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 more recent collaborations is with Geoffrey Mueller in the London group studying crystal structures of allergens. Peanut allergies affect 1% of the population and are responsible for many of the fatal food-related anaphylactic reactions. This past year, we were able to solve the crystal structure of Ara h 2, the major peanut allergen, utilizing our MBP/SER (maltose binding protein/ Surface Entropy Reduction) system. The crystal structure revealed the protein to be a five-helix bundle most similar in structure to other amylase and trypsin inhibitors. These studies revealed that the IgE antibodies from different subpopulations of patients recognize different surfaces of the Ara h 2 protein. This information may be helpful for the design of hypo-allergens for the treatment of severely allergic patients. We have also collaborated with the London group studying EndA, a sequence non-specific endonuclease, which functions as a virulence factor during Streptococcus pneumoniae infection. S. pneumoniae infection in the lungs triggers neutrophil recruitment followed by extrusion of neutrophil extracellular traps that physically entrap the bacteria limiting the infection. EndA is believed to degrade the NETs, freeing the bacteria and thereby increasing the virulence. We have solved the crystal structure of EndA, with the aid of our MBP/SER system. The crystal structure reveals the protein to be a distant homolog of NucA, EndoG, and Serratia nuclease. Using site-directed mutagenesis we have delineated potential residues involved in catalysis and substrate binding. We hope this information will be useful for the development of antibiotics specific for EndA that will be useful for the treatment of this S. pneumoniae lung infection. In addition, we have extensive collaborations with the Wilson and Kunkel laboratories studying crystal structures of polymerases involved in DNA replication and repair processes. We continue to collaboratively aid in determining structures of human X-family polymerases beta and lambda. Recently, the focus has been on understanding how mutations are created by these enzymes, through mispairing and by ribonucleotide incorporation in base excision repair and non-homologous end-joining processes. 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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