Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Protein Eng Des Sel. 2011 Jan;24(1-2):185-95. doi: 10.1093/protein/gzq053. Epub 2010 Aug 16.
Internal symmetry in proteins is likely to be the footprint of evolution by gene duplication and fusion. Like other symmetrical proteins, β-propellers, which are made of 4-10 β-sheet units (blades) circularly arranged around a central tunnel, have probably evolved by duplication and fusion of a rudimentary repetitive unit. However, reproducing the evolution of functional β-propellers by duplication and fusion of repeated units remains a challenge, in particular, because the repeated units must jointly pack to form one hydrophobic core while maintaining intact active sites. As model for generating repeat propellers, we chose tachylectin-2--a highly symmetrical five-bladed β-propeller lectin with five sugar-binding sites. We report the engineering of folded and functional lectins by duplication and fusion of repetitive sequence modules taken from tachylectin-2. The repeated modules comprise three strands of one blade plus one strand of the next blade, thus enabling the closure of the propeller's ring via strand-strand Velcro-like interactions. Duplication and fusion of five modules with the same sequence gave rise to a highly aggregated protein, yet its soluble fraction exhibited lectin function. Subsequently, a library of diversified sequence modules fused in tandem was selected by phage display for glycoprotein binding. A range of new lectins were isolated with binding and biophysical properties that resemble those of wild-type tachylectin-2. These results demonstrate the ability to construct folded and functional globular repeat proteins, and support the role of duplication and fusion of elementary modules in the evolutionary routes that led to the β-propellers fold.
蛋白质内部的对称性可能是基因复制和融合进化的痕迹。与其他对称蛋白质一样，β-发夹（β-propellers）由 4-10 个β-折叠单元（叶片）围绕中央隧道呈圆形排列组成，可能是通过基本重复单元的复制和融合而进化而来的。然而，通过重复单元的复制和融合来再现功能性β-发夹的进化仍然是一个挑战，特别是因为重复单元必须共同包装形成一个疏水性核心，同时保持完整的活性位点。作为生成重复发夹的模型，我们选择了 tachylectin-2——一种高度对称的五叶β-发夹凝集素，具有五个糖结合位点。我们报告了通过从 tachylectin-2 中重复序列模块的复制和融合来工程化折叠和功能性凝集素。重复模块由一个叶片的三条链和下一个叶片的一条链组成，从而通过链-链 Velcro 样相互作用实现发夹环的闭合。相同序列的五个模块的复制和融合导致高度聚集的蛋白质，但它的可溶部分表现出凝集素功能。随后，通过噬菌体展示对糖蛋白结合进行串联融合的多样化序列模块文库进行了选择。分离出一系列具有与野生型 tachylectin-2 相似的结合和生物物理特性的新型凝集素。这些结果证明了构建折叠和功能性球状重复蛋白的能力，并支持基本模块复制和融合在导致β-发夹折叠的进化途径中的作用。
