Krishnan Rajaraman, Tsubery Haim, Proschitsky Ming Y, Asp Eva, Lulu Michal, Gilead Sharon, Gartner Myra, Waltho Jonathan P, Davis Peter J, Hounslow Andrea M, Kirschner Daniel A, Inouye Hideyo, Myszka David G, Wright Jason, Solomon Beka, Fisher Richard A
Neurophage Pharmaceuticals, 222 Third Street, Suite 3120, Cambridge, MA 02142, USA.
Neurophage Pharmaceuticals, 222 Third Street, Suite 3120, Cambridge, MA 02142, USA.
J Mol Biol. 2014 Jun 26;426(13):2500-19. doi: 10.1016/j.jmb.2014.04.015. Epub 2014 Apr 22.
Misfolded protein aggregates, characterized by a canonical amyloid fold, play a central role in the pathobiology of neurodegenerative diseases. Agents that bind and sequester neurotoxic intermediates of amyloid assembly, inhibit the assembly or promote the destabilization of such protein aggregates are in clinical testing. Here, we show that the gene 3 protein (g3p) of filamentous bacteriophage mediates potent generic binding to the amyloid fold. We have characterized the amyloid binding and conformational remodeling activities using an array of techniques, including X-ray fiber diffraction and NMR. The mechanism for g3p binding with amyloid appears to reflect its physiological role during infection of Escherichia coli, which is dependent on temperature-sensitive interdomain unfolding and cis-trans prolyl isomerization of g3p. In addition, a natural receptor for g3p, TolA-C, competitively interferes with Aβ binding to g3p. NMR studies show that g3p binding to Aβ fibers is predominantly through middle and C-terminal residues of the Aβ subunit, indicating β strand-g3p interactions. A recombinant bivalent g3p molecule, an immunoglobulin Fc (Ig) fusion of the two N-terminal g3p domains, (1) potently binds Aβ fibers (fAβ) (KD=9.4nM); (2); blocks fAβ assembly (IC50~50nM) and (3) dissociates fAβ (EC50=40-100nM). The binding of g3p to misfolded protein assemblies is generic, and amyloid-targeted activities can be demonstrated using other misfolded protein systems. Taken together, our studies show that g3p(N1N2) acts as a general amyloid interaction motif.
以典型淀粉样折叠为特征的错误折叠蛋白聚集体在神经退行性疾病的病理生物学中起着核心作用。结合并隔离淀粉样蛋白组装神经毒性中间体、抑制组装或促进此类蛋白聚集体去稳定化的药物正在进行临床试验。在此，我们表明丝状噬菌体的基因3蛋白（g3p）介导与淀粉样折叠的有效通用结合。我们使用了一系列技术，包括X射线纤维衍射和核磁共振，来表征淀粉样蛋白结合和构象重塑活性。g3p与淀粉样蛋白结合的机制似乎反映了其在感染大肠杆菌过程中的生理作用，这取决于g3p的温度敏感型结构域间解折叠和顺反脯氨酰异构化。此外，g3p的天然受体TolA-C竞争性干扰Aβ与g3p的结合。核磁共振研究表明，g3p与Aβ纤维的结合主要通过Aβ亚基的中部和C末端残基，表明存在β链-g3p相互作用。一种重组二价g3p分子，即两个N末端g3p结构域的免疫球蛋白Fc（Ig）融合体，（1）能有效结合Aβ纤维（fAβ）（KD = 9.4nM）；（2）阻断fAβ组装（IC50约为50nM）；（3）使fAβ解离（EC50 = 40 - 100nM）。g3p与错误折叠蛋白聚集体的结合具有通用性，并且可以使用其他错误折叠蛋白系统证明其针对淀粉样蛋白的活性。综上所述，我们的研究表明g3p（N1N2）作为一种通用的淀粉样蛋白相互作用基序。
