Soteropoulos P, Valiakhmetov A, Kashiwazaki R, Perlin D S
Public Health Research Institute, New York, New York 10016, USA.
J Biol Chem. 2001 May 11;276(19):16265-70. doi: 10.1074/jbc.M011115200. Epub 2001 Feb 5.
The stalk segments of P-type ion-translocating enzymes are presumed to play important roles in energy coupling. In this work, stalk segments S4 and S5 of the yeast H(+)-ATPase were examined for helical character, optimal length, and segment orientation by a combination of proline substitution, insertion/deletion mutagenesis, and second-site suppressor analyses. The substitution of various residues for helix-disrupting proline in both S4 (L353P,L353G; A354P; and G371P) and S5 (D676P and I684P) resulted in highly defective or inactive enzymes supporting the importance of helical character and/or the maintenance of essential interactions. The contiguous helical nature of transmembrane segment M5 and stalk element S5 was explored and found to be favorable, although not essential. The deletion or addition of one or more amino acids at positions Ala(354) in S4 and Asp(676) in S5, which were intended to either rotate helical faces or extend/reduce the length of helical segments, resulted in enzyme destabilization that abolished most enzyme assembly. Second-site suppressor mutations were obtained to primary site mutations G371A (S4) and D676G (S5) and were analyzed with a molecular structure model of the H(+)-ATPase. Primary site mutations were predicted to alter the site of phosphorylation either directly or indirectly. The suppressor mutations either directly changed packing around the primary site or altered the environment of the site of phosphorylation. Overall, these data support the view that stalk segments S4 and S5 of the H(+)-ATPase are helical elements that are optimized for length and interactions with other stalk elements and can influence the phosphorylation domain.
P型离子转运酶的柄段被认为在能量偶联中发挥重要作用。在本研究中，通过脯氨酸取代、插入/缺失诱变和第二位点抑制分析相结合的方法，对酵母H(+)-ATP酶的柄段S4和S5的螺旋特性、最佳长度和片段方向进行了研究。在S4（L353P、L353G；A354P；和G371P）和S5（D676P和I684P）中，用各种残基取代破坏螺旋的脯氨酸，导致酶高度缺陷或无活性，这支持了螺旋特性和/或维持必要相互作用的重要性。尽管并非必需，但对跨膜段M5和柄元件S5的连续螺旋性质进行了探索，发现是有利的。在S4的Ala(354)和S5的Asp(676)位置缺失或添加一个或多个氨基酸，旨在旋转螺旋面或延长/缩短螺旋段的长度，导致酶不稳定，从而消除了大多数酶的组装。获得了针对主要位点突变G371A（S4）和D676G（S5）的第二位点抑制突变，并使用H(+)-ATP酶的分子结构模型进行了分析。预测主要位点突变会直接或间接改变磷酸化位点。抑制突变要么直接改变主要位点周围的堆积，要么改变磷酸化位点的环境。总体而言，这些数据支持这样一种观点，即H(+)-ATP酶的柄段S4和S5是螺旋元件，其长度和与其他柄元件的相互作用已得到优化，并可影响磷酸化结构域。
