Mechanism Of Activating Guanylate Binding Proteins In A Functional Dimer

Guanylate binding proteins(GBPs)are an interferon(IFN)-inducible subfamily of guanosine triphosphatases(GTPases),which are dynamin-related and characterized with an N-terminus GTPase domain and a C-terminus helical domian.GBPs are important IFN-inducible effectors in cell-autonomous immunity and could provide resistance against a broad spectrum of intracellular pathogens,such as bacteria,parasites and viruses.In particular,GBP5 has recently been identified as restriction factor against HIV-1 by interfering with the processing and virion incorporation of the envelope glycoprotein.In addition,GBP5 promotes selective NLRP3 inflammasome responses to pathogenic bacteria through its interaction with a pyrin domain(PYD).Although it has been reported that oligomerization of GBP5 is indispensable for these functions,the detailed molecular mechanism is still elusive due to lack of the structural information.In this study,we launched structural research on GBP5 and its oligomerization mechanism.In the first part,we determined the crystal structure of GBP5 in nucleotidefree form,and found the domain architecture is similar to that of dynamin-superfamily proteins.The globular GTPase domain and elongated helical domain together exhibited a closed conformation.In the second part,we determined the crystal structure of its GTPase domain in nucleotide–bound form.Through interaction analysis between protein and nucleotide,we revealed the catalytic mechanism at atomic level,identified the different catalytic residues from GBP1,and demonstrated the conformational change in nucleotide-loading.In the last part,we determined the crystal structure of GBP5 in nucleotide-bound form.Comparing to the structure of GBP5 in nucleotidefree form,we found that nucleotide-binding could induce large conformational change of the GTPase domain,which can then be transducted through the linker region,ultimately resulting in the association of the helical domain and assembly of GBPs into oligomers.Combined with our biochemical and biophysical data,our structures elaborated the mechanism of nucleotide-induced assembly for GBP family proteins and provide bona-fide models to study the functions of GBP5 in immune responses.