OMS II, Kirksville College of Osteopathic Medicine, A.T. Still University, Kirksville, MO 63501, USA.
Department of Biochemistry, Kirksville College of Osteopathic Medicine, A.T. Still University, Kirksville, MO 63501, USA.
Int J Biol Macromol. 2018 Sep;116:977-982. doi: 10.1016/j.ijbiomac.2018.05.118. Epub 2018 May 18.
The current study establishes the necessity of Glu-381, Glu-384, and Glu-385 in the βDELSEED-motif of Escherichia coli ATP synthase for peptide binding and inhibition. Inhibitory profiles of wild type and mutant E. coli membrane bound FF ATP synthase were studied in the presence and absence of C-terminal NH bound melittin. Melittin-NH caused almost complete inhibition of wild type ATPase, while partial inhibition was observed for mutants where Glu was replaced with Ala, Arg, or Gln. Additionally, melittin-NH caused insignificant inhibition of a triple mutant where all three Glu residues were replaced with Ala residues, changing βDELSEED-motif to βDALSAAD-motif. Little or partial loss of oxidative phosphorylation in mutant strains corroborates their distinct location away from the catalytic site of ATP synthase. Moreover, abrogation of wild type E. coli cell growth and normal growth of mutant stains in the presence of melittin-NH further validated the necessity of Glu residues in the βDELSEED-motif for peptide binding. Overall, while loss of one Glu residue at a time may allow partial peptide binding, loss of three Glu residues together-βE381, βE384, and βE385-is detrimental for peptide binding and inhibition of ATP synthase.
本研究确立了大肠杆菌 ATP 合酶βDELSEED 基序中 Glu-381、Glu-384 和 Glu-385 对肽结合和抑制的必要性。在存在和不存在 C 端 NH 结合的蜂毒素的情况下，研究了野生型和突变型大肠杆菌膜结合 FF ATP 合酶的抑制谱。蜂毒素-NH 导致野生型 ATP 酶几乎完全抑制，而对于 Glu 被 Ala、Arg 或 Gln 取代的突变体，观察到部分抑制。此外，蜂毒素-NH 对所有三个 Glu 残基都被 Ala 残基取代的三重突变体几乎没有抑制作用，将βDELSEED 基序改变为βDALSAAD 基序。突变株中氧化磷酸化的减少或部分丢失与其远离 ATP 合酶催化位点的位置相符。此外，在蜂毒素-NH 的存在下，野生型大肠杆菌细胞生长的中止和突变株的正常生长进一步验证了βDELSEED 基序中 Glu 残基对肽结合的必要性。总的来说，虽然一次损失一个 Glu 残基可能允许部分肽结合，但一次损失三个 Glu 残基（βE381、βE384 和βE385）对肽结合和 ATP 合酶抑制是有害的。
