The main focus of the Structure Function Group is to use X-ray crystallography to support research interest of principal investigators within the intramural community. One of our collaborations is with Dr. Geoffrey Mueller in Dr. Robert Londons group studying crystal structures of allergens. This past year we have been working on the crystal structure of the cockroach allergen Bla g 1. Sensitization to cockroach allergens is a major risk factor for asthma. The prevalence of sensitization to Bla g 1 is 20-40% of cockroach allergic patients. Sensitization occurs by inhalation of fecal particles released into the environment. Bla g 1 consists of at least two tandem repeats were the repeats share at least 25% sequence identity. To solve the structure of Bla g 1 we expressed in bacteria one of these tandem repeats attached to the C-terminus of the green fluorescent protein (GFP) and crystallized it as a fusion protein. This domain of Bla g 1 represents a novel fold with each repeat consisting of 6 helices that encapsulate a large hydrophobic pocket. Based on the crystallography we identified lipids binding in the pocket. Mass spectrometry analysis revealed the lipids to be PG and PE. Lipids binding to Bla g 1 could function as adjuvants enhancing ones sensitization to the protein. Currently we are working on trying to understand what the natural ligand purified from cockroach is to better understand the function of this protein. Obtaining the structure of this protein will hopefully aid in the design of hypo-allergens for therapeutic purposes. In collaboration with the Kunkel lab we have been trying to understand the mechanism at the atomic level for how ribonucleotides can be inserted into DNA. Incorporations of ribonucleotides may result in genomic instability as ribonucleotides can promote hydrolytic cleavage of the DNA backbone resulting in strand-breaks. For the most part, DNA polymerases discriminate against ribonucleotide versus deoxribonucleotides by selecting against the additional 2 hydroxyl group on the sugar. However the evidence is clear that ribonucleotides are inserted into the DNA and this may be particularly relevant during DNA repair in non-proliferating cells when ribonucleotides to deoxyribonucleotides ratios are high. To better understand how ribonucleotides can be incorporated into the DNA we solved four different crystal structures of a repair DNA polymerase (pol lambda) at various stages of the catalytic cycles for ribonucleotide incorporation and extension off of a ribonucleotide primer. These structures revealed that ribonucleotides can be accommodated in the active site. However, they bind at a higher energy due to the 2OH. Once incorporated the ribonucleotide is expelled from the active site. Yet, it is easily transferred to the primer terminus position where it can accept a deoxyribonucleoitde during chain elongation. This is consistent with the biochemical evidence obtained in Dr. Samuel Wilsons group for DNA polymerase beta. Independently, our lab is focused on the development of specific sulfotransferases to be used in enzymatic production of therapeutic heparan sulfate. Heparan sulfates (HS) are linear sulfated polysaccharides present on the cell surface and in the extra cellular matrix that play important roles in blood coagulation, inflammation response, cell differentiation and assist in bacterial and viral infection. The specific sulfation pattern of HS determines its functional selectivity. Different sulfotransferases are required for sulfation of specific hydroxyl groups or amines along the polysaccharide. Heparin is a highly sulfated form of HS. Therapeutic heparin is a 3 billion dollar a year industry as an anticoagulant. In addition, low molecular weight heparin/HS mimics show promise as potential anti-cancer/anti-metastasis drugs, possibly due to their role in growth factor regulation and as heparanase inhibitors. Currently, therapeutic heparin is purified from mast cells of mammalian sources. This can lead to contamination problems as well as-- and perhaps more importantly-- heterogeneity problems. One of the major side-effects of administration of heparin is thrombocytopenia due to interaction of the heparin with platelet factor 4 (PF4). Chemical synthesis of homogeneous polysaccharides larger than a hexasaccharide is extremely difficult and currently too challenging for mass production. We have been working with the Liu lab (UNC-CH) to obtain structures of proteins involved in heparan biosynthesis bound to heparan sulfate substrates to better understand the substrate specificity of these enzymes. Information gained from these studies thus far have been useful in the re-designing of these enzymes to create heparan sulfates with greater homogeneity as well as unique structures that may be useful in the design of better therapeutics for use as anti-coagulants, anti-inflammatory, as well as anti-cancer agents.
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