Ruddock L W, Webb H M, Ruston S P, Cheesman C, Freedman R B, Hirst T R
Research School of Biosciences, University of Kent at Canterbury, U.K.
Biochemistry. 1996 Dec 17;35(50):16069-76. doi: 10.1021/bi961865l.
The non-covalently associated B-subunit moieties of AB5 toxins, such as cholera toxin and related diarrheagenic enterotoxins, exhibit exceptional pH stability and remain pentameric at pH values as low as 2.0. Here, we investigate the structural basis of a pH-dependent conformational change which occurs within the B5 structure of Escherichia coli heat-labile enterotoxin (EtxB) at around pH 5.0. The use of far-UV CD and fluorescence spectroscopy showed that EtxB pentamers undergo a fully reversible pH-dependent conformational change with a pKa of 4.9 +/- 0.1 (R2 = 0.999) or 5.13 +/- 0.01 (R2 = 0.999), respectively. This renders the pentamer susceptible to SDS-mediated disassembly and decreases its thermal stability by 18 degrees C. A comparison of the pH-dependence of the structural change in EtxB5, with that of a mutant containing a Ser substitution at His 57, revealed that the pKa of the conformational change was shifted from ca. 5.1 to 4.4. This finding suggests that protonation of the imidazole side chain of His 57 might facilitate disruption of a spatially adjacent salt bridge, located between Glu 51 and Lys 91 in each B-subunit, thus triggering the conformational change in the pentameric structure. The pH-dependent conformational change was found to be inhibited when B-subunits bound to monosialoganglioside, GMI; and to have no effect on the stability of interaction between A- and B-subunits within the AB5 complex. This suggests that the conformational change is unlikely to have a direct involvement in toxicity. Conservation of the pH-dependent conformational change in the AB5 toxin family, combined with the potential exposure of the hydrophobic core of beta-barrel in the monomeric units, leads to the proposal that the conformational change may be the common feature that ensures the secretion of these proteins from the Vibrionaceae.
AB5毒素（如霍乱毒素和相关致腹泻性肠毒素）的非共价结合B亚基部分表现出非凡的pH稳定性，在低至2.0的pH值下仍保持五聚体状态。在此，我们研究了大肠杆菌不耐热肠毒素（EtxB）的B5结构在pH值约为5.0时发生的pH依赖性构象变化的结构基础。远紫外圆二色光谱和荧光光谱的结果表明，EtxB五聚体经历了完全可逆的pH依赖性构象变化，其pKa分别为4.9±0.1（R2 = 0.999）或5.13±0.01（R2 = 0.999）。这使得五聚体易于被SDS介导的解离，并使其热稳定性降低18℃。将EtxB5结构变化的pH依赖性与在His 57处含有Ser取代的突变体的pH依赖性进行比较，发现构象变化的pKa从约5.1移至4.4。这一发现表明，His 57的咪唑侧链质子化可能有助于破坏每个B亚基中Glu 51和Lys 91之间的空间相邻盐桥，从而引发五聚体结构的构象变化。当B亚基与单唾液酸神经节苷脂GMI结合时，发现pH依赖性构象变化受到抑制；并且对AB5复合物中A亚基和B亚基之间相互作用的稳定性没有影响。这表明构象变化不太可能直接参与毒性作用。AB5毒素家族中pH依赖性构象变化的保守性，加上单体单元中β桶疏水核心的潜在暴露，使得人们提出构象变化可能是确保这些蛋白质从弧菌科分泌的共同特征。
