Critical Factors in Human Antizymes that Determine the Differential Binding, Inhibition, and Degradation of Human Ornithine Decarboxylase

Abstract

Antizyme (AZ) is a protein that negatively regulates ornithine decarboxylase (ODC). AZ achieves this inhibition by binding to ODC to produce AZ-ODC heterodimers, abolishing enzyme activity and targeting ODC for degradation by the 26S proteasome. In this study, we focused on the biomolecular interactions between the C-terminal domain of AZ (AZ_95-228) and ODC to identify the functional elements of AZ that are essential for binding, inhibiting and degrading ODC, and we also identified the crucial factors governing the differential binding and inhibition ability of AZ isoforms toward ODC. Based on the ODC inhibition and AZ-ODC binding studies, we demonstrated that amino acid residues reside within the α1 helix, β5 and β6 strands, and connecting loop between β6 and α2 (residues 142-178), which is the posterior part of AZ_95-228, play crucial roles in ODC binding and inhibition. We also identified the essential elements determining the ODC-degradative activity of AZ; amino acid residues within the anterior part of AZ_95-228 (residues 120-145) play crucial roles in AZ-mediated ODC degradation. Finally, we identified the crucial factors that govern the differential binding and inhibition of AZ isoforms toward ODC. Mutagenesis studies of AZ1 and AZ3 and their binding and inhibition revealed that the divergence of amino acid residues 124, 150, 166, 171, and 179 results in the differential abilities of AZ1 and AZ3 in the binding and inhibition of ODC.

Keywords: AZ isoform; binding affinity; protein–protein interaction; ubiquitin-independent degradation.

Figure 1

Crystal structure of the human AZ_95–228-ODC heterodimer. (A) Complex structure of the AZ_95–228-ODC heterodimer (PDB: 4ZGY); this figure was generated using PyMOL [47]. (B) Secondary structure of the human AZ_95–228 peptide consists of two α-helices and eight β-strands and their connecting loops. (C) Amino acid residues for the mutagenesis study. Amino acid residues colored in black were AZ1 study for ODC binding and inhibition, colored in green were AZ1 study for ODC degradation, and colored in red were AZ1 study for ODC inhibition and degradation; while amino acid residues colored in blue were AZ3 study for ODC binding and inhibition. (D) Multiple sequence alignments of AZ isoforms. The multiple sequence alignment was generated by ClustalW2 [48].

Figure 2

Inhibition plots of the ODC enzyme with multiple mutants of AZ_95–228. The enzyme activity of ODC was inhibited by various multiple mutants of AZ_95–228. The IC₅₀ values of multiple mutants of AZ_95–228 presented in Table 1 were derived by curve-fitting the inhibition plots. The molar ratio refers to AZ versus the ODC monomer. (A) AZ_95–228_K153A/E164A/E165A (AZ_95–228-3Mα1), (B) AZ_95–228-3Mα1_D154A, (C) AZ_95–228-3Mα1_E161A, (D) AZ_95–228_K153A/D154A/E161A/E164A/E165A (AZ_95–228-5Mα1), (E) AZ_95–228-3Mα1_E142A, (F) AZ_95–228-3Mα1_H171A, (G) AZ_95–228-3Mα1_K178A, (H) AZ_95–228_E142A/H171A/K178A, and (I) AZ_95–228_E142A/K153A/D154A/E161A/E164A/E165A/H171A/K178A (AZ_95–228-8M).

Figure 3

Size distribution plots of multiple mutants of AZ_95–228-ODC heterodimers. (A) AZ_95–228-ODC, (B) AZ_95–228-3Mα1-ODC, (C) AZ_95–228-3Mα1_D154A-ODC, (D) AZ_95–228-3Mα1_E161A-ODC, (E) AZ_95–228-5Mα1-ODC, (F) AZ_95–228-3Mα1_E142A-ODC, (G) AZ_95–228-3Mα1_H171A-ODC, (H) AZ_95–228-3Mα1_K178A-ODC, (I) AZ_95–228_E142A/H171A/K178A-ODC, and (J) AZ_95–228-8M-ODC. The sedimentation velocity data in each figure were globally fitted with the SEDPHAT program to acquire K_d values for the AZ_95–228-ODC heterodimers shown in Table 2.

Figure 4

AZ-mediated ODC in vitro degradation with AZ mutant peptides in rabbit reticulocyte lysates. ODC can be effectively degraded by AZ binding, and protein degradation was detected by anti-ODC antibody (n = 3). (A) ODC degradation with AZ_95–228, AZ_95–228_N110A, and AZ_95–228_N117A, (B) ODC degradation with AZ_95–228, AZ_95–228_S120A and AZ_95–228_N129A, (C) ODC degradation with AZ_95–228, AZ_95–228_G136A, and AZ_95–228_G137A, (D) ODC degradation with AZ_95–228, AZ_95–228_R131A, and AZ_95–228_G145A. A residual amount of ODC protein at a different time was indicated under the ODC blotting gel in each figure.

Figure 5

Inhibition plots of the ODC enzyme with single or multiple mutants of AZ1 and AZ3. The enzyme activity of ODC was inhibited by various single or multiple mutants of AZ1 or AZ3. The IC₅₀ values of AZ1 or AZ3 mutants presented in Table 3 and Table 4 were derived by curve-fitting the inhibition plots. The molar ratio refers to AZ1 or AZ3 versus the ODC monomer. (A) AZ3_S124D, (B) AZ3_Q150E, (C) AZ3_K166Q, (D) AZ3_S171H, (E) AZ3_D179N, (F) AZ3_S124D/Q150E/K166Q/D179N (AZ3_4M), (G) AZ3_S124D/Q150E/K166Q/S171H/D179N (AZ3_5M), (H) AZ1_D124S/E150Q/Q166K/N179D (AZ1_4M), and (I) AZ1_D124S/E150Q/Q166K/H171S/N179D (AZ1_5M).

Figure 6

Size distribution plots of multiple mutants of AZ1-ODC or AZ3-ODC heterodimers. (A) AZ3_WT-ODC, (B) AZ3_4M-ODC, (C) AZ3_5M-ODC, (D) AZ1_WT-ODC, (E) AZ1_4M-ODC, and (F) AZ1_5M-ODC. The sedimentation velocity data in each figure were globally fitted with the SEDPHAT program to acquire K_d values for the AZ-ODC heterodimers shown in Table 4.

Figure 7

Structural elements of AZ responsible for binding, inhibition and degradation toward ODC. Black labels, amino acid residues of AZ1 critical for ODC binding and inhibition; green labels, amino acid residues of AZ1 essential for ODC degradation; red labels, amino acid residues of AZ1 and AZ3 governing the differential binding and inhibition toward ODC.