| G proteins are involved in almost all aspects of the cellular regulatory pathways through their ability to bind and hydrolyze GTP.With respect to heterotrimeric G proteins,small G proteins are monomeric guanine nucleotide binding proteins.The YchF(ribosome-binding ATPase)subfamily,belonging to the Obg(spoOB-associated GTP-binding protein)family,are the core universally conserved G proteins and are present in all kingdoms of life.In YchF subfamily proteins,the noncanonical G4 motif is correlated with the loss of nucleotide-binding specificities.YchF proteins derived from bacterial,yeast,and human are all defined as an ATPase.Rice is one of the main food crops in the world.Pathogen infection and saline alkali stress can lead to sharp declines in crop production.Rice YchF-type G Protein OsYchFl can negatively regulate plant defense responses and salt stress responses.OsYchFl significantly differs from other YchF homologous proteins in that it has the same ability to bind and hydrolyze GTP and ATP.However,the structure basis,molecular mechanisms and biological significance of OsYchFl having the dual binding specificities for both ATP and GTP is still unknown.Therefore,elucidating the molecular mechanism of involving in signal transduction for OsYchFl has an important significance on the use of regulators of signal transduction pathways to achieve broad-spectrum and durable disease resistance to pathogens and abiotic stresses.In this thesis,firstly,through extensive screening,the OsYchFl protein crystal was obtained.The crystal structures of apo-OsYchF1 at pH 6.5/6.35 were successfully resolved by the molecular replacement method using hOLAl(human Obg-like ATPase 1)as the search model.The OsYchFl structure,as the first crystal structure of the plant YchF homolog,shares the conserved domains of YchF subfamily proteins,including the G domain,helical domain and TGS domain.Compared with other YchF homologs,OsYchFl possesses significantly different functional areas:G2/switch I,G3/switch II and Loop A.The nucleotide-binding titration and hydrolysis activity experiments demonstrated the heterologously expressed OsYchFl had low and similar ATPase/GTPase activity.Subsequently,the complex structures of OsYchFl-AMPPNP(an ATP analog)and OsYchFl-GMPPNP(a GTP analog)were achieved using co-crystallization.The analysis of the two complex structures reveals the binding of OsYchFl to AMPPNP mainly depends on the recognition of the adenine base;the binding of OsYchFl to GMPPNP mainly depends on the interaction of the phosphate groups.Such a binding mode of OsYchFl to GTP is dramatically distinct from that found in other GTPases.Specifically,the guanine base bound in the OsYchFI’s binding pocket was shallower,and the interactions between the guanine base and the G4 and G5 motifs of OsYchFl are mainly water-mediated hydrogen bonds.Moreover,significant conformational changes occur to the OsYchF1 G3/switch II region in the OsYchFl-GMPPNP structure due to the binding to GMPPNP.Based on the two solved complex structures,an OsYchFl-G4’ was constructed,which loses ATP binding capacity and ATPase activity,but retains GTP binding capacity and GTPase activity.Finally,the structures of the apo-OsYchF1 at pH 7.85/7.2 were resolved.The comparison of the structures of the apo-form OsYchF1 at pH 6.5/6.35 and pH 7.85/7.2 shows that merely the change of pH from 6.5 to 7.2 could induce the formation of helix α3 and the swinging of helix a4 toward the nucleotide-binding site.Therefore,the G3/switch Ⅱ region of OsYchFl has a pH-dependent conformational change process.In summary,the structures of OsYchFl in complex with AMPPNP and GMPPNP explain the structural basis of the dual nucleotide-binding specificity of YchF proteins.The OsYchF1-G4’ mutant designed based on the complex structures provides advantages for studying the involvement of OsYchFl in diverse plant stress responses through binding to different nucleotides.Structural changes of apo-OsYchFl under the weakly acidic and weakly alkaline conditions illustrate that cytoplasm pH value may affect the function of OsYchFl in vivo.All of the above results provide a structural basis for using the signal transduction modulator OsYchFl to improve the tolerance of rice to biotic and abiotic stresses. |
