Baldwin Enoch P, Martin Shelley S, Abel Jonas, Gelato Kathy A, Kim Hanseong, Schultz Peter G, Santoro Stephen W
Section of Molecular and Cellular Biology, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, USA.
Chem Biol. 2003 Nov;10(11):1085-94. doi: 10.1016/j.chembiol.2003.10.015.
The basis for the altered DNA specificities of two Cre recombinase variants, obtained by mutation and selection, was revealed by their cocrystal structures. The proteins share similar substitutions but differ in their preferences for the natural LoxP substrate and an engineered substrate that is inactive with wild-type Cre, LoxM7. One variant preferentially recombines LoxM7 and contacts the substituted bases through a hydrated network of novel interlocking protein-DNA contacts. The other variant recognizes both LoxP and LoxM7 utilizing the same DNA backbone contact but different base contacts, facilitated by an unexpected DNA shift. Assisted by water, novel interaction networks can arise from few protein substitutions, suggesting how new DNA binding specificities might evolve. The contributions of macromolecular plasticity and water networks in specific DNA recognition observed here present a challenge for predictive schemes.
通过突变和筛选获得的两种Cre重组酶变体,其DNA特异性改变的基础由它们的共晶体结构揭示。这两种蛋白质有相似的取代,但对天然LoxP底物和对野生型Cre无活性的工程化底物LoxM7的偏好不同。一种变体优先重组LoxM7,并通过新型互锁的蛋白质 - DNA接触的水合网络与取代碱基接触。另一种变体利用相同的DNA主链接触但不同的碱基接触来识别LoxP和LoxM7,这是由意想不到的DNA移位促成的。在水的辅助下,少量蛋白质取代就能产生新的相互作用网络,这表明新的DNA结合特异性可能是如何进化的。这里观察到的大分子可塑性和水网络在特定DNA识别中的作用,对预测方案提出了挑战。