Crystal structure of human type II inosine monophosphate dehydrogenase: implications for ligand binding and drug design

Abstract

Inosine monophosphate dehydrogenase (IMPDH) controls a key metabolic step in the regulation of cell growth and differentiation. This step is the NAD-dependent oxidation of inosine 5' monophosphate (IMP) to xanthosine 5' monophosphate, the rate-limiting step in the synthesis of the guanine nucleotides. Two isoforms of IMPDH have been identified, one of which (type II) is significantly up- regulated in neoplastic and differentiating cells. As such, it has been identified as a major target in antitumor and immunosuppressive drug design. We present here the 2.9-A structure of a ternary complex of the human type II isoform of IMPDH. The complex contains the substrate analogue 6-chloropurine riboside 5'-monophosphate (6-Cl-IMP) and the NAD analogue selenazole-4-carboxamide adenine dinucleotide, the selenium derivative of the active metabolite of the antitumor drug tiazofurin. The enzyme forms a homotetramer, with the dinucleotide binding at the monomer-monomer interface. The 6 chloro-substituted purine base is dehalogenated, forming a covalent adduct at C6 with Cys-331. The dinucleotide selenazole base is stacked against the 6-Cl-IMP purine ring in an orientation consistent with the B-side stereochemistry of hydride transfer seen with NAD. The adenosine end of the ligand interacts with residues not conserved between the type I and type II isoforms, suggesting strategies for the design of isoform-specific agents.

Figure 1

Human Type II IMPDH tetramer with bound dinucleotide analogue SAD (circled, red) and substrate analogue 6-Cl-IMP (circled, green). The dinucleotide binds at the monomer–monomer interface (dotted lines). The following structures are illustrated: catalytic β-barrel domain (blue), flanking domain (magenta), active site loop (yellow) and active site flap fragments (white).

Figure 2

(A) Comparison of flanking domain positions in the human (magenta) and hamster (cyan) (32) complexes. The two conformations are related by a rotation (arrow) about a vector through the hinge region (dotted black line). Flanking domains are 75% and 44% complete in the human and hamster models, respectively. Also shown are the core domain (blue), active site loop (yellow), and ligands (red and green). (B) Comparison of the active site loop observed in the human (yellow) and hamster (cyan) complexes. Purine rings for IMP (cyan) and 6-Cl-IMP (green) are shown. The IMP C2-Cys-331 adduct (cyan dotted line) seen in the hamster complex (32) is replaced by a C6-Cys-331 adduct here (green dotted line). This displaces the loop (yellow) to the opposite side of the purine ring.

Figure 3

σ_A-weighted Fo-Fc omit map illustrating SAD and 6-Cl-IMP binding. The dinucleotide selenazole ring stacks in the anti position against the 6-Cl-IMP purine ring. Analogous binding of cofactor NAD would favorably position the nicotinamide ring for B-side specific hydride transfer. The map was computed at the 2-σ level with both ligands omitted. Bond colors are: Se, magenta; P, yellow; O, red; N, blue; C, white.

Figure 4

Environment of the dinucleotide analogue adenine ring. Interacting residues are highlighted in yellow and outlined by van der Waals surfaces. In the human type II complex, the adenine ring is tightly stacked between Phe-282 and His-253, and makes close edge-on contacts with Thr-45 (Right) and Thr-252 (Left, not labeled). In the human type I isoform, residues 45, 253 and 282 are substituted as indicated. Thr-45 and adjacent Gln-469 (right, not labeled) are contributed by the neighboring monomer.