Nucleotide Specificity Analysis in Universally Conserved NTPases

Abstract

P-loop GTPases and related ATPases play important roles in many cellular processes. These processes include translation, motility and ribosome biogenesis. P-loop NTPases often serve to regulate these processes by acting as “molecular switches” that change between active NTP-bound states and inactive NDP-bound states (1,2). The YchF subfamily of the Obg family of P-loop GTPases is characterized based on its homology to bacterial YchF (1). This subfamily is notable because it differentially binds and hydrolyzes ATP and/or GTP based on small changes to the G4 motif, or “nucleotide differentiation motif”. This motif varies between protiens and it has been shown that changes to its sequence affect the nucleotide specifity of the protein(1). Most protiens of the YchF family have not been characterized as ATPases or GTPases. To this end we intend to characterize the nucleotide binding and hydrolysis activity the G4 motif of multiple YchF protiens. Characterizing the G4 motifs will help to predict the nucleotide specificity of the protiens as a whole. An understanding of how changes to the G4 motif affect nucleotide binding is important for building understanding of ATPase/GTPase activity. This will be useful in the development of synthetic purine binding proteins and the examination of other similar proteins. Examination of the changes to the G4 motif in YchF homologues in multiple organisms will also help to develop an understanding of what evolutionary pressures cause the unusual variation in substrate for these proteins. Previously created Escherichia coli (E. coli) BL21-DE3 cells containing pET28a plasmids coding for YchF G4 variants were used to analyze the purine binding and hydrolysis properties. These G4 varriants consist of E. coli YchF with a G4 motif altered to match a different YchF homologue. Protien variants were analyzed using fluorescence titration and multiple turnover NTPase assays. Data were analyzed and used to determine differences in the binding and hydrolysis of purine nucleotide phosphates for each G4 motif variant. Analysis showed that each variant displayed differential binding and hydrolysis of purine phosphates. Furthermore the variant based on a previously characterized YchF homologue (OsYchF) showed dual ATPase and GTPase activity matching the original protein (3).

References:

1.      Bourne, H. R. (1995)
2.      Scott, J., et al. (2000)
3.      Cheung, M., et al. (2016)

*Indicates presenter