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 the Kunkel lab we have been working on understanding the subtle differences of function between X family polymerases. In this review cycle we published in Nature Structure and Molecular Biology on work that we carried out on DNA polymerase Mu (pol mu). Family X DNA polymerase mu is the only template-dependent human DNA polymerase known to participate in non-homologous end joining (NHEJ) of double strand DNA breaks whose 3ʹ-ends are not base paired. As such, pol mu is involved in preserving gene sequence during immunoglobulin V(D)J recombination and plays a role in repair of DJH rearrangements during embryonic development and DNA breaks due to ionizing radiation. To probe this unique function, we characterized X-ray crystal structures corresponding to four steps in the Pol mu catalytic cycle for single nucleotide incorporation. These structures indicate that in contrast to most DNA polymerases, including Family X siblings Pol beta and Pol lambda, there are no large-scale conformational changes in protein subdomains, amino acid side chains, or DNA seen upon dNTP binding or catalysis by Pol mu. Instead, the only major conformational change is seen earlier in the catalytic cycle, when Pol mu binds to the primer-template. DNA binding requires repositioning of a flexible loop (Loop1) that occludes the DNA binding cleft in the apoenzyme. Pol mu variants with changes in Loop1 have altered catalytic properties and are partially defective in NHEJ. Our results reveal that Loop1 plays a critical role in conferring upon pol mu the unique ability to catalyze template-dependent NHEJ of double strand DNA breaks whose 3-ends are not base paired, and more generally, they suggest that the more unstable the DNA substrate the fewer and less extensive the conformational changes of the polymerase. 2) This year our lab has also been working on solving the structure of the virulence factor Nuclease A (NucA) from Streptococcus agalactiae. The Group B pathogen Streptococcus agalactiae commonly populates the human gut and urogenital tract, and is a major cause of infection-based mortality in neonatal infants and in elderly or immunocompromised adults. Severe infections can lead to sepsis, meningitis, and death. Secreted by the bacteria, NucA facilitates evasion of the human innate immune response by degraded the DNA matrix within the neutrophil extracellular traps (NETS) which function to inhibit bacterial infection. We recently solved the structure of this DNA/RNA sequence non-specific nuclease revealing structural similarities to other DRGH nucleases. Using site directed mutagenesis and DNA modeling we outlined the DNA binding pocket and revealed residues important for DNA binding and catalytic activity. It is the hope that by studying these enzymes we can design specific inhibitors that will function as antibiotics, decreasing the impact of infection on the host while at the same time minimizing the impact on the hosts natural bacterial flora and fauna. 3) 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. This year we published in the Journal of Biological Chemistry on the crystal structure of the 2-O-sulfotransferase in complex with a specific heptamer HS substrate. 2-O-sulfotransferase is responsible for sulfation on either idoronic acids or glucoronic acids with preference for iudoronic 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. In collaboration with Lalith Perera high level computer simulations were carried out to help interpret the ring conformation of the acceptor iduronic acid. The results from this modeling study supported the crystallography which suggested the acceptor iduronic acid has an unusual 4C1 ring conformation within the HS chain. 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.
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