Department of Biophysical Chemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakitamachi, Higashinada-ku, Kobe 658-8558, Japan.
Biochemistry. 2009 Mar 24;48(11):2529-37. doi: 10.1021/bi802317v.
The tertiary structures of human and mouse apolipoprotein A-I (apoA-I) are comprised of an N-terminal helix bundle and a separate C-terminal domain. To define the possible intramolecular interaction between the N- and the C-terminal domains, we examined the effects on protein stability and lipid-binding properties of exchanging either the C-terminal domain or helix between human and mouse apoA-I. Chemical denaturation experiments demonstrated that replacement of the C-terminal domain or helical segment in human apoA-I with the mouse counterparts largely destabilizes the N-terminal helix bundle. Removal of the C-terminal domain or alpha-helix in human apoA-I had a similar effect on the destabilization of the helix bundle against urea denaturation, indicating that the C-terminal helical segment mainly contributes to stabilizing the N-terminal helix bundle structure in the apoA-I molecule. Consistent with this, KI quenching experiments indicated that removal or replacement of the C-terminal domain or helix in human apoA-I causes Trp residues in the N-terminal domain to become exposed to solvent. Measurements of the heats of binding to egg phosphatidylcholine (PC) vesicles and the kinetics of solubilization of dimyristoyl PC vesicles demonstrated that the destabilized human N-terminal helix bundle can strongly interact with lipids without the hydrophobic C-terminal helix. In addition, site-specific labeling of the N- and C-terminal helices by acrylodan to probe the conformational stability and the spatial proximity of the two domains indicated that the C-terminal helix is located near the N-terminal helix bundle, leading to a relatively less solvent-exposed, more organized conformation of the C-terminal domain. Taken together, these results suggest that interaction between the N- and C-terminal tertiary structure domains in apoA-I modulates the stability and lipid-binding properties of the N-terminal helix bundle.
人载脂蛋白 A-I（apoA-I）和鼠载脂蛋白 A-I 的三级结构均由 N 端螺旋束和独立的 C 端结构域组成。为了确定 N 端和 C 端结构域之间可能存在的分子内相互作用，我们研究了在人 apoA-I 中交换 C 端结构域或螺旋时对蛋白质稳定性和脂质结合特性的影响。化学变性实验表明，用鼠 apoA-I 的 C 端结构域或螺旋片段置换人 apoA-I 的 C 端结构域或螺旋片段在很大程度上使 N 端螺旋束不稳定。人 apoA-I 的 C 端结构域或α螺旋的缺失对尿素变性导致的螺旋束失稳也有类似的影响，表明 C 端螺旋片段主要有助于稳定 apoA-I 分子中 N 端螺旋束结构。与此一致的是，KI 猝灭实验表明，人 apoA-I 的 C 端结构域或螺旋的缺失或置换导致 N 端结构域中的色氨酸残基暴露于溶剂中。与卵磷酯（PC）囊泡结合的热测量和二肉豆蔻酰 PC 囊泡的溶解动力学表明，不稳定的人 N 端螺旋束可以在没有疏水性 C 端螺旋的情况下与脂质强烈相互作用。此外，用丙烯酰胺对 N 端和 C 端螺旋进行定点标记以探测两个结构域的构象稳定性和空间接近性的实验表明，C 端螺旋位于 N 端螺旋束附近，导致 C 端结构域的溶剂暴露度降低，构象更有序。综上所述，这些结果表明 apoA-I 中 N 端和 C 端三级结构域之间的相互作用调节了 N 端螺旋束的稳定性和脂质结合特性。
