Weininger Ulrich, Modig Kristofer, Geitner Anne-Juliane, Schmidpeter Philipp A M, Koch Johanna R, Akke Mikael
Department of Biophysical Chemistry, Center for Molecular Protein Science, Lund University , P.O. Box 124, SE-22100 Lund, Sweden.
Institute of Physics, Biophysics, Martin-Luther-University Halle-Wittenberg , D-06120 Halle (Saale), Germany.
Biochemistry. 2017 Jan 10;56(1):334-343. doi: 10.1021/acs.biochem.6b01157. Epub 2016 Dec 22.
FKBP12, a small human enzyme, aids protein folding by catalyzing cis-trans isomerization of peptidyl-prolyl bonds, and is involved in cell signaling pathways, calcium regulation, and the immune response. The underlying molecular mechanisms are not fully understood, but it is well-known that aromatic residues in the active site and neighboring loops are important for substrate binding and catalysis. Here we report micro- to millisecond exchange dynamics of aromatic side chains in the active site region of ligand-free FKBP12, involving a minor state population of 0.5% and an exchange rate of 3600 s, similar to previous results for the backbone and methyl-bearing side chains. The exchange process involves tautomerization of H87. In the major state H87 is highly flexible and occupies the common HNε2 tautomer, while in the minor state it occupies the rare HNδ1 tautomer, which typically requires stabilization by specific interactions, such as hydrogen bonds. This finding suggests that the exchange process is coupled to a rearrangement of the hydrogen bond network around H87. Upon addition of the active-site inhibitor FK506 the exchange of all aromatic residues is quenched, with exception of H87. The H87 resonances are broadened beyond detection, suggesting that interconversion between tautomers prevail in the FK506-bound state. While key active-site residues undergo conformational exchange in the apo state, the exchange rate is considerably faster than the catalytic turnover, as determined herein by Michaelis-Menten type analysis of NMR line shapes and chemical shifts. We discuss alternative interpretations of this observation in terms of FKBP12 function.
FKBP12是一种小型人体酶，通过催化肽基脯氨酰键的顺反异构化来辅助蛋白质折叠，并参与细胞信号通路、钙调节和免疫反应。其潜在的分子机制尚未完全明确，但众所周知，活性位点及相邻环中的芳香族残基对于底物结合和催化作用很重要。在此，我们报告了无配体FKBP12活性位点区域芳香族侧链从微秒到毫秒的交换动力学，涉及0.5%的次要状态群体和3600 s的交换速率，这与之前关于主链和含甲基侧链的结果相似。交换过程涉及H87的互变异构。在主要状态下，H87高度灵活，占据常见的HNε2互变异构体，而在次要状态下，它占据罕见的HNδ1互变异构体，这通常需要通过特定相互作用（如氢键）来稳定。这一发现表明，交换过程与H87周围氢键网络的重排相关。加入活性位点抑制剂FK506后，除H87外，所有芳香族残基的交换均被淬灭。H87的共振峰变宽至无法检测，这表明在FK506结合状态下互变异构体之间的相互转化占主导。虽然关键活性位点残基在无apo状态下会发生构象交换，但如本文通过对NMR线形和化学位移的米氏类型分析所确定的，交换速率比催化周转快得多。我们从FKBP12功能的角度讨论了这一观察结果可能的其他解释。
