Characterization of the universally conserved ATPase YchF using an in vitro and in silico approach
Rosler, Kirsten S
University of Lethbridge. Faculty of Arts and Science
Lethbridge, Alta : University of Lethbridge, Dept. of Chemistry and Biochemistry
GTPases perform a myriad of functions within the cell, from protein synthesis to cellular signaling. Of all known GTPases, only eight are conserved across all three domains of life. YchF is one of the eight universally conserved GTPases, however its cellular function is poorly understood. YchF differs from the classical GTPases in that it has a higher affinity for ATP than for GTP and functions as an ATPase. As a HAS-GTPase, YchF does not possess the canonical catalytic glutamine required for nucleotide hydrolysis, urging the question of how does YchF hydrolyze ATP. Here we have used molecular dynamics simulations (in silico) and biochemical experiments (in vitro) to identify an amino acid, histidine 114, essential for ATP hydrolysis in YchF. His 114 is located in a flexible loop of the G-domain of YchF, which shows nucleotidedependent conformations in silico. The findings reported indicate that the 70S ribosome can stimulate the ATPase activity of YchF by directly participating in catalysis as well as helping to position the catalytic histidine residue. Additionally, nucleotide binding and dissociation rate constants have been determined in the presence and absence of Mg2+ in order to further understand the functional cycle of YchF.
YchF , HAS-GTPase , ATPase , catalytic mechanism , nucleotide binding