Interferon-induced dynamin-like GTPases

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The interferon-induced Myxovirus-resistance (Mx) GTPases are central players of innate immunity. They mediate resistance against a wide range of pathogens, including influenza viruses and many other negative-strand viruses. As typical members of the dynamin superfamily, they oligomerize in ring-like structures around tubular membranes, but may in addition also bind directly to viral components.

We determined the structure of the MxA stalk and the structural basis of its assembly which involves three stalk interfaces (Gao et al., Nature, 2010). We also determined the structure of the full-length MxA molecule and proposed a model for ring-like MxA oligomers. Furthermore, we suggested a molecular pathway of how nucleotide-driven rearrangements in the MxA oligomer are transmitted from the GTPase domain to the stalk (Gao et al., Immunity, 2011).

Scientific illustration — **Figure 1**: a) Crystal structure (pdb 3SZR) and b) oligomerization model for MxA. c) We postulate that the nucleocapsids are the targeted by oligomers of MxA. Shown below is our model for the RNP structure of Hantavirus NP (based on our model pdb 5FSG).

We also characterized the molecular mechanism of GTP hydrolysis in MxA which is based on dimerization of the GTPase domains (Dick et al., J Biol Chem, 2015). Furthermore, together with the group of Georg Kochs (University of Freiburg), we analysed the consequence of allelic variations on the biochemical and antiviral properties of the MxA proteins (Graf et al., J Biol Chem, 2018).

To understand the antiviral mechanism of MxA, we also determined crystal structures of putative targets of MxA, the nucleoproteins (NPs) of the negative-strand RNA viruses. These NPs wrap around the viral genome, protect it from degradation by cellular RNAses, and allow transcription of the genome via the viral RNA-dependent RNA polymerase. Thus, we solved the crystal structure of Toscana virus NP in the presence and absence of RNA and suggested an RNA-induced oligomerization model (Olal et al., Nucleic Acids Research, 2014). We also determined the crystal structure of Hantavirus NP, elucidated similarities and differences in the structure compared to the NPs of other negative-strand RNA viruses, and proposed an oligomerization model involving N- and C-terminal arms (Olal and Daumke, Cell Rep, 2016).

Immunity-related GTPases (IRGs) comprise a second interferon-induced dynamin-like GTPase family. These proteins target intracellular bacteria that survive in eukaryotic cells within membranous compartments. Recruitment of IRGs to these compartments leads to their disruption, and the release and destruction of the pathogen.

It has previously been suggested by others that the GTPases domains of IRGs dimerize in an antiparallel fashion leading to stimulation of the GTPase activity. Using X-ray crystallography, we, however, found that IRGs, similar to other dynamin GTPases, dimerize in a parallel head-to-head fashion via their GTPase domains (Schulte et al., BMC Biol, 2016).

Guanylate-binding proteins (GBPs) comprise a family of interferon-induced, dynamin-related GTPases (reviewed in (Daumke and Praefcke, Biopolymers, 2016)). Its functionally best characterized member in human, hGBP1, conveys host defense against intracellular bacteria and parasites. During infection, hGBP1 is recruited to pathogen-containing vacuoles, such as Chlamydia trachomatis inclusions, restricts pathogenic growth, and induces the activation of the inflammasome pathway. hGBP1 has a unique catalytic activity to hydrolyze guanosine triphosphate (GTP) to guanosine monophosphate (GMP) in two consecutive cleavage steps. However, the functional significance of this activity in host defense remained elusive.

To study the role of this unusual biochemical activity, we generate a structure-guided mutant that specifically abrogates GMP production, while maintaining fast cooperative GTP hydrolysis (Xavier et al., Cell Rep, 2020). Complementation experiments in human monocytes and macrophages showed that hGBP1-mediated GMP production is dispensable for restricting Chlamydia trachomatis growth but is necessary for inflammasome activation. GMP was catabolized to uric acid, which activated the NLRP3 inflammasome. In this way, our work demonstrates how the unique enzymology of hGBP1 coordinates bacterial growth restriction and inflammasome signaling.

Publications

Gao, S., von der Malsburg, A., Paeschke, S., Behlke, J., Haller, O., Kochs, G., and Daumke, O. (2010). Structural basis of oligomerization in the stalk region of dynamin-like MxA. Nature 465, 502-506. https://doi.org/10.1038/nature08972.
Gao, S., von der Malsburg, A., Dick, A., Faelber, K., Schroder, G.F., Haller, O., Kochs, G., and Daumke, O. (2011). Structure of myxovirus resistance protein a reveals intra- and intermolecular domain interactions required for the antiviral function. Immunity 35, 514-525. https://doi.org/10.1016/j.immuni.2011.07.012.
Dick, A., Graf, L., Olal, D., von der Malsburg, A., Gao, S., Kochs, G., and Daumke, O. (2015). Role of nucleotide binding and GTPase domain dimerization in dynamin-like myxovirus resistance protein A for GTPase activation and antiviral activity. J Biol Chem 290, 12779-12792. https://doi.org/10.1074/jbc.M115.650325.
Graf, L., Dick, A., Sendker, F., Barth, E., Marz, M., Daumke, O., and Kochs, G. (2018). Effects of allelic variations in the human myxovirus resistance protein A on its antiviral activity. J Biol Chem 293, 3056-3072. https://doi.org/10.1074/jbc.M117.812784.
Olal, D., Dick, A., Woods, V.L., Jr., Liu, T., Li, S., Devignot, S., Weber, F., Saphire, E.O., and Daumke, O. (2014). Structural insights into RNA encapsidation and helical assembly of the Toscana virus nucleoprotein. Nucleic acids research 42, 6025-6037. https://doi.org/10.1093/nar/gku229.
Olal, D., and Daumke, O. (2016). Structure of the Hantavirus Nucleoprotein Provides Insights into the Mechanism of RNA Encapsidation. Cell Rep 14, 2092-2099. https://doi.org/10.1016/j.celrep.2016.02.005.
Schulte, K., Pawlowski, N., Faelber, K., Frohlich, C., Howard, J., and Daumke, O. (2016). The immunity-related GTPase Irga6 dimerizes in a parallel head-to-head fashion. BMC Biol 14, 14. https://doi.org/10.1186/s12915-016-0236-7.
Daumke, O., and Praefcke, G.J. (2016). Invited review: Mechanisms of GTP hydrolysis and conformational transitions in the dynamin superfamily. Biopolymers 105, 580-593. https://doi.org/10.1002/bip.22855.
Xavier, A., Al-Zeer, M.A., Meyer, T.F., and Daumke, O. (2020). hGBP1 Coordinates Chlamydia Restriction and Inflammasome Activation through Sequential GTP Hydrolysis. Cell Rep 31, 107667. https://doi.org/10.1016/j.celrep.2020.107667.