Crystal structure of the worm NitFhit Rosetta Stone protein reveals a Nit tetramer binding two Fhit dimers

Background: The nucleotide-binding protein Fhit, among the earliest and mos t frequently inactivated proteins in lung cancer, suppresses tumor formatio n by inducing apoptosis. In invertebrates, Fhit is encoded as a fusion prot ein with Nit, a member of the nitrilase superfamily. In mice, the Nit1 and Fhit genes have nearly identical expression profiles. According to the Rose tta Stone hypothesis, if the separate Nit and Fhit genes could be shown to occur in the same subset of genomes (that is, to share a phylogenetic profi le), then the existence of a fusion protein in invertebrates and the coordi nated expression of separate mRNAs in mouse suggest that Nit and Fhit funct ion in the same pathway and that the structure of invertebrate NitFhit may reflect the nature of Nit-Fhit interactions. Results: To satisfy the phylogenetic profile criterion for functional signi ficance of protein fusion events, we cloned additional Nit homologs from or ganisms with Fhit homologs, We used fluorescent nucleotide analogs of ApppA to follow the purification and to characterize the nucleotide specificity of NitFhit from Caenorhabditis elegans, crystallized the 200 kDa tetrameric complex, and solved the structure of NitFhit from a single mercury derivat ive phased by two-wavelength anomalous diffraction. Conclusions: Nit monomers possess a new a-P-P-a sandwich fold with a presum ptive Cys-Glu-Lys catalytic triad. Nit assembles into a tetrameric, 52-stra nded beta box that binds Fhit dimers at opposite poles and displays Nit act ive sites around the middle of the complex. The most carboxy-terminal beta strand of each Nit monomer exits the core of the Nit tetramer and interacts with Fhit, Residence in the NitFhit complex does not alter the nucleotide specificity of Fhit dimers, which are oriented with ApppA-binding surfaces away from Nit. (C) 2000 Elsevier Science Ltd. All rights reserved.