Hoyer Wolfgang, Härd Torleif
Department of Medical Biochemistry, University of Gothenburg, Box 440, SE-405 30 Göteborg, Sweden.
J Mol Biol. 2008 Apr 25;378(2):398-411. doi: 10.1016/j.jmb.2008.02.040. Epub 2008 Mar 4.
The oligomerization and aggregation of the amyloid-beta (A beta) peptide, a cleavage product of the amyloid precursor protein predominantly 40 or 42 amino acids in length, has been implicated in the pathogenesis of Alzheimer's disease. The identification of A beta-binding agents, e.g., antibodies or peptides, constitutes a promising therapeutic approach. However, the amount of structural and biophysical data on the underlying A beta interactions is currently very limited. We have earlier determined the structure of A beta (1-40) in complex with the affibody protein Z(A beta 3), a selected binding protein based on a three-helix bundle scaffold (Z domain). Z(A beta 3) is a dimer of affibody subunits linked via a disulfide bridge involving a selected cysteine mutation at position 28. Z(A beta 3) binds to the central and C-terminal part of A beta (residues 17-36), which adopts a beta-hairpin conformation in the complex. Here we present a detailed biophysical analysis of the Z(A beta 3):A beta (1-40) interaction, employing NMR, circular dichroism spectroscopy, 8-anilino-1-naphthalenesulfonic acid and tyrosine fluorescence, size-exclusion chromatography, thermal denaturation profiles and isothermal titration calorimetry. We conclude that (i) free Z(A beta 3) is characterized by conformational exchange and the loss of helix 1 of the three-helix bundle scaffold; (ii) a high-energy barrier is associated with the conversion of an initial Z(A beta 3):A beta (1-40) recognition complex into the native complex structure, entailing slow binding kinetics; (iii) both A beta and Z(A beta 3) fold upon binding, which, e.g., becomes manifest in the binding thermodynamics that feature a large negative change in heat capacity; (iv) the C28-disulfide does not merely afford dimerization, but its impact on the binding interfaces of the affibody subunits and A beta is a prerequisite for tight binding. The extensive folding coupled to binding observed here likely constitutes an obligate feature of biomolecular interactions involving the central and C-terminal part of A beta. Options for improvement of Z(A beta) binding proteins are discussed.
淀粉样前体蛋白的裂解产物β淀粉样蛋白（Aβ）肽主要长度为40或42个氨基酸，其寡聚化和聚集与阿尔茨海默病的发病机制有关。鉴定Aβ结合剂，如抗体或肽，是一种有前景的治疗方法。然而，目前关于潜在Aβ相互作用的结构和生物物理数据非常有限。我们之前已经确定了Aβ（1 - 40）与亲和体蛋白Z（Aβ3）复合物的结构，Z（Aβ3）是基于三螺旋束支架（Z结构域）选择的一种结合蛋白。Z（Aβ3）是通过涉及28位特定半胱氨酸突变的二硫键连接的亲和体亚基二聚体。Z（Aβ3）与Aβ的中央和C末端部分（残基17 - 36）结合，该部分在复合物中呈β发夹构象。在此，我们采用核磁共振、圆二色光谱、8 - 苯胺基 - 1 - 萘磺酸和酪氨酸荧光、尺寸排阻色谱、热变性曲线和等温滴定量热法，对Z（Aβ3）：Aβ（1 - 40）相互作用进行了详细的生物物理分析。我们得出以下结论：（i）游离的Z（Aβ3）具有构象交换特征，且三螺旋束支架的螺旋1会丢失；（ii）初始的Z（Aβ3）：Aβ（1 - 40）识别复合物转变为天然复合物结构存在高能垒，导致结合动力学缓慢；（iii）Aβ和Z（Aβ3）在结合时都会折叠，例如这在结合热力学中表现为热容有很大的负变化；（iv）C28 - 二硫键不仅实现二聚化，其对亲和体亚基和Aβ结合界面的影响是紧密结合的前提。此处观察到的与结合相关的广泛折叠可能是涉及Aβ中央和C末端部分的生物分子相互作用的一个必然特征。讨论了改进Z（Aβ）结合蛋白的方法。
