Ananthanarayanan V S, Saint-Jean A, Cheesman B V, Hughes D W, Bain A D
Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
J Biomol Struct Dyn. 1993 Dec;11(3):509-28. doi: 10.1080/07391102.1993.10508012.
With a view to understanding the structural requirement for tyrosine phosphorylation, we have examined the free and Ca(2+)-bound conformations of the synthetic peptide tBoc-Leu-Pro-Tyr-Ala-NHCH3, a substrate for a protein tyrosine kinase, using circular dichroism (CD), 1H and 13C nuclear magnetic resonance (NMR) and molecular modeling methods. CD spectrum of the free peptide in water showed a random coil structure, while the spectrum in acetonitrile was indicative of a folded structure containing a type III beta-turn. Dihedral angle data derived from JNH-CH coupling constants, as well as two-dimensional 1H-COSY and NOESY spectral analyses, showed that the peptide adopts a conformation close to the 3(10)-helix. Ca2+ binding by the peptide, as monitored by CD spectral changes, was quite weak in water. However, substantial CD spectral changes were observed in the peptide on addition of Ca2+ in acetonitrile suggestive of major conformational alterations due to Ca2+ binding. Analysis of the binding isotherms at 25 degrees C obtained from CD data in acetonitrile indicated a 2:1 peptide:Ca2+ ("sandwich") complex to be the dominant species with a Kd of about 30 microM. A 1:1 complex was also present and became significant at Ca2+:peptide ratios above 1. By comparison, the peptide formed a predominantly 1:1 complex with Mg2+ with a Kd of about 40 microM. 13C-NMR data showed that a mixture of cis and trans conformers (arising from rotation around the Leu-Pro bond) in the free peptide changes over to the all-trans form on coordination of the peptide carbonyl groups to the Ca2+ ion. 1H-NOESY data of the Ca2+ complex revealed several interactions involving the sidechains of two peptide molecules in the sandwich. Molecular modeling and energy minimization with and without the input of NOESY-derived distance constraints showed the sandwich complex to be an energetically very favourable conformation. Besides its relevance in terms of the possible involvement of divalent cations in substrate-tyrosine kinase interaction, the conformational characterization of tBoc-Leu-Pro-Tyr-Ala-NHCH3 and its Ca2+ complex should help understand the conformational determinants for Ca(2+)-binding by linear peptides.
为了理解酪氨酸磷酸化的结构要求，我们使用圆二色性（CD）、1H和13C核磁共振（NMR）以及分子建模方法，研究了合成肽tBoc-Leu-Pro-Tyr-Ala-NHCH3（一种蛋白质酪氨酸激酶的底物）的游离和Ca(2+)结合构象。游离肽在水中的CD光谱显示为无规卷曲结构，而在乙腈中的光谱表明存在包含III型β-转角的折叠结构。由JNH-CH耦合常数以及二维1H-COSY和NOESY光谱分析得出的二面角数据表明，该肽采用接近3(10)-螺旋的构象。通过CD光谱变化监测，肽在水中与Ca2+的结合非常弱。然而，在乙腈中加入Ca2+后，肽的CD光谱发生了显著变化，这表明由于Ca2+结合导致了主要的构象改变。对从乙腈中的CD数据获得的25℃下的结合等温线分析表明，2:1的肽:Ca2+（“三明治”）复合物是主要物种，其Kd约为30μM。也存在1:1的复合物，并且在Ca2+:肽比例高于1时变得显著。相比之下，该肽与Mg2+形成主要为1:1的复合物，其Kd约为40μM。13C-NMR数据表明，游离肽中顺式和反式构象异构体（由Leu-Pro键周围的旋转产生）的混合物在肽羰基与Ca2+离子配位时转变为全反式形式。Ca2+复合物的1H-NOESY数据揭示了三明治中涉及两个肽分子侧链的几种相互作用。有和没有NOESY衍生距离约束输入的分子建模和能量最小化表明三明治复合物是一种能量上非常有利的构象。除了其在二价阳离子可能参与底物-酪氨酸激酶相互作用方面的相关性外，tBoc-Leu-Pro-Tyr-Ala-NHCH3及其Ca2+复合物构象特征的表征应有助于理解线性肽与Ca(2+)结合的构象决定因素。
