Structural basis of subtilase cytotoxin SubAB assembly.
Pathogenic strains of Escherichia coli produce a number of toxins that belong to the AB5 toxin family, which comprise a catalytic A-subunit that induces cellular dysfunction and a B-pentamer that recognizes host glycans. Although the molecular actions of many of the individual subunits of AB5 toxins are well understood, how they self-associate and the effect of this association on cytotoxicity are poorly understood. Here we have solved the structure of the holo-SubAB toxin that, in contrast to other AB5 toxins whose molecular targets are located in the cytosol, cleaves the endoplasmic reticulum chaperone BiP. SubA interacts with SubB in a similar manner to other AB5 toxins via the A2 helix and a conserved disulfide bond that joins the A1 domain with the A2 helix. The structure revealed that the active site of SubA is not occluded by the B-pentamer, and the B-pentamer does not enhance or inhibit the activity of SubA. Structure-based sequence comparisons with other AB5 toxin family members, combined with extensive mutagenesis studies on SubB, show how the hydrophobic patch on top of the B-pentamer plays a dominant role in binding the A-subunit. The structure of SubAB and the accompanying functional characterization of various mutants of SubAB provide a framework for understanding the important role of the B-pentamer in the assembly and the intracellular trafficking of this AB5 toxin.
Australian Research Council (ARC) Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800, Australia; Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia.
Jérôme
J
Le Nours
Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, South Australia 5005, Australia.
Adrienne W
AW
Paton
Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia.
Emma
E
Byres
Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia.
Sally
S
Troy
Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, South Australia 5005, Australia.
Brock P
BP
Herdman
Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia.
Matthew D
MD
Johnson
Research Centre for Infectious Diseases, School of Molecular and Biomedical Science, University of Adelaide, South Australia 5005, Australia.
James C
JC
Paton
Australian Research Council (ARC) Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800, Australia; Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia; Institute of Infection and Immunity, Cardiff University, School of Medicine, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom. Electronic address: Jamie.Rossjohn@monash.edu.
Jamie
J
Rossjohn
Australian Research Council (ARC) Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800, Australia; Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia. Electronic address: Travis.Beddoe@monash.edu.
Travis
T
Beddoe
D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004220:Disulfides; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001427:Bacterial Toxins; D004220:Disulfides; D029968:Escherichia coli Proteins; D013381:Subtilisins; C527234:subtilase cytotoxin, E coli
AB5 Toxins; Carbohydrate-binding Protein; Cellular Trafficking; Disassembly/Assembly; Infectious Diseases; Intracellular Trafficking; Structural Biology; Toxins