Structural basis of conformational transitions in the active site and 80's loop in the FK506-binding protein FKBP12.
Structural basis of conformational transitions in the active site and 80's loop in the FK506-binding protein FKBP12.
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DOI：
10.1042/bj20131429
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发表时间：
2014-03-15
期刊：
影响因子：
--
通讯作者：
Hernández G
中科院分区：
文献类型：
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作者：
Mustafi SM;Brecher M;Zhang J;Li H;Lemaster DM;Hernández G
亲免疫蛋白FKBP12 （fk506结合蛋白12）所表现出的广泛的核磁共振双重效应是由于80 's环顶端附近Gly89的缓慢过渡到顺肽结构，这是FKBP12和其他FKBP结构域蛋白的许多蛋白质识别相互作用的位点。80 's环也表现出线宽，表明微秒到毫秒的构象动力学，但仅在反肽状态下。G89A变体将顺式肽平衡从88:12转变为33:67，而脯氨酸残基取代则完全诱导了顺式肽结构。在1.50 Å分辨率下，G89P晶体结构中的80 's环构象与野生型FKBP12主要在残基88、89和90处存在差异，与FKBP52的报道非常相似。基于结构的化学位移预测表明，80年代环路中的微秒到毫秒动力学可能是由协调的主链（ψ88和ϕ89）扭转角转变引起的。在G89P晶体结构中，Trp59活性位点裂口底部的吲哚侧链定向重定向~90°，相邻的主链发生移位。野生型FKBP12的NOE分析表明，该吲哚在垂直方向上占20%。15N弛豫分析与吲哚重定向在纳秒时间内发生一致。在1.20 Å分辨率下对G89P晶体数据的回忆显示，吲哚环具有较弱的野生型取向。作为Trp59吲哚环基础的残基的差异以及连接50 's环与活性位点的相互作用的改变表明，该环的重新定向可能在FKBP结构域家族中具有该活性位点色氨酸残基的其他6个成员中不受欢迎。广泛的共振倍增产生于Gly89的顺-反肽转变，而线增宽则是由于邻近扭转角的一致移动。活性位点Trp59环在20%的种群中采用垂直取向。
The extensive set of NMR doublings exhibited by the immunophilin FKBP12 (FK506-binding protein 12) arose from a slow transition to the cis-peptide configuration at Gly89 near the tip of the 80′s loop, the site for numerous protein-recognition interactions for both FKBP12 and other FKBP domain proteins. The 80′s loop also exhibited linebroadening, indicative of microsecond to millisecond conformational dynamics, but only in the trans-peptide state. The G89A variant shifted the trans–cis peptide equilibrium from 88:12 to 33:67, whereas a proline residue substitution induced fully the cis-peptide configuration. The 80′s loop conformation in the G89P crystal structure at 1.50 Å resolution differed from wild-type FKBP12 primarily at residues 88, 89 and 90, and it closely resembled that reported for FKBP52. Structure-based chemical-shift predictions indicated that the microsecond to millisecond dynamics in the 80′s loop probably arose from a concerted main chain (ψ88 and ϕ89) torsion angle transition. The indole side chain of Trp59 at the base of the active-site cleft was reoriented ~90o and the adjacent backbone was shifted in the G89P crystal structure. NOE analysis of wild-type FKBP12 demonstrated that this indole populates the perpendicular orientation at 20%. The 15N relaxation analysis was consistent with the indole reorientation occurring in the nanosecond timeframe. Recollection of the G89P crystal data at 1.20 Å resolution revealed a weaker wild-type-like orientation for the indole ring. Differences in the residues that underlie the Trp59 indole ring and altered interactions linking the 50′s loop to the active site suggested that reorientation of this ring may be disfavoured in the other six members of the FKBP domain family that bear this active-site tryptophan residue. Extensive resonance doubling arises from a cis–trans peptide transition at Gly89, whereas linebroadening appears due to a concerted shift in the neighbouring torsion angles. The active site Trp59 ring adopts a perpendicular orientation at a population of 20%.