Role of helix B residues in interfacial activation of a bacterial phosphatidylinositol-specific phospholipase C
Role of helix B residues in interfacial activation of a bacterial phosphatidylinositol-specific phospholipase C
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DOI：
10.1021/bi702269u
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发表时间：
2008-04-08
期刊：
影响因子：
2.9
通讯作者：
Roberts, Mary F.
中科院分区：
文献类型：
--
作者：
Guo, Su;Zhang, Xin;Roberts, Mary F.
苏云金芽孢杆菌磷脂酰肌醇特异性磷脂酶C（PI-PLC）是一种与原核生物感染性相关的界面酶，通过与两性离子表面，特别是磷脂酰胆碱（PC）结合而激活。桶结构边缘的两个色氨酸残基（两圈螺旋B中的Trp 47和无序环中的Trp 242）对这种相互作用至关重要。野生型PI-PLC中的螺旋B区（Ile 43至Gly 48）使Ile 43和Trp 47的侧链定向，使得它们聚集在一起并从蛋白质表面形成疏水突起，这可能促进初始膜结合。在以前的研究中，我们报道了在二聚体W 47 A/W242 A突变体的晶体结构中，它不能与PC结合，螺旋B区域已被突变重组成一个延长的环。在这里，我们报告的建设和表征（催化活性，荧光和NMR研究）的一系列PI-PLC突变体的螺旋B残基和周围地区的目标，探索什么是需要稳定的“膜活性”构象的螺旋B区。结果强烈表明，虽然疏水基团和可能是一个完整的螺旋B的PI-PLC膜的初始结合是至关重要的，螺旋B的破坏，允许酶二聚化是什么导致激活的PI-PLC构象。
The Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), an interfacial enzyme associated with prokaryotic infectivity, is activated by binding to zwitterionic surfaces, particularly phosphatidycholine (PC). Two tryptophan residues (Trp47 in the two-turn helix B and Trp242 in a disordered loop) at the rim of the barrel structure are critical for this interaction. The helix B region (Ile43 to Gly48) in wild-type PI-PLC orients the side chains of Ile43 and Trp47 so that they pack together and form a hydrophobic protrusion from the protein surface that likely facilitates initial membrane binding. In previous studies we reported that in the crystal structure of the dimeric W47A/W242A mutant, which is unable to bind to PC, the helix B region has been reorganized by the mutation into an extended loop. Here we report the construction and characterization (catalytic activity, fluorescence, and NMR studies) of a series of PI-PLC mutants targeting helix B residues and surrounding regions to explore what is needed to stabilize the "membrane-active" conformation of the helix B region. Results strongly suggest that, while hydrophobic groups and presumably an intact helix B are critical for the initial binding of PI-PLC to membranes, disruption of helix B to allow enzyme dimerization is what leads to the activated PI-PLC conformation.