Probing the phosphoinositide 4,5-bisphosphate binding site of human profilin I
Probing the phosphoinositide 4,5-bisphosphate binding site of human profilin I
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DOI：
10.1016/s1074-5521(98)90620-2
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发表时间：
1998-05-01
影响因子：
--
通讯作者：
Prestwich, GD
中科院分区：
文献类型：
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作者：
Chaudhary, A;Chen, J;Prestwich, GD
背景：Profilin是一种广泛高表达的14 kDa蛋白质，可与肌动蛋白单体、多(L-脯氨酸)和多磷肌醇脂类结合。Profilin I参与调节肌动蛋白-细丝动力学，而肌动蛋白是多种细胞运动所必需的，我们试图研究profilin与磷脂酰肌醇相互作用的部位。结果：三种含氚标记的4-苯甲酰二氢肉桂酰(BZDC)磷脂酰肌醇4，5-二磷酸(PtdIns(4，5)P-2)的光亲和类似物共价修饰人profilin I。P-1-三磷酸肌醇(Ins(1，4，5)P-3)能有效和特异地修饰Profilin I，与过量的PtdIns(4，5)P-2共孵育可取代共价标记，但不能与Ins(1，4，5)P-3共孵育取代共价标记。酰基修饰的PtdIns(4，5)P-2类似物即使在很低的浓度下也几乎没有蛋白质标记，而头基修饰的PtdIns(4，5)P-2磷酸三酯标记的单体和寡聚Profilin。对[H-3]BZDC-ins(1，4，5)P-3修饰的重组Profilin的CNBr酶切质谱分析表明，修饰发生在氨基末端的螺旋CNBr片段上。Edman降解证实Profilin I的Ala1(重组蛋白的4个残基)被修饰。结论：Profilin 1的PtdIns(4，5)P-2结合部位存在与羧基末端螺旋富碱基序的双磷酸盐相互作用，以及PtdIns(4，5)P-2与Profilin氨基末端螺旋疏水区的相互作用。这是磷脂酰肌醇二磷酸类似物的脂部分与Profilin相互作用的第一个直接证据。
Background: Profilin is a widely and highly expressed 14 kDa protein that binds actin monomers, poly(L-proline) and polyphosphoinositol lipids. It participates in regulating actin-filament dynamics that are essential for many types of cell motility, We sought to investigate the site of interaction of profilin with phosphoinositides.Results: Human profilin I was covalently modified using three tritium-labeled 4-benzoyldihydrocinnamoyl (BZDC)-containing photoaffinity analogs of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P-2). The P-1-tethered D-myoinositol 1,4,5-trisphosphate (Ins(1,4,5)P-3) modified profilin I efficiently and specifically; the covalent labeling could be displaced by co-incubation with an excess of PtdIns(4,5)P-2 but not with Ins(1,4,5)P-3. The acyl-modified PtdIns(4,5)P-2 analog showed little protein labeling even at very low concentrations, whereas the head-group-modified PtdIns(4,5)P-2 phosphotriester-labeled monomeric and oligomeric profilin. Mass spectroscopic analyses of CNBr digests of [H-3]BZDC-Ins(1 ,4,5)P-3-modified recombinant profilin suggested that modification was in the amino-terminal helical CNBr fragment. Edman degradation confirmed Ala1 of profilin I (residue 4 of the recombinant protein) was modified. Molecular models show a minimum energy conformation in which the hydrophobic region of the ligand contacts the amino-terminal helix whereas the 4,5-bisphosphate interacts with Arg135 and Arg136 of the carboxy-terminal helix.Conclusions: The PtdIns(4,5)P-2-binding site of profilin 1 includes a bisphosphate interaction with a base-rich motif in the carboxy-terminal helix and contact between the lipid moiety of PtdIns(4,5)P-2 and a hydrophobic region of the aminoterminal helix of profilin. This is the first direct evidence for a site of interaction of the lipid moiety of a phosphoinositide bisphosphate analog with profilin.