Baxter Daniel, Perry Samuel R, Hill Timothy A, Kok W Mei, Zaccai Nathan R, Brady R Leo, Fairlie David P, Mason Jody M
Department of Biology & Biochemistry, University of Bath , Claverton Down, Bath BA2 7AY, U.K.
Division of Chemistry and Structural Biology, Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Institute for Molecular Bioscience, University of Queensland , Brisbane, Queensland 4072, Australia.
ACS Chem Biol. 2017 Aug 18;12(8):2051-2061. doi: 10.1021/acschembio.7b00303. Epub 2017 Jun 21.
The oncogenic transcription factor activator protein-1 (AP-1) is a DNA-binding protein that assembles through dimerization of Fos and Jun protein subunits, their leucine-rich helical sequences entwining into a coiled-coil structure. This study reports on downsizing the proto-oncogene cFos protein (380 residues) to shorter peptides (37-25 residues) modified with helix-inducing constraints to enhance binding to Jun. A crystal structure is reported for a 37-residue Fos-derived peptide (FosW) bound to Jun. This guided iterative downsizing of FosW to shorter peptide sequences that were constrained into stable water-soluble α-helices by connecting amino acid side chains to form cyclic pentapeptide components. Structural integrity in the presence and absence of Jun was assessed by circular dichroism spectroscopy, while the thermodynamics of binding to cFos was measured by isothermal titration calorimetry. A 25-residue constrained peptide, one-third shorter yet 25% more helical than the structurally characterized 37-residue Fos-derived peptide, retained 80% of the binding free energy as a result of preorganization in a Jun-binding helix conformation, with the entropy gain (TΔS = +3.2 kcal/mol) compensating for the enthalpy loss. Attaching a cell-penetrating peptide (TAT) and a nuclear localization signal (SV40) promoted cell uptake, localization to the nucleus, and inhibition of the proliferation of two breast cancer cell lines.
致癌转录因子激活蛋白-1（AP-1）是一种DNA结合蛋白，它通过Fos和Jun蛋白亚基的二聚化组装而成，其富含亮氨酸的螺旋序列缠绕形成卷曲螺旋结构。本研究报道了将原癌基因cFos蛋白（380个残基）缩减为较短的肽段（37 - 25个残基），这些肽段通过诱导螺旋的限制修饰来增强与Jun的结合。报道了一种与Jun结合的37个残基的Fos衍生肽（FosW）的晶体结构。这指导了FosW迭代缩减为更短的肽序列，这些序列通过连接氨基酸侧链形成环状五肽成分而被约束成稳定的水溶性α螺旋。通过圆二色光谱评估有无Jun时的结构完整性，同时通过等温滴定量热法测量与cFos结合的热力学。一种25个残基的约束肽，比结构表征的37个残基的Fos衍生肽短三分之一，但螺旋度高25%，由于预先形成了与Jun结合的螺旋构象，保留了80%的结合自由能，熵增（TΔS = +3.2 kcal/mol）补偿了焓减。连接细胞穿透肽（TAT）和核定位信号（SV40）促进了细胞摄取、细胞核定位以及对两种乳腺癌细胞系增殖的抑制。
