Perbandt M, Bruns O, Vallazza M, Lamla T, Betzel Ch, Erdmann V A
Department of Biochemistry and Molecular Biology, University of Hamburg, 20146 Hamburg, Germany.
Proteins. 2007 Jun 1;67(4):1147-53. doi: 10.1002/prot.21236.
A novel peptide was designed which possesses nanomolar affinity of less than 20 nM for streptavidin. Therefore it was termed Nano-tag and has been used as an affinity tag for recombinant proteins. The minimized version of the wild type Nano-tag is a seven-amino acid peptide with the sequence fMDVEAWL. The three-dimensional structure of wild type streptavidin in complex with the minimized Nano-tag was analyzed at atomic resolution of 1.15 A and the details of the binding motif were investigated. The peptide recognizes the same pocket of streptavidin where the natural ligand biotin is bound, but the peptide requires significantly more space than biotin. Therefore the binding loop adopts an "open" conformation in order to release additional space for the peptide. The conformation of the bound Nano-tag corresponds to a 3(10) helix. However, the analysis of the intermolecular interactions of the Nano-tag with residues of the binding pocket of streptavidin reveals astonishing similarities to the biotin binding motif. In principle the three-dimensional conformation of the Nano-tag mimics the binding mode of biotin. Our results explain why the use of the Nano-tag in fusion with recombinant proteins is restricted to their N-terminus and we describe the special significance of the fMet residue for the high affinity binding mode.
设计了一种新型肽，它对链霉亲和素具有小于20 nM的纳摩尔亲和力。因此它被称为纳米标签，并已用作重组蛋白的亲和标签。野生型纳米标签的最小化版本是一种七氨基酸肽，序列为fMDVEAWL。在1.15 Å的原子分辨率下分析了野生型链霉亲和素与最小化纳米标签复合物的三维结构，并研究了结合基序的细节。该肽识别链霉亲和素上天然配体生物素结合的同一口袋，但该肽比生物素需要更多的空间。因此，结合环采用“开放”构象，以便为该肽释放额外的空间。结合的纳米标签的构象对应于一个3(10)螺旋。然而，对纳米标签与链霉亲和素结合口袋残基的分子间相互作用的分析揭示了与生物素结合基序惊人的相似性。原则上，纳米标签的三维构象模仿了生物素的结合模式。我们的结果解释了为什么纳米标签与重组蛋白融合使用仅限于其N端，并且我们描述了fMet残基对于高亲和力结合模式的特殊意义。
