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 A 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 5'-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.

Figure 1.

Crystal structure of AzI. (A) Ribbon representation of the AzI homodimer. Helices are colored in red and β strands in yellow in monomer A (left) and in cyan and magenta, respectively, in monomer B (right). The N termini are colored blue, and the C termini are gray. (B) Monomer of AzI showing its two domains: TIM α/β-barrel domain (residues 45–280) (orange) and a modified Greek key β-sheet domain (residues 8–44 and 281–435, with its helices in green and two sheets—S1 and S2—in blue and cyan, respectively). The figure was created using PyMOL (DeLano Scientific).

Figure 2.

Sequence alignment of mAzI, mODC, hODC, and tODC (numbering refers to the sequence of AzI). AzI secondary-structure elements are labeled above the corresponding sequence; α-helices are indicated by spirals, and β strands by arrows. Residues conserved in all four proteins appear in red blocks. The figure was created using ESPript (Gouet et al. 1999).

Figure 3.

Comparison of AzI and mODC structures. (A) Superposition of the AzI crystallographic dimer (orange) and the mODC dimer (green) (PDB code 7ODC). (B) Superposition of the interface of mAzI and mODC showing the variable loops between monomers A and B and between monomers B and A (AzI residues 355–362 and 387–401). AzI loops are in cyan, and ODC loops are in magenta. (C) Comparison of the N and C termini in the structures of AzI and mODC. AzI N-terminal β1 is in red, and C-terminal β18 in cyan; mODC N-terminals β−1 and β1 are in green and C-terminal α11 in blue. β-1 designates the additional β strand present in the N terminus of ODC, whereas β18 designates the extra β strand at the C terminus of AzI.

Figure 4.

Comparison of homodimer interfaces of mODC (PDB code 7ODC) and AzI structures showing side chains that form contacts (up to 3.5 Å) (monomer A in cyan, and monomer B in purple). The figure was created using PyMOL (DeLano Scientific). (A) mODC residues that form the hydrophobic zipper. (B) mAzI showing the absence of the hydrophobic zipper.

Figure 5.

AzI exists as a monomer in solution. (A) SDS-PAGE of AzI and ODC following cross-linking. (B) Size-exclusion chromatogram of mAzI compared to mODC. Each protein (50 μg) was injected into a Superdex 200 HR 10/30 column (GE Healthcare) in a buffer containing 50 mM Tris pH 7.5, 0.1 M NaCl, and 1 mM DTT. The elution positions of monomers and dimers of ODC and AzI are indicated by arrows (molecular weight standards [Amersham Biosciences] migrated under the same conditions, as follows: aldolase [M _r 158,000] at 12.5 mL, albumin [M _r 67,000] at 13.7 mL, and ovalbumin [M _r 43,000] at 14.8 mL).

Figure 6.

Comparison of PLP-binding site in ODC to the corresponding residues in AzI. Residues are depicted by sticks; PLP is in yellow, ODC residues D88, R154, R277, and Y389 are in green, and corresponding AzI residues A88, H154, S274, and D387 are in magenta. The figure was created using PyMOL (DeLano Scientific).

Figure 7.

AzI does not bind PLP. (A) Equal amounts of in vitro-translated ODC and AzI were bound to PMP beds and eluted with PLP as described in Materials and Methods. The eluted material was fractionated by SDS–polyacrylamide gel electrophoresis, and the radioactive bands were visualized using a Fuji BAS 2500 phosphorimager. (B) Pyridoxal phosphate was released from recombinant ODC and AzI, and determined fluorometrically as described in Materials and Methods.