Minimizing a binding domain from protein A
Minimizing a binding domain from protein A
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DOI：
10.1073/pnas.93.12.5688
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发表时间：
1996-06-11
影响因子：
11.1
通讯作者：
Wells, JA
中科院分区：
文献类型：
--
作者：
Braisted, AC;Wells, JA
我们提出了一种系统性方法来最大限度地减少蛋白A的Z结构域，蛋白A是一个三螺旋束，总共159个残基，与免疫球蛋白IgG的F-c部分紧密结合（Kd = 10 nM）（1）。尽管在复合物的X射线结构中看到的与IgG的所有接触都来自前两个螺旋中的残基，但当螺旋3缺失时，结合亲和力降低> 10（5）倍通过使用基于结构的设计和噬菌体展示方法，我们已经迭代地提高了双螺旋衍生物的稳定性和结合亲和力，33个残基的长度，使得其以43 nM的Kd结合IgG（1）。这通过从截短的Z-肽的三个区域逐步选择随机突变来实现：螺旋1和螺旋2的4个疏水残基与螺旋3接触（外表面），随后是螺旋1和螺旋2之间的5个残基最后是在与F-c相互作用的界面处或附近的19个残基（界面），因为从每个区域选择的突变被编译（总共12个），它们导致对IgG的亲和力的逐渐增加，并且伴随着α-螺旋含量的增加，反映了双螺旋支架的稳定性增加，因此，通过结构稳定性的连续增加和分子间接触质量的改进，可以将较大的结合域减小为较小的结合域，如此迷你-蛋白质结合结构域更适合于合成化学并且因此可以是用于设计较小有机模拟物的有用起点，较小的结合基序也提供了简化和更易于处理的模型，了解蛋白质的功能和稳定性的决定因素。
We present a systematic approach to minimizing the Z-domain of protein A, a three-helix bundle 159 residues total) that binds tightly (K-d = 10 nM) to the F-c portion of an immunoglobin IgG(1). Despite the fact that all the contacts seen in the x-ray structure of the complex with the IgG are derived from residues in the first two helices, when helix 3 is deleted, binding affinity is reduced > 10(5)-fold (K-d > 1 mM), By using structure-based design and phage display methods, we have iteratively improved the stability and binding affinity for a two-helix derivative, 33 residues in length, such that it binds IgG(1) with a K-d Of 43 nM. This was accomplished by stepwise selection of random mutations from three regions of the truncated Z-peptide: the 4 hydrophobic residues from helix 1 and helix 2 that contacted helix 3 (the exoface), followed by 5 residues between helix 1 and helix 2 (the intraface), and lastly by 19 residues at or near the interface that interacts with F-c (the interface), As selected mutations from each region were compiled (12 in total), they led to progressive increases in affinity for IgG, and concomitant increases in alpha-helical content reflecting increased stabilization of the two-helix scaffold, Thus, by sequential increases in the stability of the structure and improvements in the quality of the intermolecular contacts, one can reduce larger binding domains to smaller ones, Such mini-protein binding domains are more amenable to synthetic chemistry and thus may be useful starting points for the design of smaller organic mimics, Smaller binding motifs also provide simplified and more tractable models for understanding determinants of protein function and stability.