Zhorov B S, Ananthanarayanan V S
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Biophys J. 1996 Jan;70(1):22-37. doi: 10.1016/S0006-3495(96)79561-9.
Grove et al. have demonstrated L-type Ca2+ channel activity of a synthetic channel peptide (SCP) composed of four helices (sequence: DPWNVFDFLI10VIGSIIDVIL20SE) tethered by their C-termini to a nanopeptide template. We sought to obtain the optimal conformations of SCP and locate the binding sites for Ca2+ and for the dihydropyridine ligand nifedipine. Eight Ca2+ ions were added to neutralize the 16 acidic residues in the helices. Eight patterns of the salt bridges between Ca2+ ions and pairs of the acidic residues were calculated by the Monte Carlo-with-energy-minimization (MCM) protocol. In the energetically optimal conformation, two Ca2+ ions were bound to Asp-1 residues at the intracellular side of SCP, and six Ca2+ ions were arrayed in two files at the diametrically opposite sides of the pore, implying a Ca2+ relay mechanism. Nine modes of nifedipine binding to SCP were simulated by the MCM calculations. In the energetically optimal mode, the ligand fits snugly in the pore. The complex is stabilized by Ca2+ bound between two Asp-17 residues and hydrophilic groups of the ligand. The latter substitute water molecules adjacent to Ca2+ in the ligand-free pore and thus do not obstruct Ca2+ relay. The ligand-binding site is proximal to a hydrophobic bracelet of Ile-10 residues whose rotation is sterically hindered. In some conformations, the bracelet is narrow enough to block the permeation of the hydrated Ca2+ ions. The bracelet may thus act as a "gate" in SCP. Nifedipine and (R)-Bay K 8644, which act as blockers of the SCP, extend a side-chain hydrophobic moiety toward the Ile-10 residues. This would stabilize the pore-closing conformation of the gate. In contrast, the channel activator (S)-Bay K 8644 exposes a hydrophilic moiety toward the Ile-10 residues, thus destabilizing the pore-closing conformation of the gate.
格罗夫等人已经证明了一种由四个螺旋（序列：DPWNVFDFLI10VIGSIIDVIL20SE）组成的合成通道肽（SCP）的L型钙通道活性，这些螺旋通过其C末端连接到一个纳米肽模板上。我们试图获得SCP的最佳构象，并确定Ca2+和二氢吡啶配体硝苯地平的结合位点。添加了八个Ca2+离子以中和螺旋中的16个酸性残基。通过蒙特卡罗能量最小化（MCM）协议计算了Ca2+离子与成对酸性残基之间的八种盐桥模式。在能量最优构象中，两个Ca2+离子结合在SCP细胞内侧的Asp-1残基上，六个Ca2+离子以两列排列在孔的直径相对侧，这意味着存在Ca2+中继机制。通过MCM计算模拟了硝苯地平与SCP结合的九种模式。在能量最优模式下，配体紧密地契合在孔中。该复合物通过结合在两个Asp-17残基之间的Ca2+和配体的亲水基团而稳定。后者取代了无配体孔中与Ca2+相邻的水分子，因此不会阻碍Ca2+中继。配体结合位点靠近Ile-10残基的疏水环，其旋转受到空间位阻。在某些构象中，该环足够窄以阻止水合Ca2+离子的渗透。因此，该环可能在SCP中充当“门”。作为SCP阻滞剂的硝苯地平和（R）-Bay K 8644向Ile-10残基延伸侧链疏水部分。这将稳定门的孔关闭构象。相反，通道激活剂（S）-Bay K 8644向Ile-10残基暴露亲水部分，从而使门的孔关闭构象不稳定。
