EUROISMAR 2019

Cholera is an acute, diarrheal illness caused by infection of the intestine by the bacterium Vibrio cholerae. An estimated 2.9 million cases and 95,000 deaths occur each year around the world. During infection, the bacterium releases a toxin. The cholera toxin is an oligomeric complex made up of six protein subunits: one copy of the enzymatic A subunit and five copies of the receptor binding B subunit (CTB). Cholera toxin has been suggested to bind to cells in the human intestine via the GM1 ganglioside. The x-ray structures of cholera toxin in the apo form, with the bound GM1 ligand as well as the structure of the highly homologous heat-labile enterotoxin (LT) are known. 
We used NMR as a tool to study the CTB protein-ligand interaction. A prerequisite is backbone and preferably side chain assignments. Some kind of protonated samples are required and we used uniformly {1H,13C,15N} CTB , expressed in Vibrio cholera. Direct heteronuclear (13C and 15N) 2D and 3D experiments (2D NH, 2D NCA, 2D NCO, 3D HCACO) (5 mm TXO / 800 MHz) were compared to 1H-detected standard triple resonance experiments (3mm TCI / 800 MHz) when we obtained the complete backbone and methyl group assignment of the pentameric apo-CTB as well as the GM1-CTB. Differential scanning calorimetry shows two transitions at 65° C and 82° C, respectively. At 45° C, the estimated rotational correlation time is sufficiently fast to allow observation of most of the Cb signals in backbone experiments. Chemical shift perturbation on ligand binding indicates an allosteric mechanism in accordance with previously reported ligand binding cooperativity. The backbone dynamics (15N NOE, T1 and T2 relaxation as well as T2 relaxation dispersion) was analyzed for both CTB-apo and CTB-GM1 forms in order to correlate changes in dynamics with allosteric changes on ligand binding.