Graduate School of Frontier Bioscience, Osaka University, Osaka, Japan.
Biophys J. 2012 Sep 5;103(5):970-8. doi: 10.1016/j.bpj.2012.06.054.
F(1)-ATPase is an ATP-driven rotary motor that generates torque at the interface between the catalytic β-subunits and the rotor γ-subunit. The β-subunit inwardly rotates the C-terminal domain upon nucleotide binding/dissociation; hence, the region of the C-terminal domain that is in direct contact with γ-termed the DELSEED loop-is thought to play a critical role in torque transmission. We substituted all the DELSEED loop residues with alanine to diminish specific DELSEED loop-γ interactions and with glycine to disrupt the loop structure. All the mutants rotated unidirectionally with kinetic parameters comparable to those of the wild-type F(1), suggesting that the specific interactions between DELSEED loop and γ is not involved in cooperative interplays between the catalytic β-subunits. Glycine substitution mutants generated half the torque of the wild-type F(1), whereas the alanine mutant generated comparable torque. Fluctuation analyses of the glycine/alanine mutants revealed that the γ-subunit was less tightly held in the α(3)β(3)-stator ring of the glycine mutant than in the wild-type F(1) and the alanine mutant. Molecular dynamics simulation showed that the DELSEED loop was disordered by the glycine substitution, whereas it formed an α-helix in the alanine mutant. Our results emphasize the importance of loop rigidity for efficient torque transmissions.
F(1)-ATP 合酶是一种 ATP 驱动的旋转分子马达，它在催化β亚基和转子γ亚基之间的界面产生扭矩。β亚基在核苷酸结合/解离时向内旋转 C 端结构域；因此，与γ直接接触的 C 端结构域区域被认为在扭矩传递中起着关键作用。我们用丙氨酸取代了 DELSEED 环的所有残基，以减少特定的 DELSEED 环-γ相互作用，并用甘氨酸破坏环结构。所有突变体都单向旋转，其动力学参数与野生型 F(1)相当，这表明 DELSEED 环与γ之间的特定相互作用不参与催化β亚基之间的合作相互作用。甘氨酸取代突变体产生的扭矩只有野生型 F(1)的一半，而丙氨酸突变体产生的扭矩相当。甘氨酸/丙氨酸突变体的波动分析表明，与野生型 F(1)和丙氨酸突变体相比，γ亚基在甘氨酸突变体的α(3)β(3)-定子环中结合得不够紧密。分子动力学模拟表明，甘氨酸取代使 DELSEED 环失序，而在丙氨酸突变体中形成α-螺旋。我们的结果强调了环刚性对于有效扭矩传递的重要性。
