Zhang Y L, Marepalli H R, Lu H F, Becker J M, Naider F
Department of Chemistry, The College of Staten Island, The Graduate School of The City University of New York 10314, USA.
Biochemistry. 1998 Sep 8;37(36):12465-76. doi: 10.1021/bi980787u.
Biochemical and biophysical investigations on the Saccharomyces cerevisiae alpha-factor indicate that this tridecapeptide mating pheromone (WHWLQLKPGQPMY) might adopt a type II beta-turn in the center of the peptide when it binds to its G protein-coupled receptor. To test this hypothesis we synthesized analogues of alpha-factor incorporating a (R or S)-gamma-lactam conformational constraint [3-(R or S)-amino-2-oxo-1-pyrrolidineacetamido] in place of the Pro-Gly at residues 8 and 9 of the peptide and tested their biological activities and receptor binding. Analogues were purified to >99% homogeneity as evidenced by high-performance liquid chromatography and capillary electrophoresis and characterized by amino acid analysis, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. The restricted alpha-factor analogue WHWLQLK[(R)-gamma-lactam]QP[Nle]Y was more active than its lactam-containing diastereomeric homologue WHWLQLK[(S)-gamma-lactam]QP[Nle]Y and about equally active with the [Nle12]-alpha-factor in growth arrest and FUS1-lacZ gene induction assays. Both lactam analogues competed with tritiated [Nle12]-alpha-factor for binding to the alpha-factor receptor (Ste2p) with the (R)-gamma-lactam-containing peptide having 7-fold higher affinity than the (S)-gamma-lactam-containing homologue. Two-dimensional NMR spectroscopy and modeling analysis gave evidence that the (R)-gamma-analogue is a flexible peptide that assumes a transient gamma-turn structure around the lactam moiety. The results represent the first example of an alpha-factor analogue containing a peptidomimetic constraint that is as active as the native pheromone. The correlation between activity and structure provides further evidence that the biologically active conformation of the molecule contains a turn in the middle of the pheromone. This study provides new insights into the structural basis of alpha-factor activity and adds to the repertoire of conformationally biasing constraints that can be used to maintain and even enhance biological activity in peptide hormones.
对酿酒酵母α-因子的生化和生物物理研究表明，这种十三肽交配信息素（WHWLQLKPGQPMY）在与G蛋白偶联受体结合时，可能在肽的中心形成II型β-转角。为了验证这一假设，我们合成了α-因子的类似物，在肽的第8和9位残基处用（R或S）-γ-内酰胺构象限制基团[3-（R或S）-氨基-2-氧代-1-吡咯烷乙酰胺]取代Pro-Gly，并测试了它们的生物活性和受体结合能力。通过高效液相色谱和毛细管电泳证明类似物的纯度>99%，并通过氨基酸分析、质谱和核磁共振（NMR）光谱进行表征。受限的α-因子类似物WHWLQLK[（R）-γ-内酰胺]QP[Nle]Y比其含内酰胺的非对映异构体同系物WHWLQLK[（S）-γ-内酰胺]QP[Nle]Y更具活性，并且在生长抑制和FUS1-lacZ基因诱导试验中与[Nle12]-α-因子的活性大致相同。两种内酰胺类似物都能与氚标记的[Nle12]-α-因子竞争结合α-因子受体（Ste2p），含（R）-γ-内酰胺的肽的亲和力比含（S）-γ-内酰胺的同系物高7倍。二维NMR光谱和建模分析表明，（R）-γ-类似物是一种柔性肽，在酰胺部分周围呈现瞬态γ-转角结构。这些结果代表了含有拟肽限制基团且活性与天然信息素相当的α-因子类似物的首个实例。活性与结构之间的相关性进一步证明，该分子的生物活性构象在信息素中间含有一个转角。这项研究为α-因子活性的结构基础提供了新的见解，并增加了可用于维持甚至增强肽类激素生物活性的构象偏向限制基团的种类。
