Structural and molecular mechanism for autoprocessing of MARTX toxin of Vibrio cholerae at multiple sites
K. Prochazkova 1, L. A. Shuvalova 2,3, G. Minasov 2,3, Z. Voburka 4, W. F. Anderson 2,3, K. J.Satchell 1
2Center for Structural Genomics of Infectious Diseases
Medicine, Northwestern University, Chicago, Illinois 60611, USA
4Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague 161-10 Praha 6, Czech Republic
The Vibrio cholerae MARTX toxin is associated with rounding of eukaryotic cells through destruction of the actin cytoskeleton by covalent cross-linking of actin and inactivation of Rho GTPases . The effector domains responsible for these activities are here shown to be independent proteins released from the large toxin by autoproteolysis catalyzed by an embedded cysteine protease domain (CPD) . The CPD is activated upon binding inositol hexakisphosphate (InsP6), which is present predominantly in eukaryotic cells. We demonstrated that InsP6 is not simply an allosteric cofactor, but rather binding of InsP6 stabilized the CPD structure, facilitating formation of the enzyme-substrate complex. The 1.95-A crystal structure of this InsP6-bound unprocessed form of CPD was determined and revealed the scissile bond Leu(3428)-Ala(3429) captured in the catalytic site. Upon processing at this site, CPD was converted to a form with reduced affinity for InsP6, but was reactivated for high affinity binding of InsP6 by cooperative binding of both a new substrate and InsP6. This allowed CPD to cleave MARTX toxin between cytopathic domains by hydrolyzing specifically at Leu-Xaa bonds. Thus we uncovered the mechanism of MARTX autoprocessing, which results in release of cytopathic domains from large holotoxin to reach their targets within eukaryotic cells.
1. K. J. Satchell, Infect Immun., 75, (2007), 5079.
2. K. Prochazkova, L. A. Shuvalova, G. Minasov, Z. Voburka, W. F. Anderson, K. J. Satchell, J Biol Chem., 284, (2009), 26557.
This work was supported in part by a development project from National Institute of Health Grant U54 AI057153 to Great Lakes (Region V) Regional Center for Excellence in Biodefense and Emerging Infectious Diseases Research.