PROJECT SUMMARY
Interferon-inducible GTPases are among the most potent effectors in cell-autonomous immunity. They are
dynamin-like large GTPases highly induced by proinflammatory cytokines, especially type I and II interferons.
Interferon-inducible GTPases eliminate or restrict intracellular bacteria and protozoan parasites through a variety
of strategies. The most prominent strategies are rupture of pathogen-containing vacuoles (PCVs) to expose
pathogens to cytosolic pattern recognition receptors (PRRs), and direct attack and lysis of bacterial membranes.
On the other hand, interferon-inducible GTPases are under tight control to avoid indiscriminate attack on host
cell endomembranes. The critical roles of IFN-inducible GTPases are gaining attention and appreciation recently,
yet the molecular mechanisms of their activation, their effector functions on target membranes, and their
regulation by cellular and microbial factors remain evasive. In this proposal, we study the activation and
regulation mechanisms of interferon-inducible GTPases using guanylate-binding protein 2 (GBP2) as the
prototype. In our preliminary studies, we purified GBP2, determined its crystal structures, and gained initial
insights into its structures and functions. In this research, we will take advantage of these preliminary data to
determine the structural and mechanistic basis of GBP activation in solution and on target membrane, and the
regulation mechanism by cellular and microbial factors. We will first elucidate GBP2 oligomerization status and
GTPase activity in solution. We will then determine the atomic structures of GBP2 in various nucleotide-bound
states using X-ray crystallography and cryogenic electron microscopy (cryo-EM). Next, we have modified GBP2
with farnesyl group and will characterize its interaction with lipids and liposomes. We will then determine the
remodeling/lysis effect of farnesylated GBP2 on its target membranes using fluorescence-based liposome
leakage assay and EM techniques. We also plan to characterize the higher-order assembly mode of membrane-
attached GBP2. Finally, we will define how GBP2 is self-inhibited and regulated by other cellular and pathogen-
derived proteins. Successful accomplishment of this proposed research will fill a major gap in our knowledge of
the molecular mechanisms of intracellular pathogen detection and restriction. This work will also expand our
understanding of activation and assembly of large GTPases in general. Ultimately, these findings will facilitate
the development of novel therapeutic strategies for microbial infections and autoinflammatory diseases.