Kohl Andreas, Amstutz Patrick, Parizek Petra, Binz H Kaspar, Briand Christophe, Capitani Guido, Forrer Patrik, Plückthun Andreas, Grütter Markus G
Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Structure. 2005 Aug;13(8):1131-41. doi: 10.1016/j.str.2005.04.020.
Aminoglycoside phosphotransferase (3')-IIIa (APH) is a bacterial kinase that confers antibiotic resistance to many pathogenic bacteria and shares structural homology with eukaryotic protein kinases. We report here the crystal structure of APH, trapped in an inactive conformation by a tailor-made inhibitory ankyrin repeat (AR) protein, at 2.15 A resolution. The inhibitor was selected from a combinatorial library of designed AR proteins. The AR protein binds the C-terminal lobe of APH and thereby stabilizes three alpha helices, which are necessary for substrate binding, in a significantly displaced conformation. BIAcore analysis and kinetic enzyme inhibition experiments are consistent with the proposed allosteric inhibition mechanism. In contrast to most small-molecule kinase inhibitors, the AR proteins are not restricted to active site binding, allowing for higher specificity. Inactive conformations of pharmaceutically relevant enzymes, as can be elucidated with the approach presented here, represent powerful starting points for rational drug design.
氨基糖苷磷酸转移酶（3'）-IIIa（APH）是一种细菌激酶，可赋予许多病原菌抗生素抗性，并且与真核蛋白激酶具有结构同源性。我们在此报告APH的晶体结构，它被一种特制的抑制性锚蛋白重复序列（AR）蛋白捕获在无活性构象中，分辨率为2.15埃。该抑制剂是从设计的AR蛋白组合文库中筛选出来的。AR蛋白与APH的C末端叶结合，从而使三个对于底物结合必不可少的α螺旋稳定在明显移位的构象中。BIAcore分析和动力学酶抑制实验与所提出的变构抑制机制一致。与大多数小分子激酶抑制剂不同，AR蛋白不限于活性位点结合，因而具有更高的特异性。如本文所介绍的方法那样阐明的药学相关酶的无活性构象，是合理药物设计的有力起点。
