Critical factors governing the difference in antizyme-binding affinities between human ornithine decarboxylase and antizyme inhibitor

Abstract

Both ornithine decarboxylase (ODC) and its regulatory protein, antizyme inhibitor (AZI), can bind with antizyme (AZ), but the latter has a higher AZ-binding affinity. The results of this study clearly identify the critical amino acid residues governing the difference in AZ-binding affinities between human ODC and AZI. Inhibition experiments using a series of ODC mutants suggested that residues 125 and 140 may be the key residues responsible for the differential AZ-binding affinities. The ODC_N125K/M140K double mutant demonstrated a significant inhibition by AZ, and the IC(50) value of this mutant was 0.08 µM, three-fold smaller than that of ODC_WT. Furthermore, the activity of the AZ-inhibited ODC_N125K/M140K enzyme was hardly rescued by AZI. The dissociation constant (K(d)) of the [ODC_N125K/M140K]-AZ heterodimer was approximately 0.02 µM, which is smaller than that of WT_ODC by approximately 10-fold and is very close to the K(d) value of AZI_WT, suggesting that ODC_N125K/M140K has an AZ-binding affinity higher than that of ODC_WT and similar to that of AZI. The efficiency of the AZI_K125N/K140M double mutant in the rescue of AZ-inhibited ODC enzyme activity was less than that of AZI_WT. The K(d) value of [AZI_K125N/K140M]-AZ was 0.18 µM, nine-fold larger than that of AZI_WT and close to the K(d) value of ODC_WT, suggesting that AZI_K125N/K140M has an AZ-binding affinity lower than that of AZI_WT and similar to that of ODC. These data support the hypothesis that the differences in residues 125 and 140 in ODC and AZI are responsible for the differential AZ-binding affinities.

Figure 1. Sequence alignment and structures of ODC and AZI.

(A) Pairwise sequence alignment between ODC and AZI in the putative AZ-binding element. (B) Structure of human ODC monomer (PDB code: 1D7K). (C) Structure of mouse AZI monomer (PDB code: 3BTN). The putative AZ-binding site in ODC is colored in deep green and that in AZI is colored in hot pink. This figure was generated with PyMOL (DeLano Scientific LLC, San Carlos, CA, USA).

Figure 2. Inhibition of wild-type and mutant ODC enzyme in the presence of AZ.

Open circles: ODC_WT; closed circles: mutant ODC enzyme. (A) ODC_N125K. (B) ODC_N126V. (C) ODC_F133C. (D) ODC_S135N. (E) ODC_M140K. (F) ODC_N125K/M140K. In the inhibition assay, the enzyme concentration was fixed at 20 µg/mL.

Figure 3. Rescue of AZ-mediated inhibition of the activity of wild-type and mutant ODC by AZI.

ODC (20 µg/mL) was preincubated with AZ (34 µg/mL) and was then treated with various concentrations of AZI. Open circles: ODC_WT; closed circles: mutant ODC enzyme. (A) ODC_N125K. (B) ODC_M140K. (C) ODC_N125K/M140K. The molar ratio of AZ monomer versus ODC monomer was fixed at 4.

Figure 4. Rescue of AZ-mediated inhibition of the activity of ODC by wild-type and mutant AZI.

ODC (20 µg/mL) was preincubated with AZ (30 µg/mL) and then treated with various concentrations of AZI. Open circles: AZI_WT; closed circles: mutant AZI. (A) AZI_K125N. (B) AZI_ K140M. (C) AZI_K125N/K140M. The molar ratio of AZ monomer versus ODC monomer was fixed at 3.5.

Figure 5. Continuous sedimentation coefficient distribution of human ODC and AZI in the presence of AZ.

The concentration of ODC or AZI was fixed at 0.3 mg/mL with four concentrations of AZ at 0.015, 0.03, 0.06, or 0.09 mg/mL (the molar ratios of AZ/ODC were 0.12, 0.24, 0.47 and 0.73). The sedimentation velocity data were globally fitted with SEDPHAT to acquire the K _d values of the ODC-AZ and AZI-AZ complexes (Table 2). (A) ODC_WT. (B) ODC_N125K. (C) ODC_M140K. (D) ODC_N125K/M140K. (E) AZI_WT. (F) AZI_K125N. (G) AZI_ K140M. (H) AZI_K125N/K140M.