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. Listed below are highlights from this past year. 1) In collaboration with Dr. Geoffrey Mueller in Dr. Robert Londons group at NIEHS we published 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 that share 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 to the pocket. Mass spectrometry analysis revealed the lipids to be PG and PE which could function as adjuvants enhancing ones sensitization to Bla g 1. Obtaining the structure of this protein will hopefully aid in the design of hypo-allergens for therapeutic purposes. This work was published in J Allergy Clin Immunol 2013. 2) In collaboration with Dr Linda Birnbaum at NIEHS we determined the crystal structures of the flame retardant TBBPA and a metabolite of BDE-47, 3-OH-BDE-47, to the active site of the human estrogen sulfotransferase, SULT1E1. SULT1E1 is an important enzyme in the metabolism of estradiol as sulfation of estradiol inhibits binding of estradiol to the estrogen receptor as well as increases solubility for renal excretion. Brominated flame retardants are pervasive chemicals in consumer products and are currently being investigated for their role as potential endocrine disruptors. Previously, it has been shown that TBBPA and 3-OH-BDE-47 are potent inhibitors of SULT1E1 with IC50s near the Km for the endogenous substrate estradiol. Our crystal structures suggest that these compounds inhibit estrogen sulfotransferase by binding at the estradiol binding site. These results suggest that brominated flame retardants can mimic estradiol binding to SULT1E1 and thus their role in disrupting estrogen metabolism should be considered. This work was published in Environmental Health Perspectives 2013. 3) In collaboration with the Korach lab at NIEHS we tried to understand at a molecular level how diharylheptanoids bind to the estrogen receptor. Diarylheptanoids are phytoestrogens isolated from plants that are used as dietary supplements as a treatment for hormone replacement therapy in menopausal women. Dr Korachs group demonstrated that D3, a diarylheptanoid isolated from Curcuma comosa, is a weak estrogenic agonist to ERalpha. Using the crystal structure of ERalpha with TFMPV-E2 bound we were able to model D3 binding to ERalpha providing insight as to how this molecule can function as an ER agonist. This work was published in Environmental Health Perspectives 2013. 4) In collaboration with the Kunkel lab at NIEHS we have been studying the mechanism by which ribonucleotides incorporated into DNA during replication or repair may result in stalling of DNA polymerases during ribonucleotide bypass. The inability of cells to remove these errors results in replication stress and may be relevant in human diseases such as Aicardi-Goutieres syndrome due to mutations in the RNH201A/B/C genes encoding the three domains of RNase H2, the protein thought to be responsible for the bulk of ribonucleotide removal from genomic DNA. To carry out this work we obtained crystal structures of the bacteriophage polymerase RB69 in complex with DNA containing ribonucleotides at various positions during various stages of bypass. These structures reveal subtle changes in the protein and the template strand due to the presence of ribonucleotides that may be responsible for stalling of the polymerase. These structures also reveal how stalling increases as the number of consecutive template ribonucleotides increases due to greater distortion in the templating strand. The Kunkel lab also demonstrated that a similar behavior is observed for the replicative yeast polymerase epsilon with some stalling also observed for delta. This work has been accepted for publication into PNAS 2013. 5) 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. 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. The Liu lab at UNC-CH has been working on a chemoenzymatic synthesis approach using enzymes in the biosynthesis pathway that shows great promise in the production of specific heparan sulfates. 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. Over the last two years we have been working on the crystal structure of the 2-O-sulfotransferase in complex with a specific heptamer heparan sulfate substrate. 2-O-sulfotransferase is responsible for sulfation on either idoronic acids or glucoronic acids with preference for Idoronic acid. This crystal structure reveals the specific pattern of sulfation on the heparan required for 2-O-sulfotransferase binding as well as why 6-O-sulfation cannot occur prior to 2-O sulfation in the biosynthesis pathway. It also suggests the preference for idoronic acid sulfation is due to interactions with a specific arginine residue R189. This crystal structure may provide ways the protein can be manipulated to allow for sulfation of novel substrates using chemoenzymatic synthesis 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.

Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2013
Total Cost
$944,572
Indirect Cost
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State
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Moon, Andrea F; Pryor, John M; Ramsden, Dale A et al. (2014) Sustained active site rigidity during synthesis by human DNA polymerase ?. Nat Struct Mol Biol 21:253-60
Mueller, Geoffrey A; Ankney, John A; Glesner, Jill et al. (2014) Characterization of an anti-Bla g 1 scFv: epitope mapping and cross-reactivity. Mol Immunol 59:200-7
Bebenek, Katarzyna; Pedersen, Lars C; Kunkel, Thomas A (2014) Structure-function studies of DNA polymerase ?. Biochemistry 53:2781-92
Hewitt, Sylvia C; Li, Leping; Grimm, Sara A et al. (2014) Novel DNA motif binding activity observed in vivo with an estrogen receptor ? mutant mouse. Mol Endocrinol 28:899-911
Mueller, Geoffrey A; Maleki, Soheila J; Pedersen, Lars C (2014) The molecular basis of peanut allergy. Curr Allergy Asthma Rep 14:429
Zheng, Xunhai; Pedersen, Lars C; Gabel, Scott A et al. (2014) Selective unfolding of one Ribonuclease H domain of HIV reverse transcriptase is linked to homodimer formation. Nucleic Acids Res 42:5361-77
Moon, Andrea F; Gaudu, Philippe; Pedersen, Lars C (2014) Structural characterization of the virulence factor nuclease A from Streptococcus agalactiae. Acta Crystallogr D Biol Crystallogr 70:2937-49
Liu, Chunhui; Sheng, Juzheng; Krahn, Juno M et al. (2014) Molecular mechanism of substrate specificity for heparan sulfate 2-O-sulfotransferase. J Biol Chem 289:13407-18
Pedersen, Lars C; Birnbaum, Linda S; Gosavi, Rajendrakumar A et al. (2014) Crystallographic analysis and mimicking of estradiol binding: Pedersen et al. Respond. Environ Health Perspect 122:A91-2
Batra, Vinod K; Perera, Lalith; Lin, Ping et al. (2013) Amino acid substitution in the active site of DNA polymerase * explains the energy barrier of the nucleotidyl transfer reaction. J Am Chem Soc 135:8078-88

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