No abstract

GTP triphosphatases (GTPases) have been meticulously documented for their roles to regulate multiple cellular processes spanning from cell mobility, membrane fusion and fission, to cytokinesis and vesicle transport. Interferon (IFN)‐inducible GTPases, consisting of more than forty members in human and mice, are among the most highly expressed interferon stimulated genes (ISGs), sometimes accounting for twenty percent of all proteins induced by IFN‐γ. Recent genetic and cell studies start to reveal the important roles of IFN‐inducible GTPases in restriction and elimination of pathogens, yet the molecular mechanisms are largely unclear. Using a combination of biochemical, structural, biophysical, and computational tools, we are investigating the functional forms of two IFN‐inducible GTPases, GBP2 and IRGM, and the mechanisms governing their activation. We established the relationship between nucleotide binding and the oligomeric status of GBP2. A combination of X‐ray crystallography, electron microscopy, and molecular dynamics captured GBP2 conformational changes along the full trajectory of GTP binding and hydrolysis. Structure‐guided mutagenesis validated the roles of key residues in nucleotide binding pocket. In contrast to the dynamic nature of GBP2, IRGM assumed a highly stable oligomeric state that possessed intrinsic affinity towards lipids. In conclusion, GTP binding and hydrolysis regulate the oligomeric status of interferon‐inducible GTPases and subsequential effector molecule binding.Support or Funding InformationNIAID R00AI108793 “Dissecting inflammasome anatomy: mechanistic studies and potential intervention”; Florida State University startup fundsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Guanylate-binding proteins (GBPs) are interferon-inducible GTPases that confer protective immunity against a variety of intracellular pathogens including bacteria, viruses, and protozoan parasites. GBP2 is one of the two highly inducible GBPs, yet the precise mechanisms underlying the activation and regulation of GBP2, in particular the nucleotide-induced conformational changes in GBP2, remain poorly understood. In this study, we elucidate the structural dynamics of GBP2 upon nucleotide binding through crystallographic analysis. GBP2 dimerizes upon GTP hydrolysis and returns to monomer state once GTP is hydrolyzed to GDP. By determining the crystal structures of GBP2 G domain (GBP2GD) in complex with GDP and nucleotide-free full-length GBP2, we unveil distinct conformational states adopted by the nucleotide-binding pocket and distal regions of the protein. Our findings demonstrate that the binding of GDP induces a distinct closed conformation both in the G motifs and the distal regions in the G domain. The conformational changes in the G domain are further transmitted to the C-terminal helical domain, leading to large-scale conformational rearrangements. Through comparative analysis, we identify subtle but critical differences in the nucleotide-bound states of GBP2, providing insights into the molecular basis of its dimer-monomer transition and enzymatic activity. Overall, our study expands the understanding of the nucleotide-induced conformational changes in GBP2, shedding light on the structural dynamics governing its functional versatility. These findings pave the way for future investigations aimed at elucidating the precise molecular mechanisms underlying GBP2's role in the immune response and may facilitate the development of targeted therapeutic strategies against intracellular pathogens.