Crystallographic and biochemical studies revealing the structural basis for antizyme inhibitor function

Abstract

Antizyme inhibitor (AzI) regulates cellular polyamine homeostasis by binding to the polyamine-induced protein, Antizyme (Az), with greater affinity than ornithine decarboxylase (ODC). AzI is highly homologous to ODC but is not enzymatically active. In order to understand these specific characteristics of AzI and its differences from ODC, we determined the 3D structure of mouse AzI to 2.05 Å resolution. Both AzI and ODC crystallize as a dimer. However, fewer interactions at the dimer interface, a smaller buried surface area, and lack of symmetry of the interactions between residues from the two monomers in the AzI structure suggest that this dimeric structure is nonphysiological. In addition, the absence of residues and interactions required for pyridoxal 59-phosphate (PLP) binding suggests that AzI does not bind PLP. Biochemical studies confirmed the lack of PLP binding and revealed that AzI exists as a monomer in solution while ODC is dimeric. Our findings that AzI exists as a monomer and is unable to bind PLP provide two independent explanations for its lack of enzymatic activity and suggest the basis for its enhanced affinity toward Az.Keywords: structure/function studies; protein crystallization; protein structures-new; antizyme inhibitor; antizyme; ornithine decarboxylase Polyamines are small organic polycations that are essential for cell proliferation and play an important role in regulating other fundamental cellular processes. Elevated polyamine levels are observed in rapidly growing cells including transformed cells; thus, polyamine metabolism has been suggested as a potential target for cancer therapy (Pegg 1988;Marton and Pegg 1995;Wallace and Fraser 2004). The range of intracellular polyamines is determined at the lower limit by their absolute requirement for cellular proliferation and at the upper limit by their cytotoxicity (Poulin et al. 1993;Tobias and Kahana 1995), indicating a need for strict regulation of their intracellular concentration. Multiple pathways such as synthesis, uptake, degradation, and efflux regulate cellular polyamine levels. Ornithine decarboxylase (ODC) is the first and rate-limiting enzyme in the polyamine biosynthesis pathway (Pegg 2006). It is a pyridoxal 59-phosphate (PLP)-dependent enzyme that provides the only route for converting ornithine to putrescine. ODC, which is characterized by a short intracellular half-life, is part of an autoregulatory circuit mediated by a polyamine-induced protein, termed Antizyme (Az). An increased intracellular polyamine concentration increases the synthesis of Az by stimulating ribosomal frameshifting (Rom and Kahana 1994;Matsufuji et al. 1995). Az, in turn, binds to transient ODC monomer subunits with high affinity, preventing their re-association into active homodimers and targeting them for rapid degradation by the 26S proteasome (Murakami et al. 1992). Az also regulates polyamine transport across the plasma membrane via an unknown mechanism Mitchell et al. 1994;Suzuki et al. 1994;Sakata et al. 2000). 3 These authors contri...

Structural basis of antizyme-mediated regulation of polyamine homeostasis

“…To understand how AzIN may interact more strongly with Az 1 , we have produced crystals of AzIN in complex with an Az 1 C-terminal fragment (Az 110-228 1 ; covering residues 110-228) that diffract to 5.8 Å resolution (Table S1). Initial phases were obtained by molecular replacement using a polyalanine search model derived from the mouse AzIN structure (26), which yielded an unbiased electron density suitable for the placement of Az 110-228 1 (Fig. 4A).…”

Non-synonymous single-nucleotide polymorphisms associated with blood pressure and hypertension

“…The structure of human ODC protein has been elucidated to 2.1 angstrom resolution. 23 ODC is predicted to form a homodimer (PDB_ID: 3btn) (Figure 3b). Ala287 of ODC, the amino-acid equivalent of A288 in ADC, is likely to be buried in the homodimer, on the basis of the low percentage of wateraccessible surface on the residue (1% for the water-accessible surface of A287 in ODC, according to PDB prediction; data not shown).…”

Polyamine Antagonist Therapies Inhibit Neuroblastoma Initiation and Progression

“…Both Odc1 and Amd1 have the shortest half-lives of any mammalian enzymes (10-30 minutes). Odc activity is further regulated posttranslationally by Odc antizymes that mediate its degradation (9), which are themselves regulated by antizyme inhibitors (10). Catabolism is regulated through polyamine acetylation by the spermidine/spermine-N-acetyltransferase, SAT1, which controls flux through the pathway (11), whereas spermine can also be oxidized via SMOX activity to spermidine.…”