Peggion E, Mammi S, Schievano E, Behar V, Rosenblatt M, Chorev M
Department of Organic Chemistry, Biopolymer Research Center, University of Padua, Via Marzolo 1, 35131 Padua, Italy.
Biopolymers. 1999 Oct 15;50(5):525-35. doi: 10.1002/(SICI)1097-0282(19991015)50:5<525::AID-BIP6>3.0.CO;2-8.
The N-terminal 1-34 segments of both parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences between the two hormones in their amino acid sequence. Recently, it was shown that in (1-34)PTH/PTHrP segmental hybrid peptides, the N-terminal 1-14 segment of PTHrP is incompatible with the C-terminal 15-34 region of PTH leading to substantial reduction in potency. The sites of incompatibility were identified as positions 5 in PTH and 19 in PTHrP. In the present paper we describe the synthesis, biological evaluation, and conformational characterization of two point-mutated PTH/PTHrP 1-34 hybrids in which the arginine residues at positions 19 and 21 of the native sequence of PTHrP have been replaced by valine (hybrid V(21)) and glutamic acid (hybrid E(19)), respectively, taken from the PTH sequence. Hybrid V(21) exhibits both high receptor affinity and biological potency, while hybrid E(19) binds weakly and is poorly active. The conformational properties of the two hybrids were studied in aqueous solution containing dodecylphosphocholine (DPC) micelles and in water/2,2, 2-trifluoroethanol (TFE) mixtures. Upon addition of TFE or DPC micelles to the aqueous solution, both hybrids undergo a coil-helix transition. The maximum helix content in 1 : 1 water/TFE, obtained by CD data for both hybrids, is approximately 80%. In the presence of DPC micelles, the maximum helix content is approximately 40%. The conformational properties of the two hybrids in the micellar system were further investigated by combined 2D-nmr, distance geometry (DG), and molecular dynamics (MD) calculations. The common structural motif, consisting of two helical segments located at N- and C-termini, was observed in both hybrids. However, the biologically potent hybrid V(21) exhibits two flexible sites, centered at residues 12 and 19 and connecting helical segments, while the flexibility sites in the weakly active hybrid E(19) are located at position 11 and in the sequence 20-26. Our findings support the hypothesis that the presence and location of flexibility points between helical segments are essential for enabling the active analogs to fold into the bioactive conformation upon interaction with the receptor.
甲状旁腺激素（PTH）和甲状旁腺激素相关蛋白（PTHrP）的N端1 - 34片段尽管在氨基酸序列上存在重大差异，但它们能结合并激活相同的膜受体。最近研究表明，在（1 - 34）PTH/PTHrP片段杂交肽中，PTHrP的N端1 - 14片段与PTH的C端15 - 34区域不兼容，导致活性大幅降低。不兼容位点被确定为PTH中的第5位和PTHrP中的第19位。在本文中，我们描述了两种点突变的PTH/PTHrP 1 - 34杂交肽的合成、生物学评估和构象表征，其中PTHrP天然序列中第19位和第21位的精氨酸残基分别被来自PTH序列的缬氨酸（杂交肽V(21)）和谷氨酸（杂交肽E(19)）取代。杂交肽V(21)表现出高受体亲和力和生物学活性，而杂交肽E(19)结合较弱且活性较差。在含有十二烷基磷酸胆碱（DPC）胶束的水溶液以及水/2,2,2 - 三氟乙醇（TFE）混合物中研究了这两种杂交肽的构象性质。向水溶液中加入TFE或DPC胶束后，两种杂交肽都发生了从无规卷曲到螺旋的转变。通过圆二色光谱（CD）数据得到，在1:1水/TFE中两种杂交肽的最大螺旋含量约为80%。在DPC胶束存在下，最大螺旋含量约为40%。通过二维核磁共振（2D - nmr）、距离几何（DG）和分子动力学（MD）计算相结合的方法，进一步研究了这两种杂交肽在胶束体系中的构象性质。在两种杂交肽中都观察到了由位于N端和C端的两个螺旋段组成的共同结构基序。然而，具有生物学活性的杂交肽V(21)在残基12和19处有两个柔性位点，连接着螺旋段，而活性较弱的杂交肽E(19)的柔性位点位于第11位以及20 - 26序列处。我们的研究结果支持这样的假设，即螺旋段之间柔性位点的存在和位置对于使活性类似物在与受体相互作用时折叠成生物活性构象至关重要。
