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首页> 外文期刊>Biochemistry >Mapping a Ketosynthase:Acyl Carrier Protein Binding Interface via Unnatural Amino Acid-Mediated Photo-Cross-Linking
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Mapping a Ketosynthase:Acyl Carrier Protein Binding Interface via Unnatural Amino Acid-Mediated Photo-Cross-Linking

机译：通过非天然氨基酸介导的光交叉连接定位酮合酶：酰基载体蛋白结合界面

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摘要

Probing and interrogating protein interactions that involve acyl carrier proteins (ACPs) in fatty acid synthases and polyketide synthases are critical to understanding the molecular basis for the programmed assembly of complex natural products. Here, we have used unnatural amino acid mutagenesis to site specifically install photo-cross-linking functionality into acyl carrier proteins from diverse systems and the ketosynthase FabF from the Escherichia coli type II fatty acid synthase. Subsequently, a photo-cross-linking assay was employed to systematically probe the ability of FabF to interact with a broad panel of ACPs, illustrating the expected orthogonality of ACP:FabF interactions and the role of charged residues in helix II of the ACP. In addition, FabF residues involved in the binding interaction with the cognate carrier protein were identified via surface scanning mutagenesis and photo-cross-linking. Furthermore, the ability to install the photo-cross-linking amino acid at virtually any position allowed interrogation of the role that carrier protein acylation plays in determining the binding interface with FabF. A conserved carrier protein motif that includes the phosphopantetheinylation site was also shown to play an integral role in maintenance of the AcpP:FabF binding interaction. Our results provide unprecedented insight into the molecular details that describe the AcpP:FabF binding interface and demonstrate that unnatural amino acid based photo-cross-linking is a powerful tool for probing and interrogating protein interactions in complex biosynthetic systems.
机译：探查和询问涉及脂肪酸合酶和聚酮化合物合酶中的酰基载体蛋白（ACP）的蛋白相互作用对于理解复杂天然产物程序组装的分子基础至关重要。在这里，我们使用了非天然氨基酸诱变来将光交联功能特异地安装在来自各种系统的酰基载体蛋白中以及来自大肠杆菌II型脂肪酸合酶的酮合酶FabF中。随后，采用光交联测定法来系统地探测FabF与各种ACP相互作用的能力，从而说明了ACP：FabF相互作用的预期正交性以及ACP螺旋II中带电残基的作用。另外，通过表面扫描诱变和光交联来鉴定参与与同源载体蛋白的结合相互作用的FabF残基。此外，将光交联氨基酸安装在几乎任何位置的能力允许询问载体蛋白酰化在确定与FabF的结合界面中所起的作用。还显示了包括磷酸泛肽基化位点的保守的载体蛋白基序在维持AcpP：FabF结合相互作用中起着不可或缺的作用。我们的结果为描述AcpP：FabF结合界面的分子细节提供了空前的洞察力，并证明了基于非天然氨基酸的光交联是探测和审视复杂生物合成系统中蛋白质相互作用的有力工具。

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