Peptide inhibitors use two related mechanisms to alter the apparent calcium affinity of the sarcoplasmic reticulum calcium pump
Peptide inhibitors use two related mechanisms to alter the apparent calcium affinity of the sarcoplasmic reticulum calcium pump
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DOI：
10.1021/bi800880q
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发表时间：
2008-09-09
期刊：
影响因子：
2.9
通讯作者：
Young, Howard S.
中科院分区：
文献类型：
--
作者：
Afara, Michael R.;Trieber, Catharine A.;Young, Howard S.
受磷蛋白（PLB）的一级序列为肌浆网钙ATP酶（SERCA）肽抑制剂的合理设计提供了模板。 PLB的跨膜结构域中极性残基很少，只有一个是必需的(Asn(34))。我们之前使用合成肽研究了 Asn34 在简单疏水性跨膜肽中的作用。在此，我们提出 Asn 在 SERCA 抑制中的作用是位置敏感的并且依赖于疏水残基的分布。为了检验这一假设，我们基于 24 个氨基酸的聚丙氨酸序列合成了一系列跨膜肽，该序列具有交替的 Leu-A1a 序列 (Leu(12)) 或 PLB 中天然位置的亮氨酸残基 (Leu(9))。含有 Asn 的 Leu(9) 和 Leu12 肽是用单个 Asn 残基合成的，该残基位于一个氨基酸 (N +/- 1) 或从其天然位置沿任一方向的一圈螺旋 (N +/- 4)。将这些肽与 SERCA 共重构为脂蛋白体，揭示了对与 Asn 和 Leu 残基位置相关的 SERCA 表观钙亲和力和协同性的影响。正如 Hill 系数所示，最具抑制性的肽增加了 SERCA 的协同性，表明钙依赖性可逆性是抑制机制的固有部分。动力学模拟与肽和 SERCA 之间相互作用的分子模型相结合，揭示了两种相关的抑制机制。类似于 PLB 的肽使用相同的抑制机制，而与 PLB 更不同的肽会改变钙转运循环中的额外步骤。
The primary sequence of phospholamban (PLB) has provided a template for the rational design of peptide inhibitors of the sarcoplasmic reticulum calcium ATPase (SERCA). In the transmembrane domain of PLB, there are few polar residues and only one is essential (Asn(34)). Using synthetic peptides, we have previously investigated the role of Asn34 in the context of simple hydrophobic transmembrane peptides. Herein we propose that the role of Asn in SERCA inhibition is position-sensitive and dependent upon the distribution of hydrophobic residues. To test this hypothesis, we synthesized a series of transmembrane peptides based on a 24 amino acid polyalanine sequence having either an alternating Leu-A1a sequence (Leu(12)) or Leu residues at the native positions found in PLB (Leu(9)). Asn-containing Leu(9) and Leu12 peptides were synthesized with a single Asn residue located either one amino acid (N +/- 1) or one turn of the helix (N +/- 4) in either direction from its native position. Co-reconstitution of these peptides with SERCA into proteoliposomes revealed effects on the apparent calcium affinity and cooperativity of SERCA that correlated with the positions of the Asn and Leu residues. The most inhibitory peptides increased the cooperativity of SERCA as indicated by the Hill coefficients, suggesting that calcium-dependent reversibility is an inherent part of the inhibitory mechanism. Kinetic simulations combined with molecular modeling of the interaction between the peptides and SERCA reveal two related mechanisms of inhibition. Peptides that resemble PLB use the same inhibitory mechanism, whereas peptides that are more divergent from PLB alter an additional step in the calcium transport cycle.