Tracing functional evolution in P-loop NTPases

Abstract

The simplest members of the P-loop NTPase superfamily- the Ras family of GTPases act as molecular switches that regulate many cellular processes in all domains of life. The GTPase domain in small Ras-like GTPases is functionally minimalistic and hence the completion of the GTPase cycle in these GTPases requires additional proteins - GTPase Activating Proteins (GAPs) and Guanine nucleotide Exchange factors (GEFs). The project reveals that the interface in Ras-GAP and Ras-GEF complexes most often involves the nucleotide-binding pocket. Hence, these interactors predominantly employ a direct mode of regulation of the Ras GTPase. The GAPs facilitate the hydrolysis of GTP to GDP in Ras by properly positioning two catalytic residues, while GEFs reduce Ras's affinity for GDP by transiently opening up the nucleotide-binding pocket. An examination of more complex P-loop NTPase members revealed that these P-loop NTPases avoid dependence on GAPs by either modifying the existing minimal Ras core or evolving insertions to carry the stimulatory residues. The strategy adopted seems to depend on the degree of relatedness to the Ras GTPase, as closely related members incorporate point mutations in the core while further distantly related members evolve insertions that carry the stimulatory residues. The analysis of adenine binding environments revealed that adenine could position itself atleast in four different ways in P-loop ATPases by altering the tilt of the adenine plane or the relative positioning of ribose and purine planes. In all four modes of adenine positioning, insertions majorly stabilize the adenine. Analyzing residues that stabilize the adenine revealed that there is prominent utilization of main chain and water-based interactions unlike guanine recognition in Ras GTPases with a strictly conserved side-chain based recognition. The results from the project offer insights into specific parts of the small Ras-like GTPase core that have been modified by evolutionarily related NTPases to possess gains in function like hydrolytic competency and altered substrate specificity.