Nucleotide-dependent structural dynamics and domain motion in Coxiella burnetii EngA GTPases: Insights from molecular...

EngA, a ribosome-associated bacterial GTPase essential for 50S subunit maturation and bacterial growth, lacks a human ortholog, making it an attractive antibacterial target. Its activity is governed by a nucleotide-dependent molecular switch: GTP binding promotes EngA association with the immature 50S subunit and facilitates rRNA processing, whereas GTP hydrolysis to GDP triggers dissociation from the ribosome. Structural studies on Bacillus subtilis EngA revealed that GTP-analog-bound EngA disrupts the GD1-KH interface required for 45S subunit association, while GDP-bound EngA retains these interactions. However, the molecular mechanism by which nucleotides regulate these transitions and whether similar mechanisms exist in other pathogenic species remain unclear. To address this, 1000 ns molecular dynamics simulations of Coxiella burnetii EngA was performed in four nucleotide-bound states: [GDP:GDP], [GDP:GTP-Mg²⁺], [GTP-Mg²⁺:GDP], and [GTP-Mg²⁺:GTP-Mg²⁺]. Analyses of principal components, interaction energies, and distance-angle parameters revealed nucleotide-dependent domain dynamics. In the [GTP-Mg²⁺:GTP-Mg²⁺] state, GD1 and KH domains moved apart, forming an open conformation, while in [GDP:GDP] they approached each other, forming a closed conformation consistent with cryo-EM structures. Community network analysis further showed that GDP binding to GD1 extends connectivity from the nucleotide to SwI, stabilizing SwI-KH interactions and restricting GD1 motion. In contrast, GTP-Mg²⁺ binding disrupts this network, enabling SwI-GD2 interactions that weaken the GD1-KH interface and promote an open conformation. Overall, the results highlight how nucleotide charge-dependent interactions regulate EngA allosteric network and drive its conformational switching mechanism.