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oai:figshare.com:article/33868576 2026-09-16T23:04:39Z category_1 category_4 category_8 category_14 category_15 category_21 category_873 category_46 category_734 category_64 category_69 category_132 portal_63 item_type_2 month_year_09_2026

A Single Amino Acid in the A2 Linker of Cholera Toxin Affects the Efficiency of Toxin Disassembly and Resulting Toxicity Flore Kersten (17973191) Natalia Mojica (17973188) Albert Serrano (24999718) Joel B. Heim (24999721) Jessica L. Guyette (24999724) Henrique Musseli Cezar (18974200) Gabriele Cordara (2267719) Suren A. Tatulian (208721) Michele Cascella (119841) Ute Krengel (2267728) Ken Teter (208722) Biophysics Biochemistry Microbiology Molecular Biology Neuroscience Biotechnology Chemical Sciences not elsewhere classified Immunology Biological Sciences not elsewhere classified Cancer Inorganic Chemistry Infectious Diseases two proteins bind shared gsα target protein disulfide isomerase highly related ab higher conformational freedom enzymatic activity toward enterotoxigenic escherichia coli collective data uncover 6 å resolution 11 amino acids single amino acid molecular dynamics simulations cholera toxin affects glutamic acid 229 determined crystal structures catalytic a1 subunit driven toxin disassembly a2 linker exits d229e </ sub a1 subunit aspartic acid toxin disassembly structural dynamics position 229 a2 linker toxin variants toxin folding vibrio cholerae similar levels previous hypothesis less efficient labile enterotoxin key contributor intoxication process h233y ). gm1 receptor four residues essential event critical role binding homopentamer Cholera toxin (CT) from Vibrio cholerae and heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli are highly related AB₅-type protein toxins that contain a catalytic A1 subunit, an A2 linker, and a cell-binding homopentamer of B subunits. The two proteins bind the same GM1 receptor and have similar levels of enzymatic activity toward their shared Gsα target, yet CT is more potent than LT. This difference has been mapped to a sequence of 11 amino acids (residues 226–236) near the C-terminus of the A2 linker. We now identify a single amino acid, aspartic acid at position 229, as a key contributor to the function of CT: substituting it for glutamic acid 229 from LT reduced the toxicity of CT to the level of LT. Toxin disassembly by protein disulfide isomerase (PDI), which is an essential event in the intoxication process, was less efficient for CT_D229E and LT than for wild-type CT. This was further confirmed by a CT variant, in which four residues were converted into the LT sequence (D229E, I230V, T232I, and H233Y). To test our previous hypothesis that the positioning of the A1 subunit over the B pentamer critically determines toxin disassembly and thus potency, we determined crystal structures of both toxin variants to 1.6 Å resolution and calculated the angle at which the A2 linker exits the pore. The conformations of the two toxin chimeras lie between CT and LT, with the A2 C-terminus of the CT quadruple variant adopting essentially the same fold as LT. The A subunits of the toxin variants as well as that of LT showed a higher conformational freedom than CT as determined by molecular dynamics simulations, suggesting that a more rigid positioning of the A subunit drives toxin disassembly and potency. Our collective data uncover the critical role of D229 in toxin folding and structural dynamics, which in turn impacts toxin potency through effects on the efficiency of PDI-driven toxin disassembly. 2026-09-16T00:00:00Z Dataset Media 10.1021/acsomega.6c07022.s003 https://figshare.com/articles/media/A_Single_Amino_Acid_in_the_A2_Linker_of_Cholera_Toxin_Affects_the_Efficiency_of_Toxin_Disassembly_and_Resulting_Toxicity/33868576 CC BY-NC 4.0