Martins J C, Enassar M, Willem R, Wieruzeski J M, Lippens G, Wodak S J
High Resolution NMR Centrum (HNMR), Vrije Universiteit Brussel, Belgium.
Eur J Biochem. 2001 Apr;268(8):2379-89. doi: 10.1046/j.1432-1327.2001.02118.x.
The most abundant alpha-amylase inhibitor (AAI) present in the seeds of Amaranthus hypochondriacus, a variety of the Mexican crop plant amaranth, is the smallest polypeptide (32 residues) known to inhibit alpha-amylase activity of insect larvae while leaving that of mammals unaffected. In solution, 1H NMR reveals that AAI isolated from amaranth seeds adopts a major trans (70%) and minor cis (30%) conformation, resulting from slow cis-trans isomerization of the Val15-Pro16 peptide bond. Both solution structures have been determined using 2D 1H-NMR spectroscopy and XPLOR followed by restrained energy refinement in the consistent-valence force field. For the major isomer, a total of 563 distance restraints, including 55 medium-range and 173 long-range ones, were available from the NOESY spectra. This rather large number of constraints from a protein of such a small size results from a compact fold, imposed through three disulfide bridges arranged in a cysteine-knot motif. The structure of the minor cis isomer has also been determined using a smaller constraint set. It reveals a different backbone conformation in the Pro10-Pro20 segment, while preserving the overall global fold. The energy-refined ensemble of the major isomer, consisting of 20 low-energy conformers with an average backbone rmsd of 0.29 +/- 0.19 A and no violations larger than 0.4 A, represents a considerable improvement in precision over a previously reported and independently performed calculation on AAI obtained through solid-phase synthesis, which was determined with only half the number of medium-range and long-range restraints reported here, and featured the trans isomer only. The resulting differences in ensemble precision have been quantified locally and globally, indicating that, for regions of the backbone and a good fraction of the side chains, the conformation is better defined in the new solution structure. Structural comparison of the solution structure with the X-ray structure of the inhibitor when bound to its alpha-amylase target in Tenebrio molitor shows that the backbone conformation is only slightly adjusted on complexation, while that of the side chains involved in protein-protein contacts is similar to those present in solution. Therefore, the overall conformation of AAI appears to be predisposed to binding to its target alpha-amylase, confirming the view that it acts as a lid on top of the alpha-amylase active site.
墨西哥农作物苋属植物皱果苋种子中存在的最丰富的α-淀粉酶抑制剂（AAI）是已知的最小多肽（32个残基），它能抑制昆虫幼虫的α-淀粉酶活性，而不影响哺乳动物的α-淀粉酶活性。在溶液中，1H NMR显示，从苋属种子中分离出的AAI呈现出主要的反式构象（70%）和次要的顺式构象（30%），这是由Val15-Pro16肽键的缓慢顺反异构化导致的。两种溶液结构均已通过二维1H-NMR光谱和XPLOR测定，随后在一致价力场中进行受限能量精修。对于主要异构体，从NOESY光谱中获得了总共563个距离约束，包括55个中程约束和173个长程约束。对于如此小尺寸的蛋白质来说，这种相当多的约束源于通过以半胱氨酸结基序排列的三个二硫键形成的紧密折叠。次要顺式异构体的结构也已使用较小的约束集确定。它揭示了Pro10-Pro20片段中不同的主链构象，同时保留了整体全局折叠。主要异构体的能量精修集合由20个低能量构象组成，平均主链均方根偏差为0.29±0.19 Å，且没有大于0.4 Å的偏差，与之前通过固相合成获得的AAI的独立计算结果相比，在精度上有了显著提高，之前的计算仅使用了此处报道的中程和长程约束数量的一半，且仅涉及反式异构体。已对集合精度的差异进行了局部和全局量化，表明对于主链区域和相当一部分侧链，新的溶液结构中构象的定义更好。将溶液结构与该抑制剂与黄粉虫中的α-淀粉酶靶标结合时的X射线结构进行结构比较，结果表明，在形成复合物时主链构象仅略有调整，而参与蛋白质-蛋白质接触的侧链构象与溶液中的相似。因此，AAI的整体构象似乎易于与其靶标α-淀粉酶结合，这证实了它作为α-淀粉酶活性位点顶部盖子的观点。
