The Structural Biology Research On NS3 Helicase Domain Of Tembusu Virus And YoeB Toxin In Staphylococcus Aureus

In 2010,the outbreak of an acute infectious disease occurred in many duck farms in the southeast coastal provinces of China.It was caused by the Tembusu virus(TMUV),a novel member of flavivirus.The C-terminal helicase domain of nonstructural protein NS3 possesses NTP-dependent RNA unwinding activity that plays critical roles in viral replication.In this study,we solved two crystal structures of NS3 helicase of TMUV that present different conformations,i.e.,Form Ⅰ and Form Ⅱ.In the structure of Form I,the domain Ⅱ rotates～10° away from the domain I with displacement of～4.5 ? as compared to the structure of Form Ⅱ.In addition,the inner of domain Ⅱ of Form Ⅰ containing Arg461,motif V and α7 helix also possess the conformational change i.e.,the sidechain of Arg461 projects into the hydrophobic core of domain Ⅱ that forces the movement of residues in motif V,resulting in disorder ofα7 helix.Comparative structural analysis of NS3 with other flavivirus demonstrated that the structures of two Forms might present the conformational changes in the catalytic process.Moreover,we also solved the structure of Form Ⅱ-3PO(Triphosphoric Acid)-Ca2+complex,which reflects the structural rearrangement of ploop region upon ligand binding.The assay of ATP hydrolysis indicates the NS3 helicase could utilize the Ca2+ion,however,Mg2+is the optimal ion during ATP hydrolysis.The ratio of unwinding of duplex RNA/DNA could be promoted by ATP hydrolysis.Mutational analysis demonstrates that the stability of hydrophobic core of domain Ⅱ is very critical for activity of helicase,and mutation of Arg461 not only affects the activity of ATPase but also significantly reduces helicase activity.Taken together,these structural and biochemical results provide the important information for NS3 Helicase conformational dynamics and antiviral drug development.In the second part,the Staphylococcus aureus YoeB-YefM complex,one of type Ⅱtoxin-antitoxin(TA)system for the response during environmental stress,was studied.YoeB-YefM TA system adopts a strategy,termed ’conditional cooperativity’,to autoregulate its transcription.The level of TA operon transcription is controlled by the ratio of toxin to antitoxin,resulting low or high affinity for its own promoter.However,there is absent the structure of YoeB toxin and how YoeB toxin recognizes and binds to the YefM antitoxin.We solved three different crystal structures about YoeB toxin.The crystal structure of the YoeB dimer at the resolution of 1.8?.The structure adopts a canonical RNase fold that is similar with the YoeB from E.coli.Two monomers form the dimer based on the hydrophobic and hydrogen bonds interactions of residues in theβ1 sheet and α-helix(α1 and α2).In addition,we also solved the structure of YoeB monomer binding the YefM C terminal region(46-83aa)at high resolution of 0.98 ?,which forms a "U" shaped structure to bind the YoeB monomer and blocks most of the catalytic residues.Upon YefM binding,the YoeB N-and C-terminal has the conformation change.The structure suggested that YefM inhibits the toxicity of YoeB by blocking the catalytic core and disrupting the dimer formation.Moreover,we also determined the structure of YoeB dimer in complex with hairpin DNA at resolution of 1.94 A.It indicates that the YoeB could bind the backbone of DNA base on the positive surface,suggesting that this surface might be responsible for ribosome binding.Based on these three crystal structures,we elucidate the changes in oligomeric states and conformation between YoeB alone and YoeB-YefM complex and the positive surface for ribosome binding.The present study provides detailed structural information about the type II YoeB-YefM TA system from S.aureus.