NMR Structure and Functional Characterization of a Human Cancer-related Nucleoside Triphosphatase

A screen of the human cancer genome anatomy project (CGAP) database was performed to search for new proteins involved in tumorigenesis. The resulting hits were further screened for recombinant expression, solubility and protein aggregation, which led to the identification of the previously unknown human cancer-related (HCR) protein encoded by the mRNA NM_032324 as a target for structure determination by NMR. The three-dimensional structure of the protein in its complex with ATPγS forms a three-layered α/β sandwich, with a central nine-stranded β-sheet surrounded by five α-helices. Sequence and three-dimensional structure comparisons with AAA+ ATPases revealed the presence of Walker A (GPPGVGKT) and Walker B (VCVIDEIG) motifs. Using 1D 31P-NMR spectroscopy and a coupled enzymatic assay for the determination of inorganic phosphate, we showed that the purified recombinant protein is active as a non-specific nucleoside triphosphatase, with kcat = 7.6 × 10−3 s− 1. The structural basis for the enzymatic activity of HCR-NTPase was further characterized by site-directed mutagenesis of the Walker B motif, which further contributes to making the HCR-NTPase an attractive new target for further biochemical characterization in the context of its presumed role in human tumorigenesis.