Rawat Anoop, Nagaraj Ramakrishnan
Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Uppal Road, Hyderabad 500 007, India.
Biochim Biophys Acta. 2010 Oct;1798(10):1854-63. doi: 10.1016/j.bbamem.2010.06.009. Epub 2010 Jun 18.
The SH4 domain of Fyn, a member of the Src family of tyrosine kinases, though rich in polar amino acid residues, anchors to the cytosolic face of membranes upon fatty acylation. In order to probe the requirement of specific fatty acylation at the N-terminus and at the side-chain of this domain for membrane-association, we have studied the interaction of peptides corresponding to the polar segment of the SH4 domain of Fyn and its mono- and dually fatty acylated analogs with model membranes. While the polar segment without covalently linked fatty acids (KDKEATKLTEW-amide) does not interact with lipid vesicles, peptides with one covalently linked fatty acid at the N-terminus or in the side-chain, associate with zwitterionic and anionic lipids to varying degrees. The interaction of dually acylated peptides (Myr-GK(epsilon-myr)KDKEATKLTEW-amide and Myr-GC(S-pal)KDKEATKLTEW-amide) with lipids depends on the linkage between fatty acyl side-chain and peptide backbone. The peptide chain associates with membranes only when the side-chain acylation is via an amide bond and not via a thioester bond. Our investigations indicate that acylation is essential for membrane targeting and unacylated polar stretch of the SH4 domain does not have a role in membrane-anchoring. Side-chain acylation via a thioester bond not only provides membrane anchorage but also directs the peptide chain away from the bilayer which might be important to enable the full length protein to interact with other signaling partners.
Fyn是酪氨酸激酶Src家族的成员，其SH4结构域虽然富含极性氨基酸残基，但在脂肪酰化后会锚定在膜的胞质面。为了探究该结构域N端和侧链上特定脂肪酰化对于膜结合的必要性，我们研究了与Fyn的SH4结构域极性片段及其单脂肪酰化和双脂肪酰化类似物对应的肽段与模型膜的相互作用。没有共价连接脂肪酸的极性片段（KDKEATKLTEW-酰胺）不与脂质囊泡相互作用，而在N端或侧链有一个共价连接脂肪酸的肽段则与两性离子和阴离子脂质有不同程度的结合。双脂肪酰化肽段（Myr-GK(ε-myr)KDKEATKLTEW-酰胺和Myr-GC(S-pal)KDKEATKLTEW-酰胺）与脂质的相互作用取决于脂肪酰侧链与肽主链之间的连接方式。只有当侧链酰化通过酰胺键而非硫酯键时，肽链才会与膜结合。我们的研究表明，酰化对于膜靶向至关重要，SH4结构域未酰化的极性延伸在膜锚定中不起作用。通过硫酯键进行的侧链酰化不仅提供膜锚定，还会使肽链远离双层膜，这对于全长蛋白与其他信号伴侣相互作用可能很重要。
