Characterization of the Intramolecular Interactions in the Circularly Permuted GTPase Nucleostemin

Abstract

Nucleostemin is a unique protein that plays a role in cell cycle regulation, cellular stress sensing, telomere maintenance, and tumor suppression. Similar to other nucleolar proteins, compartmentalization plays a large role in regulating the activity of NS. The mechanism behind this redistribution is poorly defined but it is linked to the GTPase properties of the protein and an uncharacterized inhibitory region that directs complex formation between NS and various cellular partners. We have been studying the structural and biochemical properties of drosophilia NS1 to better understand the molecular basis of this dynamic localization process. The canonical GTPase core contains a six-stranded β sheet surrounded by 5 α helices. The nucleotide-binding site of the protein is defined by four conserved amino acid sequence motifs, the G-1 through G-4 boxes. cpGTPases are characterized by a reordering of the characteristic G boxes specifically to a G4-G1-G3 pattern, thus a circular permutation of the classic GTPase arrangement. There is little known about how altering the positioning of the G-boxes affects the nucleotide binding and regulatory properties of the protein. Work from our group and other laboratories demonstrated that cpGTPases have broad substrate specificity and can hydrolyze GTP and ATP. We have also identified several structural domains in dNS1 using limited proteolysis. Truncation constructs have been made to elucidate the role of each domain and how the physical interactions between these domains contribute to the biological properties of the protein. Taken together, these data point to a mechanism of action that differs from the larger GTPase family.