Structure-based engineering of streptavidin monomer with a reduced biotin dissociation rate
Structure-based engineering of streptavidin monomer with a reduced biotin dissociation rate
复制标题
DOI：
10.1002/prot.24320
复制
发表时间：
2013-09-01
影响因子：
2.9
通讯作者：
Park, Sheldon
中科院分区：
文献类型：
--
作者：
DeMonte, Daniel;Drake, Eric J.;Park, Sheldon
我们最近报道了单体链霉亲和素（mSA）的工程改造，对应于野生型（wt）链霉亲和素四聚体的一个亚基。该单体是通过同源建模设计的，其中链霉抗生物素蛋白和根霉抗生物素蛋白序列被组合以工程化仅包含来自单个亚基的残基的高亲和力结合口袋。尽管mSA是稳定的并且以纳摩尔亲和力结合生物素，但其快速解离速率（k（off））在应用期间产生实际挑战。我们获得了与生物素结合的mSA的1.9埃晶体结构，以详细了解它们的相互作用，并使用该结构引入靶向突变以改善其结合动力学。为此，我们将mSA与shwanavidin进行了比较，shwanavidin在结合口袋中含有含有F43的疏水盖并紧密结合生物素。然而，mSA中的T48F突变引入了相当的疏水盖，仅导致测量的k（off）适度提高20 - 40%。另一方面，在结合的生物素的双环附近引入S25 H突变使解离半衰期（t（1/2））在20 ℃下从11分钟增加到83分钟。分子动力学（MD）模拟表明，H25通过与A47相互作用来稳定结合环L3，4，并通过限制溶剂进入结合口袋来保护关键的分子间氢键。同时发生的T48F或T48W突变与H25冲突，并部分消除了H25的有益作用。两者合计，这项研究表明，稳定的结合环和溶剂化的结合口袋是重要的决定因素的解离动力学的mSA。Proteins 2013. (c)2013 Wiley Periodicals，Inc.
We recently reported the engineering of monomeric streptavidin, mSA, corresponding to one subunit of wild type (wt) streptavidin tetramer. The monomer was designed by homology modeling, in which the streptavidin and rhizavidin sequences were combined to engineer a high affinity binding pocket containing residues from a single subunit only. Although mSA is stable and binds biotin with nanomolar affinity, its fast off rate (k(off)) creates practical challenges during applications. We obtained a 1.9 angstrom crystal structure of mSA bound to biotin to understand their interaction in detail, and used the structure to introduce targeted mutations to improve its binding kinetics. To this end, we compared mSA to shwanavidin, which contains a hydrophobic lid containing F43 in the binding pocket and binds biotin tightly. However, the T48F mutation in mSA, which introduces a comparable hydrophobic lid, only resulted in a modest 20-40% improvement in the measured k(off). On the other hand, introducing the S25H mutation near the bicyclic ring of bound biotin increased the dissociation half life (t(1/2)) from 11 to 83 min at 20 degrees C. Molecular dynamics (MD) simulations suggest that H25 stabilizes the binding loop L3,4 by interacting with A47, and protects key intermolecular hydrogen bonds by limiting solvent entry into the binding pocket. Concurrent T48F or T48W mutation clashes with H25 and partially abrogates the beneficial effects of H25. Taken together, this study suggests that stabilization of the binding loop and solvation of the binding pocket are important determinants of the dissociation kinetics in mSA. Proteins 2013. (c) 2013 Wiley Periodicals, Inc.