Rouviere N, Vincent M, Craescu C T, Gallay J
Institut National de la Santé et de la Recherche Médicale U33, Hôpital de Bicêtre, Le Kremlin-Bicêtre, France.
Biochemistry. 1997 Jun 17;36(24):7339-52. doi: 10.1021/bi962289w.
The interaction of the immunophilin domain of FKBP59 (FKBP59-I) with immunosuppressant drugs was investigated by steady-state and time-resolved fluorescence of tryptophan. One of the two Trp residues present in this protein (W89), conserved in almost all immunophilins, is buried in the hydrophobic core and participates in the immunosuppressant binding. By comparison with the highly homologous protein FKBP12, containing only the buried Trp, it has been concluded that its weak fluorescence is due to an atypical H-bond interaction involving the indole nitrogen and the Phe129 benzene ring. The second Trp residue (W59) in FKBP59-I is located on the external hydrophilic side of the 50-60 beta-sheet [Craescu, C. T., Rouvière, N., Popescu, A., Cerpolini, E., Lebeau, M.-C., Baulieu, E.-E., & Mispelter, J. (1996) Biochemistry 35, 11045-11052] and is responsible for >95% of the fluorescence emission. The long lifetime of the major excited state, the large activation energy of thermal quenching, and the rotational correlation time distribution pattern suggest that its environment is not highly mobile. Binding of the immunosuppressant drugs FK506 and rapamycin leads to a approximately 60% decrease of the fluorescence intensity without any change in the fluorescence emission maximum. Time-resolved measurements show that this "quenching" is due to a conformational change which depletes the long excited-state lifetime population to the profit of a more quenched minor excited state, which becomes prominent in the complexes. This is accompanied by a strong slowing of the indole ring dynamics in the case of FK506 and by a complete immobilization in the case of rapamycin, as shown by two-dimensional (tau, theta) maximum entropy analysis of the polarized fluorescence decays. Binding of the immunosuppressant drugs therefore modifies the structure and the dynamics of the external side of the 50-60 beta-sheet in FKBP59-I, which could be relevant for the formation of ternary complexes with other protein targets.
通过色氨酸的稳态荧光和时间分辨荧光研究了FKBP59免疫亲和素结构域（FKBP59-I）与免疫抑制剂药物的相互作用。该蛋白中存在的两个色氨酸残基之一（W89），在几乎所有免疫亲和素中都保守，位于疏水核心中并参与免疫抑制剂的结合。与仅含有埋藏色氨酸的高度同源蛋白FKBP12相比，得出结论其弱荧光是由于涉及吲哚氮和苯丙氨酸129苯环的非典型氢键相互作用。FKBP59-I中的第二个色氨酸残基（W59）位于50-60β折叠的外部亲水侧[Craescu, C. T., Rouvière, N., Popescu, A., Cerpolini, E., Lebeau, M.-C., Baulieu, E.-E., & Mispelter, J. (1996) Biochemistry 35, 11045-11052]，并且负责超过95%的荧光发射。主要激发态的长寿命、热猝灭的大活化能以及旋转相关时间分布模式表明其环境流动性不高。免疫抑制剂药物FK506和雷帕霉素的结合导致荧光强度降低约60%，而荧光发射最大值没有任何变化。时间分辨测量表明这种“猝灭”是由于构象变化，使长激发态寿命的群体减少，有利于形成在复合物中占主导的猝灭程度更高的次要激发态。如极化荧光衰减的二维（τ，θ）最大熵分析所示，在FK506的情况下伴随着吲哚环动力学的强烈减慢，而在雷帕霉素的情况下则完全固定。因此，免疫抑制剂药物的结合改变了FKBP59-I中50-60β折叠外部的结构和动力学，这可能与与其他蛋白质靶点形成三元复合物有关。
