Revealing nucleotide-induced domain movements of GTP-bound Rbg1 with long-timescale MD simulations.

Abstract

Developmentally regulated GTP-binding proteins (Drgs) are highly conserved among eukaryotes and archaea and play a crucial role in such fundamental pathways as translation, differentiation, and proliferation. Ribosome-binding GTPase 1 (Rbg1), a structurally known homolog of Drgs in yeast, consists of an N-terminal HTH domain, an S5D2L domain, a typical G-domain with five G-motifs (G1-G5) responsible for binding to nucleotides, and a C-terminal TGS domain. Like other GTPases, Rbg1 is expected to undergo significant inter-domain conformational changes upon associating with nucleotides, which are transduced as cellular signals to the downstream effectors. However, nucleotide-bound structures of Rbg1 have not been resolved yet, hence the putative coupling between domain movements and the switching function of the protein remain elusive. To capture these conformational changes at an atomic level, we modeled binding of nucleotides to the protein and performed molecular dynamics simulations of GTP-bound, GDP-bound, and apo Rbg1 states for 5 µs, in 5 independent replicas for each state. We observed a significant increase in the movement of the HTH-S5D2L domains only in GTP-bound Rbg1. Close examination of trajectories shows that in GTP-bound Rbg1, the HTH-S5D2L domains move away from the G-domain resulting in a loose form of Rbg1. The angle between helix α2 of the HTH domain and helix α5 of the G-domain, representing the relative orientation of the HTH-S5D2L domains and the G-domain, exhibits a wider range of fluctuation from 50 to 122 degrees in the GTP-bound Rbg1 compared to the other systems. The above results provide valuable insights into the large-scale conformational changes of Rbg1 induced by nucleotide binding, which potentially offer a deeper understanding of the steps involved in activation of Rbg1.