Ornithine decarboxylase and antizyme interaction interface shows critical residues that are important for ornithine decarboxylase inhibition of activity and binding Ornithine decarboxylase (ODC) is a key rate limiting enzymes of polyamine biosynthesis. Polyamines are cationic molecules that play an important role in cellular proliferation, protein synthesis and gene regulation and are found in organisms ranging from bacteria to humans. In various organisms ODC activity is post-translationally regulated by its inhibitory protein antizyme (AZ), which is synthesized as a result of a ribosomal frameshifting due to increasing polyamine concentrations. AZ is expressed as a monomer, which inhibits dimeric ODC enzymatic activity by interacting with a single ODC protomer and this complex is degraded by the 26S proteasome in an ubiquitin independent manner. While ODC is highly homologous in different species, AZ protein shows a wide diversity. The crystal structure of mammalian ODC and a partial NMR structure of rat antizyme are known. Although the structural information of ODC-AZ complex is unknown, a recent in silico experiment has shown residues that are involved in mammalian ODC-AZ binding. In our recent study we have purified and characterized the ODC:AZ inhibitory complex from Saccharomyces cerevisiae. In the current experiments we present a detail study of the yeast ODC:AZ heterodimer, which are important in understanding how yeast AZ regulates ODC and in identifying the critical residues involved in heterodimer formation. We have purified yeast ODC, AZ and ODC:AZ heterodimer to homogeneity and comparisons of the proteins were performed by hydrogen deuterium exchange to identify the interface residues that are significant in forming the heterodimer. Three peptides in the AZ and four peptides in ODC were identified from H/D experiments that are present in the interface of ODC:AZ complex. We then used systematic site-directed mutagenesis to verify the critical residues of AZ that are important for heterodimer interaction in both in vivo and in vitro assays. Using a pull down assay, an equimolar AZ protein was tested for its ability to bind ODC. We observed that different AZ mutants have varying affinities to ODC. When the same samples were tested for AZ inhibition assays, various AZ mutants showed a reduced inhibition of ODC as compared to the wild type AZ showing the importance of these residues. In contrast to wild type AZ, the mutant AZ showed reduced degradation of ODC in vivo. These same in vivo samples were tested for ODC activity and we found that AZ mutants had greatly diminished ability to inhibit ODC activity. Polyamine content of the AZ mutants were also analyzed;it showed increased polyamine content compared to the wild type, further supporting the reduced activity and degradation of ODC in AZ mutants. Our study demonstrates that yeast AZ contains evolutionary conserved interface residues that are important for hetero-dimerization and are essential for its binding and inhibitory activity both in vitro and in vivo. We are currently performing a cross-linking experiment with BS3 (a chemical cross-linker) followed by mass-spectrometric analyses (MSMS and LCMS) to identify AZ and ODC interacting residues in the interface of purified ODC:AZ heterodimer.
| Chattopadhyay, Manas K; Fernandez, Cristina; Sharma, Deepak et al. (2011) Yeast ornithine decarboxylase and antizyme form a 1:1 complex in vitro: purification and characterization of the inhibitory complex. Biochem Biophys Res Commun 406:177-82 |
