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首页> 外文期刊>Biochemistry >Structure-Function Studies of Analogues of Parathyroid Hormone (PTH)-1-34 Containing #beta#-Amino Acid Residues in Positions 11-13
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Structure-Function Studies of Analogues of Parathyroid Hormone (PTH)-1-34 Containing #beta#-Amino Acid Residues in Positions 11-13

机译：11-13位含有＃beta＃-氨基酸残基的甲状旁腺激素（PTH）-1-34类似物的结构功能研究

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摘要

The 1-34 N-terminal fragments of human parathyroid hormone (PTH) and PTH-related protein (PTHrP) elicit the full spectrum of bone-relevant activities characteristic of the intact hormones. The structural elements believed to be reqired for receptor binding are biological activity are two helical segments, one N-termina and one C-terminal, connected by hinges or flexible points located around positions 12 and 19. To test this hypothesis, we synthesized and characterized the following analogues of PTH-(1-34), each containing single or double substitutions with #beta#-amino acid residues around the putative hinge located at position 12: I. [Nle~(8,18),#beta#-Ala~(11,12), Nal~23,Tyr~34]bPTH-(1-34)NH_2; II. [Nle~(8,18),#beta#-Ala~(12,13), Nal~23,Tyr~34]bPTH-(1-34)NH_2; III.[Nle~(8,18),#beta#-Ala~11,Nal~23,Tyr~34]bPTH-(1-34)NH_2; IV.[Nle~(8,18),#beta#-hLeu~11,Nal~23,Tyr~34]bPTH-(1-34)NH_2; V.[Nle~(8,18),#beta#-Ala~12,Nal~23,Tyr~34]bPTH-(1-34)NH_2;VI.[Nle~(8,18),#beta#-Ala~13,Nal~23,Tyr~34]bPTH-(1-34)NH_2 (#beta#-hLeu = #beta#-homo-leucine; #beta#-Ala = #beta#-alanine; Nal = L-2-naphtyl-alanine; Nle = norleucine). Analogues I and III exhibit very low binding affinity and are devoid of adenylyl cyclase activity. Analogue II, despite its very low binding capacity is an agonist. Biological activity and binding capacity are partially restored in analgoue IV, and completely restored in analogues V and VI. The conformational properties of the analogues were investigated in aqueous solution containing dodecylphosphocholine (DPC) micelles as a membrane-mimetic environment using CD, 2D-NMR, and molecular dynamics calculations. All peptides fold partially into the #alpha#-helical conformation in the presence of DPC micells, with a miximum helix content in the range of 30-35%. NMR analysis reveals the presence of two helical segments, one N-terminal and one C-terminal, as a common structural motif in all analogues. Incorporation of #beta#-Ala dyads at positions 11,12 and 12, 13 in analogues I and II, respectively, enhances the conformational disorder in this portion of the sequence but also destabilizes the N-terminal helix. This could be one of the possible reasons for the lack of biological activity in these analogues. The partial recovery of binding affinity and biological activity in analgoue IV, compared to the structurally similar analogue III, is clearly the consequence of the reintroduction of Leu side-chain of the native sequence. In the fully active analogues V and VI, the helix stability at the N-terminus is further incerased. Taken together, these results stress the functional importance of the conformatinal stability of the helical activation domain in PTH-(1-34). Contrary to expectation, insertion of a single #beta#-amino acid residue in positions 11, 12, or 13 in analgoues III-VI does not favor a disordered structure in this portion of the sequence.
机译：人甲状旁腺激素（PTH）和PTH相关蛋白（PTHrP）的1-34 N端片段引发了完整激素特有的骨相关活动的全谱图。被认为需要与受体结合的结构元件是生物学活性，是两个螺旋段，一个N末端和一个C末端，通过位于位置12和19周围的铰链或柔性点连接。为检验该假设，我们合成并表征了PTH-（1-34）的以下类似物，每个类似物包含位于位置12的推定铰链周围的＃beta＃-氨基酸残基的单取代或双取代：I。[Nle〜（8,18），＃beta＃- Ala〜（11,12），Nal〜23，Tyr〜34] bPTH-（1-34）NH_2;二。 [Nle〜（8,18），＃beta＃-Ala〜（12,13），Nal〜23，Tyr〜34] bPTH-（1-34）NH_2; III。[Nle〜（8,18），＃beta＃-Ala〜11，Nal〜23，Tyr〜34] bPTH-（1-34）NH_2; IV。[Nle〜（8,18），＃beta＃-hLeu〜11，Nal〜23，Tyr〜34] bPTH-（1-34）NH_2; V. [Nle〜（8,18），＃beta＃-Ala〜12，Nal〜23，Tyr〜34] bPTH-（1-34）NH_2; VI。[Nle〜（8,18），＃beta＃ -Ala〜13，Nal〜23，Tyr〜34] bPTH-（1-34）NH_2（＃beta＃-hLeu =＃beta＃-homo-leucine;＃beta＃-Ala =＃beta＃-丙氨酸; Nal = L-2-萘基丙氨酸; Nle ＝正亮氨酸）。类似物I和III表现出非常低的结合亲和力，并且没有腺苷酸环化酶活性。类似物II，尽管其结合能力很低，却是激动剂。生物活性和结合能力在模拟IV中部分恢复，而在类似物V和VI中完全恢复。使用CD，2D-NMR和分子动力学计算方法，在含有十二烷基磷酸胆碱（DPC）胶束的水溶液中模拟类似物的构象特性，从而模拟膜环境。在DPC胶束存在下，所有肽均部分折叠成＃alpha＃-螺旋构象，混合螺旋含量在30-35％的范围内。 NMR分析显示存在两个螺旋区段，一个N末端和一个C末端，作为所有类似物中的共同结构基序。分别在类似物I和II中的位置11、12和12、13处掺入＃beta＃-Ala二聚体，增强了该序列的该部分中的构象障碍，但也使N末端螺旋不稳定。这可能是这些类似物中缺乏生物活性的可能原因之一。与结构类似的类似物III相比，在类似物IV中结合亲和力和生物学活性的部分恢复显然是天然序列的Leu侧链重新引入的结果。在完全活性的类似物V和VI中，进一步确定了N末端的螺旋稳定性。综上所述，这些结果强调了在PTH-（1-34）中螺旋激活域的构象稳定性的功能重要性。与预期相反，在肛门III-VI的11、12或13位插入单个＃β＃-氨基酸残基在该序列的该部分中不利于无序结构。

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