Crystal structure of the human N-Myc downstream-regulated gene 2 protein provides insight into its role as a tumor suppressor

Abstract

Considerable attention has recently been paid to the N-Myc downstream-regulated gene (NDRG) family because of its potential as a tumor suppressor in many human cancers. Primary amino acid sequence information suggests that the NDRG family proteins may belong to the α/β-hydrolase (ABH) superfamily; however, their functional role has not yet been determined. Here, we present the crystal structures of the human and mouse NDRG2 proteins determined at 2.0 and 1.7 Å resolution, respectively. Both NDRG2 proteins show remarkable structural similarity to the ABH superfamily, despite limited sequence similarity. Structural analysis suggests that NDRG2 is a nonenzymatic member of the ABH superfamily, because it lacks the catalytic signature residues and has an occluded substrate-binding site. Several conserved structural features suggest NDRG may be involved in molecular interactions. Mutagenesis data based on the structural analysis support a crucial role for helix α6 in the suppression of TCF/β-catenin signaling in the tumorigenesis of human colorectal cancer, via a molecular interaction.

FIGURE 1.

Structure-related functional sequence conservation between hNDRG family members. Elements of the secondary structure of hNDRG2 are shown above the alignments. The numbering is based on hNDRG2, which is labeled NDRG2b in this figure. The other isoform of hNDRG2 is labeled NDRG2a. hNDRG4 also consists of two isoforms, a and b. Red stars represent residues located at the corresponding positions of the conserved catalytic residues Ser, Asp, and His, respectively, in the α/β-hydrolase family proteins. Green triangles represent residues involved in the pseudo-active site. Red open circles indicate the hydrophobic residues on the helix α6. P indicates phosphorylation sites. The long blue, open box indicates the three decapeptide sequence repeats of hNDRG1. Strictly conserved residues are highlighted with solid red boxes. Biological sources and accession codes for the sequences are as follows: NDRG2b, N-Myc downstream-regulated gene 2 isoform b (gi:42544224); NDRG2a, N-Myc downstream-regulated gene 2 isoform a (gi:42544222); NDRG1, N-Myc downstream-regulated gene 1 (gi:48145801); NDRG3, N-Myc downstream-regulated gene 3 (gi:12083721); NDRG4a, N-Myc downstream-regulated gene 4 isoform a (gi:13430864); and NDRG4b, N-Myc downstream-regulated gene 4 isoform b (gi:194440722). Sequence alignments were assembled using T-COFFEE software and visualized using ESPript software, both located on the ExPASy Proteomics Server.

FIGURE 2.

Crystal structure of NDRG2. a, schematic representation of the domain structure of human NDRG2. Phosphorylation sites are indicated. b, superimposition of each chain of the hNDRG2 and K2A structures in the asymmetric unit, as well as K2Y and mNDRG2. c, ribbon representation of K2Y. α and 3₁₀ helices are shown in cyan, β-strands in green, and loops in gray. d, hNDRG2 (cyan) and K2A (gray) molecules in the asymmetric unit.

FIGURE 3.

Comparison between NDRG2 and its structural homologues. a, structural comparison of proteins in the α/β-hydrolase family. K2Y, the B. subtilis stress-response regulator (RsbQ, PDB code 1WOM), and P. putida IFO12996 esterase (EST, PDB code 1ZOI) proteins are displayed in black, cyan, and olive, respectively. b, superposition of the pseudo-active site residues of hNDRG2 with the catalytic residues of RsbQ. The active triad of RsbQ is shown as a green carbon skeleton. The corresponding residues of hNDRG2 are displayed as a yellow carbon skeleton. c, comparison of the active site in RsbQ with the corresponding site in hNDRG2. The active site in RsbQ is displayed in green, and the pseudo-active site in hNDRG2 is shown in yellow.

FIGURE 4.

Structural analysis of the cap-like domain of NDRG2. a, comparison of the residue distribution of helix α6 of NDRG2 with the corresponding helix in RsbQ. The surface is represented in a range of colors that indicate electrostatic potential, with red being negative and blue being positive. b, analysis of interaction interface of the symmetry-related K2Y molecules.

FIGURE 5.

Impact of the helix α6 of NDRG2 on TCF/β-catenin signaling. a, luciferase reporter assay was carried out to evaluate TCF/LEF transcriptional activity. The mutant plasmids hNDRG2^Δ164–175 and hNDRG2^L172D transfected into the HEK293 and SW620 cells with or without LiCl did not affect the TCF/LEF transcriptional activity, whereas the native hNDRG2 attenuated the TCF/LEF activity. Mean ± S.D. values from three independent experiments performed in duplicate are shown (*, p < 0.05). b, target genes of TCF/LEF, cyclin D1 and fibronectin were assessed by RT-PCR. Introduction of NDRG2 into the SW620 cells resulted in down-regulation of the target genes, although no regulation of TCF/LEF target genes was observed in the mutant introduced cells. c, hNDRG2 was immunoprecipitated with an anti-NDRG2 antibody, and the precipitant was analyzed by SDS-PAGE and Western blot analysis using anti-β-catenin antibody.