Esposito Cinzia, Scrima Mario, Carotenuto Alfonso, Tedeschi Annamaria, Rovero Paolo, D'Errico Gerardino, Malfitano Anna Maria, Bifulco Maurizio, D'Ursi Anna Maria
Department of Pharmaceutical Sciences, University of Salerno, I-84084 Fisciano, Italy.
Biochemistry. 2008 Jan 8;47(1):308-19. doi: 10.1021/bi701474t. Epub 2007 Dec 13.
2,3'-Cyclic nucleotide-3'-phosphodiesterase (CNP) is a myelin-associated protein, an enzyme abundantly present in the central nervous system of mammals and some vertebrates. In vitro, CNP specifically catalyzes the hydrolysis of 2',3'-cyclic nucleotides to produce 2'-nucleotides, but the physiologically relevant in vivo substrate is still unknown. Recently, it was found that CNP is a possible linker protein between microtubules and the plasma membranes. Since CNP is modified post-translationally by an isoprenylation process at its C terminus, the prenylation is hypothesized to be a requisite process, which permanently anchors CNP to the plasma membrane. This study investigates the molecular mechanism of the interaction between CNP and the plasma membrane, proposing a general model to interpret the structural bases of prenylated proteins binding to the membrane. A 13 residue, C-terminal CNP fragment, C13, was demonstrated to be directly responsible for CNP membrane anchoring. C13 and its lipidated derivative (LIPO-C13) were subjected to conformational analysis in membrane mimetic environments, by means of CD and NMR spectroscopies. The orientation of C13 in relation to the membrane was investigated by NMR and EPR spin labeling studies. Our structural investigation shows that the presence of the lipidic tail is essential for the peptide to be folded and correctly positioned on the membrane surface. A general model is proposed in which the post-translational lipidation is an important biomolecular trick to enlarge the hydrophobic surface and to enable the contact of the protein with membrane.
2,3'-环核苷酸-3'-磷酸二酯酶（CNP）是一种髓鞘相关蛋白，是一种在哺乳动物和一些脊椎动物的中枢神经系统中大量存在的酶。在体外，CNP特异性催化2',3'-环核苷酸水解生成2'-核苷酸，但体内生理相关底物仍不清楚。最近，发现CNP是微管与质膜之间可能的连接蛋白。由于CNP在其C末端通过异戊二烯化过程进行翻译后修饰，因此推测异戊二烯化是一个必要过程，它将CNP永久锚定在质膜上。本研究调查了CNP与质膜相互作用的分子机制，提出了一个通用模型来解释异戊二烯化蛋白与膜结合的结构基础。一个由13个残基组成的C末端CNP片段C13被证明直接负责CNP的膜锚定。通过圆二色光谱和核磁共振光谱对C13及其脂化衍生物（LIPO-C13）在模拟膜环境中进行构象分析。通过核磁共振和电子顺磁共振自旋标记研究调查了C13相对于膜的取向。我们的结构研究表明，脂尾的存在对于肽在膜表面折叠和正确定位至关重要。提出了一个通用模型，其中翻译后脂化是一种重要的生物分子技巧，可扩大疏水表面并使蛋白质与膜接触。
