Localized protein interaction surfaces on the EntB carrier protein revealed by combinatorial mutagenesis and selection
Localized protein interaction surfaces on the EntB carrier protein revealed by combinatorial mutagenesis and selection
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DOI：
10.1021/ja063238h
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发表时间：
2006-08-30
影响因子：
15
通讯作者：
Walsh, Christopher T.
中科院分区：
文献类型：
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作者：
Lai, Jonathan R.;Fischbach, Michael A.;Walsh, Christopher T.
载体蛋白是80至100个残基的中途站，是聚酮合酶（PKS）和非核糖体肽合成酶（NRPS）酶促组装线的中心。由于催化操作的生物合成中间体在载体蛋白上以共价连接的硫酯形式存在（通过4 '-磷酸泛酰巯基乙胺辅基），载体蛋白与其他NRPS/PKS结构域之间的特定蛋白质-蛋白质相互作用对于高保真转化为最终产物至关重要。在这里，我们通过组合诱变和选择表明，EntB的芳基载体蛋白（EntB−ArCP）包含局部蛋白质相互作用表面。我们的策略包括对EntB−ArCP的N端区域进行随机诱变，然后通过在缺铁培养基上铺板来选择产生肠杆菌素的克隆。我们确定了几个在我们的选择中高度保守的残基，其中两个（G242和D244）构成了磷酸泛酰巯基乙胺基转移酶（PPT酶）EntD和Sfp在EntB−ArCP上的相互作用表面。这个PPTase接口是不同于以前的特征接口EntB-ArCP的下游延伸模块，EntF。这些结果表明，不同的蛋白质组分识别肠杆菌素合成酶中EntB−ArCP的不同表面，并且大多数EntB−ArCP表面残基不参与这些相互作用。因此，在PKS和NRPS系统中设计非同源载体蛋白相互作用应该是可能的，在特定载体蛋白上具有非常少的突变。
Carrier proteins are 80- to 100-residue way stations that are central to polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) enzymatic assembly lines. Because the biosynthetic intermediates for catalytic operations are presented on carrier proteins as covalently attached thioesters (via a 4‘-phosphopantetheine prosthetic group), the specific protein−protein interactions between carrier proteins and other NRPS/PKS domains are critical for high-fidelity conversion to the final product. Here we show by combinatorial mutagenesis and selection that the aryl carrier protein of EntB (EntB−ArCP) contains localized protein interaction surfaces. Our strategy involved random mutagenesis of N-terminal regions of EntB−ArCP, then selection for clones that produce enterobactin by plating onto iron-deficient media. We identified several residues that were highly conserved from our selection, two of which (G242 and D244) constitute an interaction surface on EntB−ArCP for the phosphopantetheinyl transferases (PPTases) EntD and Sfp. This PPTase interface is distinct from a previously characterized interface on EntB−ArCP for the downstream elongation module, EntF. These results suggest that different protein components recognize different faces of EntB−ArCP in the enterobactin synthetase and that the majority of EntB−ArCP surface residues are not involved in these interactions. Therefore, designing noncognate carrier protein interactions in PKS and NRPS systems should be possible with very few mutations on a particular carrier protein.