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.
Highlights
We used the truncated constructs hNDRG2 and mNDRG2 to characterize their structures. mNDRG2 was determined at a resolution of 1.7 Å by the Joint Center for Structural Genomics and provided the first structure for the N-Myc downstream-regulated gene (NDRG) family (Pfam database [36] ID: PF03096), thereby facilitating structure determination of additional family members, including hNDRG2
The structures of the hNDRG2 K2Y and K2A mutants were determined by the molecular replacement (MR) method at 2.15 and 2.0 Å resolution, respectively
The NDRG family has been studied in a number of cell and animal systems and has been shown to influence diverse cellular processes, such as the modulation of cell differentiation and proliferation, little study has been made, so far, of its function and mechanism at the molecular level
Summary
The DNA fragment encoding the human NDRG2 protein (residues 23–304) was amplified by PCR and subcloned into the pPosKJ expression vector [22] This vector produced NDRG2(23–304) fused to a hexahistidine tag and bacterial hemoglobin (His6-VHb) at its N terminus. Buffer exchange was performed to remove imidazole from the eluate, and the protein in buffer Q (20 mM Tris-HCl, pH 7.9, 5% (v/v) glycerol, 0.25 mM TCEP) containing 50 mM NaCl was applied to a Resource Q column (Amersham Biosciences) pre-equilibrated with the same buffer. The appropriate fractions were pooled, further purified using a Superdex 200 size-exclusion column (Amersham Biosciences) with elution in crystallization buffer (20 mM Tris-HCl, pH 7.9, 150 mM NaCl, 0.25 mM TCEP), and concentrated to 17.6 mg/ml for crystallization assays. 93.10 Å 93.10 Å 90.21 Å 2.15 Å (2.23–2.15 Å) 227,387 24,942 9.1(6.6) 99.9% (99.8%) 8.0% (49.6%) 28.16(2.78)
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