Structural Studies of Proteins involved in Nucleotide Metabolism : Studies of a ribonucleotide reductase from A. aeolicus and NUDT15

Sammanfattning: This thesis is separated into two parts. The first part concerns ribonucleotide reductase from Aquifex aeolicus. A. aeolicus is a hyperthermophilic bacterium that thrives at extremely high temperatures of 80-95 °C. We present the X-ray crystal structures of both the R1 and R2 subunits of this protein, which represents the first structure of a class Ia ribonucleotide reductase from a hyperthermophile and the first structure of an R1 from the NrdAh phylogenetic subclass. Several novel features were seen in the R1 structure such as the simultaneous binding of two ATP molecules in the ATP-cone domain as well as a novel “β-hairpin hook” feature which spans the dimer interface of the R1 protein. The gene encoding the R2 protein contains a self-cleaving intein domain. We examined two constructs of this protein, one with the sequence of the intein removed at the DNA level and the wild-type construct. Both crystal structures were found to be identical, showing the efficient cleavage of the intein domain in the wild-type construct. The second part of this thesis concerns the NUDIX hydrolase NUDT15. The physiological function of NUDT15 is still unknown, however certain mutations in this gene are associated with thiopurine intolerance in patients. Thiopurines are chemotherapeutic drugs used in the treatment of diseases such as acute lymphoblastic leukemia, the most common type of childhood leukemia, and inflammatory bowel disease. Thiopurine drugs are converted by the cell to the active metabolite 6-thio-dGTP which can then act as a substrate for DNA polymerase. Incorporation of these anti-metabolites into DNA produces the desired cytotoxic effects. We show that NUDT15 breaks down the active metabolites of these drugs which leads to a lowered effective dose. The absence of a functioning NUDT15 protein in patients that have inactivating mutations in the gene coding for NUDT15 results in a drastically increased effective dose of these compounds. A normal dose of a thiopurine drug can lead to severe and possibly life-threatening complications in these patients. The role of NUDT15 in thiopurine metabolism is established by in vitro and cellular data as well as the X-ray crystal structure of NUDT15 in complex with 6-thio-GMP. Acyclovir and ganciclovir are two antiviral drugs whose mechanism of action is similar to that of thiopurines. These drugs are also metabolized to their tri-phosphorylated forms and are then preferentially incorporated into viral DNA. Here again, we use in vitro, cellular and structural data to show that NUDT15 breaks down the active metabolites of these drugs. Two separate and structurally distinct lines of potent inhibitors for NUDT15 were developed with support of crystallographic studies. We show that cells are sensitized to both thiopurine and antiviral treatments in the presence of these inhibitors. Binding of our inhibitors to NUDT15 provided substantial thermal stabilization. The stabilizing effect of inhibitor binding enabled us to solve structures of the four most clinically relevant NUDT15 variants, thus elucidating the structural basis for the thiopurine sensitivity phenotype.