Targeted Reengineering of Protein Geranylgeranyltransferase Type I Selectivity Functionally Implicates Active-Site Residues in Protein-Substrate Recognition
Targeted Reengineering of Protein Geranylgeranyltransferase Type I Selectivity Functionally Implicates Active-Site Residues in Protein-Substrate Recognition
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DOI：
10.1021/bi4011732
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发表时间：
2014-01-21
期刊：
影响因子：
2.9
通讯作者：
Hougland, James L.
中科院分区：
文献类型：
--
作者：
Gangopadhyay, Soumyashree A.;Losito, Erica L.;Hougland, James L.
翻译如下：
翻译后修饰对许多蛋白质的功能至关重要。异戊二烯化就是这样一种修饰，其中蛋白质香叶基香叶基转移酶I型（GGTase - I）或蛋白质法尼基转移酶（FTase）通过分别将一个20碳或15碳的类异戊二烯基团连接到靶蛋白C末端附近的一个半胱氨酸残基上来修饰蛋白质。这些酶在其底物上需要一个C末端Ca(1)a(2)X序列，其中a(1)、a(2)和X残基作为FTase和/或GGTase - I的底物识别元件。虽然大鼠GGTase - I的晶体结构显示出一个紧密堆积且疏水的a(2)残基结合口袋，这与GGTase - I底物中对中等大小的a(2)残基的偏好一致，但该活性位点内酶 - 底物接触的功能影响仍有待确定。通过定点诱变和肽底物构效关系研究，我们已经确定了大鼠GGTase - I中参与底物识别的特定活性位点残基，并开发了具有扩展/改变的底物选择性的新型GGTase - I变体。大幅改变GGTase - I选择性的能力与在FTase中观察到的类似行为相似，但采用了一组不同的结构同源活性位点残基的突变：我们的工作表明，可调节的选择性可能是参与翻译后修饰的多特异性酶中的一种普遍现象，并提出了来自不同生物体的GGTase - I直系同源物之间底物选择性可变的可能性。此外，本文开发的GGTase - I变体可作为研究GGTase - I底物选择性以及异戊二烯化途径修饰对特定蛋白质影响的工具。
Posttranslational modifications are vital for the function of many proteins. Prenylation is one such modification, wherein protein geranylgeranyltransferase type I (GGTase-I) or protein farnesyltransferase (FTase) modify proteins by attaching a 20- or 15-carbon isoprenoid group, respectively, to a cysteine residue near the C-terminus of a target protein. These enzymes require a C-terminal Ca(1)a(2)X sequence on their substrates, with the a(1), a(2), and X residues serving as substrate-recognition elements for FTase and/or GGTase-I. While crystallographic structures of rat GGTase-I show a tightly packed and hydrophobic a(2) residue binding pocket, consistent with a preference for moderately, sized a(2) residues in GGTase-I substrates, the functional impact of enzyme substrate contacts within this active site remains to be determined. Using site-directed mutagenesis and peptide substrate structure activity studies, we have identified specific active-site residues within rat GGTase-I involved in substrate recognition and developed novel GGTase-I variants with expanded/altered substrate selectivity. The ability to drastically alter GGTase-I selectivity mirrors similar behavior observed in FTase but employs mutation of a distinct set of structurally homologous active-site residues: Our work demonstrates that tunable selectivity may be a general phenomenon among multispecific enzymes involved in posttranslational modification and raises the possibility of variable substrate selectivity among GGTase-I orthologues from different organisms. Furthermore, the GGTase-I variants developed herein can serve as tools for studying GGTase-I substrate selectivity and the effects of prenylation pathway modifications on specific proteins.