Critical factors determining dimerization of human antizyme inhibitor

Abstract

Ornithine decarboxylase (ODC) is the first enzyme involved in polyamine biosynthesis, and it catalyzes the decarboxylation of ornithine to putrescine. ODC is a dimeric enzyme, whereas antizyme inhibitor (AZI), a positive regulator of ODC that is homologous to ODC, exists predominantly as a monomer and lacks decarboxylase activity. The goal of this paper was to identify the essential amino acid residues that determine the dimerization of AZI. The nonconserved amino acid residues in the putative dimer interface of AZI (Ser-277, Ser-331, Glu-332, and Asp-389) were substituted with the corresponding residues in the putative dimer interface of ODC (Arg-277, Tyr-331, Asp-332, and Tyr-389, respectively). Analytical ultracentrifugation analysis was used to determine the size distribution of these AZI mutants. The size-distribution analysis data suggest that residue 331 may play a major role in the dimerization of AZI. Mutating Ser-331 to Tyr in AZI (AZI-S331Y) caused a shift from a monomer configuration to a dimer. Furthermore, in comparison with the single mutant AZI-S331Y, the AZI-S331Y/D389Y double mutant displayed a further reduction in the monomer-dimer K(d), suggesting that residue 389 is also crucial for AZI dimerization. Analysis of the triple mutant AZI-S331Y/D389Y/S277R showed that it formed a stable dimer (K(d) value = 1.3 microm). Finally, a quadruple mutant, S331Y/D389Y/S277R/E332D, behaved as a dimer with a K(d) value of approximately 0.1 microm, which is very close to that of the human ODC enzyme. The quadruple mutant, although forming a dimer, could still be disrupted by antizyme (AZ), further forming a heterodimer, and it could rescue the AZ-inhibited ODC activity, suggesting that the AZ-binding ability of the AZI dimer was retained.

FIGURE 1.

Crystal structure and the amino acid residues at the dimer interface of human ornithine decarboxylase (hODC) and mouse antizyme inhibitor (mAZI). A, homodimeric structure of human ODC with the cofactor PLP analog, LLP (Protein Data Bank code 1D7K). B, putative dimeric structure of mouse AZI (Protein Data Bank code 3BTN). The amino acid residues in the dimer interface are shown as a ball-and-stick model. The putative AZ-binding site is colored in cyan. This figure was generated using PyMOL (DeLano Scientific LLC, San Carlos, CA).

FIGURE 2.

Continuous sedimentation coefficient distribution of the human ODC-WT, AZI-WT, and AZI single mutants. The proteins used are at three protein concentrations, 0.3, 0.6, and 0.9 mg/ml in 50 mm Tris-HCl buffer, pH 7.4, at 20 °C. A, ODC-WT; B, AZI-WT; C, AZI-S331Y; D, AZI-D389Y; E, AZI-S277R; F, AZI-E332D. M, monomer; D, dimer.

FIGURE 3.

Continuous sedimentation coefficient distribution for the six double mutants of human AZI. The proteins used are at three protein concentrations, 0.3, 0.6, and 0.9 mg/ml in 50 mm Tris-HCl buffer, pH 7.4, at 20 °C. A, AZI-S331Y/S277R; B, AZI-S331Y/E332D; C, AZI-S331Y/D389Y; D, AZI-D389Y/S277R; E, AZI-S277R/E332D; F, AZI-D389Y/E332D. M, monomer; D, dimer.

FIGURE 4.

Continuous sedimentation coefficient distribution of the human AZI with triple and quadruple mutations. The proteins used are at three protein concentrations, 0.3, 0.6, and 0.9 mg/ml in 50 mm Tris-HCl buffer, pH 7.4, at 20 °C. A, AZI-S331Y/D389Y/S277R; B, AZI-S331Y/D389Y/E332D; C, AZI-S331Y/S277R/E332D; D, AZI-S331Y/D389Y/S277R/E332D. M, monomer; D, dimer.

FIGURE 5.

Continuous sedimentation coefficient distribution for the human ODC-WT, AZI-WT, and quadruple mutant AZI-S331Y/D389Y/S277R/E332D in the presence of AZ. ODC and the AZI quadruple mutant were used at protein concentrations of 0.3 mg/ml, and AZ was used at 0.15 mg/ml in 50 mm Tris-HCl buffer, pH 7.4, at 20 °C. A, ODC-WT; B, AZI-WT; C, AZI-S331Y/D389Y/S277R/E332D. Solid line, ODC or AZI only; dashed line, ODC or AZI in the presence of AZ.

FIGURE 6.

Rescue of the AZ-inhibited ODC enzyme activity with AZI. A, relative enzyme activities of ODC inhibited by AZ. B, AZ-inhibited ODC enzyme activity is rescued by AZI-WT (open circles) and the quadruple mutant AZI-S331Y/D389Y/S277R/E332D (closed circles).

FIGURE 7.

Enzymatic and size analysis of the ODC-AZI mixture. A, relative enzyme activities of ODC in the presence of AZI-WT (open circles) and the quadruple mutant AZI-S331Y/D389Y/S277R/E332D (closed circles); B, continuous size distribution analysis of the ODC-AZI mixture.

FIGURE 8.

LIGPLOT of the amino acid residues at the dimer interface for human ODC. Ligand interactions at the dimer interface residues Tyr-331, Tyr-389, Arg-277, and Asp-332 are shown as the LIGPLOT diagram (68). The boldface bonds indicate the specific amino acid residue; the thin bonds are the hydrogen-bonded residues, and the green dashed lines correspond to the hydrogen bonds. Spoked arcs represent hydrophobic contacts.