Evolution of class-specific peptides targeting a hot spot of the Gαs subunit

被引:24
作者
Austin, Ryan J. [1 ,2 ,3 ]
Ja, William W. [4 ]
Roberts, Richard W. [1 ,2 ,3 ]
机构
[1] Univ So Calif, Dept Chem Engn Mat Sci, Dept Chem, Los Angeles, CA 90089 USA
[2] Univ So Calif, Dept Chem Engn Mat Sci, Dept Biol, Los Angeles, CA 90089 USA
[3] Univ So Calif, Dept Chem Engn Mat Sci, Dept Mork Family, Los Angeles, CA 90089 USA
[4] CALTECH, Div Biol, Pasadena, CA 91125 USA
关键词
mRNA display; directed evolution; G protein; convergent binding site; hot spot;
D O I
10.1016/j.jmb.2008.01.032
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 [生物化学与分子生物学]; 081704 [应用化学];
摘要
The four classes of heterotrimeric G-protein alpha subunits act as molecular routers inside cells, gating signals based on a bound guanosine nucleotide (guanosine 5'-triphosphate versus guanosine 5-diphosphate). Ligands that specifically target individual subunits provide new tools for monitoring and modulating these networks, but are challenging to design due to the high sequence homology and structural plasticity of the G alpha-binding surface. Here we have created an mRNA display library of peptides based on the short G alpha-modulating peptide R6A-1 and selected variants that target a convergent protein-binding surface of G alpha s . guanosine 5'-diphosphate. After selection/evolution, the most G alpha s-specific peptide, G alpha s(s)-binding peptide (GSP), was used to design a second-generation library, resulting in several new affinity-and selectivity-matured peptides denoted as mGSPs. The two-step evolutionary walk from R6A-1 to mGSP-1 resulted in an 8000-fold inversion in binding specificity, altered seven out of nine residues in the starting peptide core, and incorporated both positive and negative design steps. The resulting mGSP-1 peptide shows remarkable selectivity and affinity, exhibiting little or no binding to nine homologous G alpha subunits or human H-Ras, and even discriminates the Gets splice variant G alpha s(1). Selected peptides make specific contacts with the effector-binding region of Ga, which may explain an interesting bifunctional activity observed in GSP. Overall, our work demonstrates a design of simple, linear, highly specific peptides. that target a protein-binding surface of G alpha s. and argues that mRNA display-based selection/evolution is a powerful route for targeting protein families with high class specificity and state specificity. (C) 2008 Elsevier Ltd. All rights reserved.
引用
收藏
页码:1406 / 1418
页数:13
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