Crystallographic studies of macromolecular structures

National Institute Of Environmental Health Sciences

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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 and antibody/antigen interactions 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) has been centered around GAG biosynthesis enzymes that are utilized for the production of potential therapeutics dealing with coagulation, inflammation, and viral infection. In this past year our work has been focused on obtaining the structure of the heparosan synthase enzyme from Pasteurella multocida pmHS2. This is a dual-functional enzyme utilized in the chemoenzymatic synthesis process to produce unmodified heparan sulfate (HS) oligosaccharides. This enzyme grows the HS chain by putting on repeating units of N-acetyl-glucosamines and glucuronic acids via alpha1-4-N-acetylglucosaminyltransferease and beta1-4 glucuronyltransferase activities, respectively. In the past year we have solved both CryoEM (in collaboration with Dr. Borgnia) and X-rays crystallographic structures of this enzyme in the presence of substrate acceptors and donor products. By understanding how these enzymes interact with their substrates at a molecular level, we hope to modify them to improve their effectiveness in generating novel therapeutics with specific lengths, sequences, and sulfation patterns. We also hope to be able to utilize the structural information to improve the enzymes stability and activity to enhance the synthesis yield in an industrial setting. In addition to understand how HSs are synthesized we are focused on understanding the specificities of HS targets to help tailor HS interactions with these targets to reduce off target binding. Two of the targets we have been studying in collaboration with Dr. Ding Xu at the University of Buffalo, are TRAIL and cathepsin K. Trail (TNF-related apoptosis-inducing ligand) is a potent inducer of tumor cell apoptosis through the TRAIL receptor. TRAIL can interact with HS to form higher order oligomers. Cell surface HS has been shown to function as a co-receptor for TRAIL and plays an important role in its apoptotic activity in both breast and myeloma cancers. This year, we have worked on the X-ray crystallographic structure of TRAIL to better understand its trimeric nature as well as how HS interacts with TRAIL with the hopes of using HS mimetics to specifically block TRAIL/receptor signaling. Cathepsin K (CtsK) is a cysteine protease with collagenase activity that is highly expressed by bone resorbing osteoclasts and plays an essential role in bone remodeling. As such it is an attractive target for therapeutics to reduce undesired bone resorption in diseases such as osteoporosis, periodontal bone loss, Pagets disease and rheumatoid arthritis. Unfortunately, due to the role of CtsK in other tissues, direct cysteine protease inhibitors have been plagued with severe side effects. Interestingly, heparin, a highly sulfated form of HS, has been shown to specifically inhibit the collagenase activity of Ctsk without inhibiting its peptidase activity for other substrates. We have been studying crystal structures of CtsK in the presence of HS and the inhibitor Cystatin C to learn how we might be able to design drugs to target HS binding without blocking the cysteine protease activity that is necessary for other physiological purposes. 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) during NHEJ. One structural determinate that we have focused on is the variable Loop 1. Deletion of this loop has been shown to strongly affect the fidelity of these polymerases. For Tdt and Pol mu, deletion of Loop1 exhibits reduced template-independent terminal transferase activity while Pol lambda becomes more permissive for misincorporation of both bases and ribonucleotides. Unexpectedly, we recently discovered that replacing Pol lambda Loop1 with Pol betas Loop1 resulted in a gain of function that allows the enzyme to engage in non-complimentary DSB, an activity that is specific for Pol mu. We have used crystallography to obtain a crystal structure of Pol lambda Loop1 deletion mutant engaged in synapsis with a non-complementary DSB substrate containing an unpaired primer terminus. We are currently examining this structure to better understand the role of this loop in noncomplementary DNA double strand break repair. 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. We have worked with Dr. Mueller to obtain numerous structures of food and other allergens alone and in-complex with patient derived antibodies. This includes allergens from walnut and multiple allergens from peanuts. We continue to examine the epitopes that we discover for ways to generate hypo-allergens that can be used in oral immunotherapy to build resistance without initiating anaphylaxis.