8G0F

Crystal structure of diphtheria toxin H223Q/H257Q double mutant (pH 5.5)

8G0F の概要

• エントリーDOI: 10.2210/pdb8g0f/pdb
• 分子名称: Diphtheria toxin (2 entities in total)
• 機能のキーワード: diptheria toxin, ph dependent conformational changes, toxin
• 由来する生物種: Corynebacterium diphtheriae
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 117449.59

構造登録者

Lovell, S.,Kashipathy, M.M.,Battaile, K.P.,Ladokhin, A.S. (登録日: 2023-01-31, 公開日: 2023-07-05, 最終更新日: 2024-11-13)

主引用文献

Rodnin, M.V.,Vasques-Montes, V.,Kyrychenko, A.,Oliveira, N.F.B.,Kashipathy, M.M.,Battaile, K.P.,Douglas, J.,Lovell, S.,Machuqueiro, M.,Ladokhin, A.S.
Histidine Protonation and Conformational Switching in Diphtheria Toxin Translocation Domain.
Toxins, 15:-, 2023

PubMed Abstract: Protonation of key histidine residues has been long implicated in the acid-mediated cellular action of the diphtheria toxin translocation (T-) domain, responsible for the delivery of the catalytic domain into the cell. Here, we use a combination of computational (constant-pH Molecular Dynamics simulations) and experimental (NMR, circular dichroism, and fluorescence spectroscopy along with the X-ray crystallography) approaches to characterize the initial stages of conformational change happening in solution in the wild-type T-domain and in the H223Q/H257Q double mutant. This replacement suppresses the acid-induced transition, resulting in the retention of a more stable protein structure in solutions at pH 5.5 and, consequently, in reduced membrane-disrupting activity. Here, for the first time, we report the pK values of the histidine residues of the T-domain, measured by NMR-monitored pH titrations. Most peaks in the histidine side chain spectral region are titrated with pKs ranging from 6.2 to 6.8. However, the two most up-field peaks display little change down to pH 6, which is a limiting pH for this protein in solution at concentrations required for NMR. These peaks are absent in the double mutant, suggesting they belong to H223 and H257. The constant-pH simulations indicate that for the T-domain in solution, the pK values for histidine residues range from 3.0 to 6.5, with those most difficult to protonate being H251 and H257. Taken together, our experimental and computational data demonstrate that previously suggested cooperative protonation of all six histidines in the T-domain does not occur.

PubMed: 37505680
DOI: 10.3390/toxins15070410

実験手法

X-RAY DIFFRACTION (2.25 Å)