4bwg
Structural basis of subtilase cytotoxin SubAB assembly
Structural highlights
FunctionSUBA_ECOLX Protease subunit of subtilase cytotoxin SubAB5 (PubMed:17024087). An endoprotease specific for host endoplasmic reticulum (ER) chaperone BiP/HSPA5, has no activity on human HSP70 or HSPA8 (PubMed:17024087). Cleaves between 'Leu-416' and 'Leu-417' of BiP/HSPA5 in the hinge between BiP's ATPase and protein-binding domains (PubMed:17024087). This induces host ER stress response and eventual cell death (PubMed:18005237, PubMed:18433465). Culture supernatant of E.coli expressing both subA and subB are toxic for Vero cells (African green monkey kidney cell line), Chinese hamster ovary cells and Hct-8 cells (human colonic epithelial cell line); the subunits are not toxic individually (PubMed:15226357). Purified SubAB5 is highly toxic, <0.1 pg is able to kill at least 50% of 30'000 Vero cells in a microtiter plate assay after 3 days; no cytotoxicity is seen at 24 hours (PubMed:15226357). Preabsorption with cells expressing a ganglioside GM2 mimic reduced cytotoxicity of SubAB5 by 93% in the Vero cytotoxicity assay (PubMed:15226357). Intraperitoneal injection of 200 ng of purified SubAB5 kills mice; the higher the dose the faster the mice die. Animals injected intraperitoneally with purified SubAB5 have microvascular thrombi in the brain and other organs, including the renal tubules and glomeruli (PubMed:15226357). Injection induces an unfolded response in mice (PubMed:17024087). Mice fed E.coli cells expressing cloned SubAB5 experience drastic weight loss and appear ill and lethargic (PubMed:15226357). Protein synthesis in Vero cells is transiently inhibited by SubAB5; both subunits are required for this effect (PubMed:17101670, PubMed:18005237, PubMed:18433465). Inhibition of protein synthesis is prevented by brefeldin A; cells are arrested in the G1 phase (PubMed:18005237). SubAB5 at 100 ng/ml induced caspase-dependent apoptosis in Vero cells through mitochondrial membrane damage (PubMed:19380466).[1] [2] [3] [4] [5] [6] Publication Abstract from PubMedPathogenic 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. Whilst 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 is 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 (ER) 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 of other AB5 toxin family members, combined with extensive mutagenesis studies on SubB, shows 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 characterisation 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. Structural basis of subtilase cytotoxin SubAB assembly.,Le Nours J, Paton AW, Byres E, Troy S, Herdman BP, Johnson MD, Paton JC, Rossjohn J, Beddoe T J Biol Chem. 2013 Aug 6. PMID:23921389[7] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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