with different substrates that help us understand the specificity of these enzymes. This work has helped explain why 6 sulfated heparan sulfates are not good substrates for isoform 3 as well as uncovered the preferred substrate for 3-OST-5 which contrary to the dogma, differs from that of isoforms 1 and 3. This work has helped to aid in the design of unique heparan sulfates by chemoenzymatic synthesis by suggesting which 3-OST to use depending on the starting material and the desired sequence. Heparin has also been suggested to possibly serve as a co-receptor for SARS-cov2 virus entry by interacting with the spike protein as well as reducing the inflammatory response and potential clotting side effects associated with SARS-cov2 infection. In this past year, we worked on trying to obtain structures of heparan sulfate oligosaccharides that showed promise in microarray experiments, binding to the spike and receptor binding domain of the spike protein utilizing X-ray crystallography and CryoEM. 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). Our work this year, in collaboration with Dr. Thomas Kunkels Replication Fidelity Group at NIEHS and Dr. Dale Ramsdens lab (UNC), has focused on understanding the contribution of the Family X Polymerase Lambda (Pol) in gap-filling of DSBs repaired by NHEJ. Andrea Kaminski in our group used X-ray crystallography to determine how Pol engages its DSB substrates, creating a synapse of two broken ends so that sequence gaps at the break site can be filled before being rejoined. We have obtained complexes of catalytically trapped Pol bound to a variety of DSB end structures, poised immediately prior to, during, and after catalysis. Comparative analysis of these crystal structures reveals that a specific loop region (Loop1), previously known to influence nucleotide incorporation fidelity, can adopt different conformations, which could help stabilize different DNA substrate configurations. In addition, chimeric variants of this loop have been found to exhibit unexpected changes in DSB substrate selectivity, allowing Pol to bind and repair broken DNA ends with no sequence complementarity at the break site. Other work involved in DNA repair was initiated including supporting work in the Doetsch and Wilson groups at NIEHS understanding functional aspects of DNA gyrase and APE1 endonuclease using X-ray crystallography. 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. Over the last year Dr. Jungki Min in my group has 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). Preliminary data from the Mueller group suggest that different patients converge on similar epitopes. Knowledge of where these epitopes are on the structure of the antigen may help to design better therapeutics. To this end, Dr. Min has obtained crystal structures of Fabs from two of the antibodies alone, as well as two crystal structures of Ara h 2 binding to two different Fabs, in each structure. These structures have provided a wealth of insight into the epitopes recognized by human IgGs and have suggested targets for the design of hypoallergens. Currently, mutations are being made on Ara h 2 to investigate their ability to reduce recognition by the antibodies. 4) Working in support of Dr. Mario Borgnias group at NIEHS, Dr. Min in our group has been trying to obtain crystal structures of nanobodies that bind to the RBD from the SARS-cov2 spike protein, both alone, and in complex with the RBD. To this end, we were able to obtain a 1.3 A crystal structures of a specific nanobody to the RBD. Currently this structure is being utilized in modeling attempts with the CryoEM map of the nanobody engaged with the spike protein to better understand its mode of recognition to the spike protein and how it blocks viral infection. This project involves research on human coronavirus, novel coronavirus, COVID-19, Severe Acute Respiratory Syndrome coronavirus disease, SARS coronavirus, SARS-coronavirus-2, SARS-cov-2, SARS-cov2, SARS-related coronavirus 2, Severe acute respiratory syndrome coronavirus 2, SARS-Associated Coronavirus, SARS-cov, or SARS-Related Coronavirus.