Structure-function analysis of inositol hexakisphosphate-induced autoprocessing in Clostridium difficile toxin A.
The action of Clostridium difficile toxins A and B depends on inactivation of host small G-proteins by glucosylation. Cellular inositol hexakisphosphate (InsP6) induces an autocatalytic cleavage of the toxins, releasing an N-terminal glucosyltransferase domain into the host cell cytosol. We have defined the cysteine protease domain (CPD) responsible for autoprocessing within toxin A (TcdA) and report the 1.6 A x-ray crystal structure of the domain bound to InsP6. InsP6 is bound in a highly basic pocket that is separated from an unusual active site by a beta-flap structure. Functional studies confirm an intramolecular mechanism of cleavage and highlight specific residues required for InsP6-induced TcdA processing. Analysis of the structural and functional data in the context of sequences from similar and diverse origins highlights a C-terminal extension and a pi-cation interaction within the beta-flap that appear to be unique among the large clostridial cytotoxins.
Pruitt
Rory N
RN
Departments of Microbiology and Immunology, Nashville, Tennessee 37232.
Chagot
Benjamin
B
Biochemistry, Nashville, Tennessee 37232.
Cover
Michael
M
Departments of Microbiology and Immunology, Nashville, Tennessee 37232.
Chazin
Walter J
WJ
Biochemistry, Nashville, Tennessee 37232.
Spiller
Ben
B
Departments of Microbiology and Immunology, Nashville, Tennessee 37232; Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232.
Lacy
D Borden
DB
Departments of Microbiology and Immunology, Nashville, Tennessee 37232; Biochemistry, Nashville, Tennessee 37232.
D001427:Bacterial Toxins / Q000737:chemistry*; D020134:Catalytic Domain; D002412:Cations; D016360:Clostridioides difficile / Q000378:metabolism*; D018360:Crystallography, X-Ray / Q000379:methods; D004768:Enterotoxins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D008958:Models, Molecular; D008968:Molecular Conformation; D010833:Phytic Acid / Q000494:pharmacology*; D017354:Point Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry / Q000379:methods; D013997:Time Factors
D001427:Bacterial Toxins; D002412:Cations; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile; D010833:Phytic Acid