Structural Study Of RID Domain Of Bacterial MARTX Toxins

Vibrio cholerae colonizes the intestinal epithelium after the consumption of contaminated food or water.After colonization,The bacterium elicits the diarrhoeal disease cholera through the secretion of cholera toxin and exotoxin.MARTX is one of exotoxins,which is secreted outside through Type I secretion systems.All MARTX toxins have regions at the amino-and carboxyl-termini that are characterized by extensive glycine rich repeats.Once secreted from the bacterium,the repeat regions of the toxins are postulated to bind to eukaryotic receptors on target cells,where they form a pore for translocation of the central portion of the toxin across the plasma membrane to the eukaryotic cell cytoplasm.The central portion of the toxin is comprised of four domains,that is ACD(actin crosslinking domain),RID(Rho inactivation domain),ABH(alpha/beta hydrolase),CPD(cysteine protease domain).CPD is activated after translocation to the eukaryotic cell cytoplasm by binding of inositol hexakisphosphate(InsP6).The protease is able to autoprocess the MARTX toxin to release ACD,RID and ABH.Among the three effector domains of MARTXVc is the Rho inactivation domain(RIDVc)known to cause cell rounding through inactivation of small RhoGTPases.The N terminal of RID has been shown to localize to the plasma membrane(PM).RID can be directed to the PM by a membrane localization domain(MLD)to access their intracellular targets.The mechanism by which the RID domain causes RhoGTPase inactivation has not been assessed experimentally.We have used crystallography and biochemistry experiments to studty the mechanism of RID inactvating RhoGTPase,in order to explain the pathogenesis of MARTXVc led to cholerae desease.Vibrio cholerae RID shares high homology with Vibrio vulnificus RID.We have amplified RID from Vibrio vulnificus genomic DNA.After expression,purification and crystallization,crystals of RID was obtained.We have successfully solved the three-dimensional structure of RID short fragment,which is VvRID(2564-2898),and RID long fragment,which is VvRID(2283-2898).RID short fragment,which only contains the catalytic domain,was appearanced as dimer in stucture.Futher experiments revealed that the dimer is artificial.The structure of RID long fragment is divided as three regions.The N-terminal projection includes one ahelix and six?sheet.The next region consists of four a-helices,which is thought to act as a membrane localization domain(MLD).The C-terminal portion has a unique circularly permuted papain-like fold surrounded by multiple helical projections.The results reported here provide a basis for futher structural studies of the complex of RID and its substrates.