Profilin interacts with the Gly-Pro-Pro-Pro-Pro-Pro sequences of vasodilator-stimulated phosphoprotein (VASP): Implications for actin-based Listeria motility
Profilin interacts with the Gly-Pro-Pro-Pro-Pro-Pro sequences of vasodilator-stimulated phosphoprotein (VASP): Implications for actin-based Listeria motility
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DOI：
10.1021/bi970065n
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发表时间：
1997-07-08
期刊：
影响因子：
2.9
通讯作者：
Purich, DL
中科院分区：
文献类型：
--
作者：
Kang, F;Laine, RO;Purich, DL
单核细胞增生性李斯特氏菌基于肌动蛋白的细胞内运动需要两个不同的富含脯氨酸的序列的蛋白质-蛋白质相互作用：第一，紧密结合的细菌表面蛋白ActA利用其多个寡核苷酸寄存器[共识序列=FE(D)FPPPPTD(E)E(D)]将血管扩张剂刺激的磷酸蛋白(Vasp)拴在细菌表面；然后，Vasp部署自己的多个GPPPPP(或GP(5))注册表来定位肌动蛋白调节蛋白profilin以促进肌动蛋白聚合，我们现在报道的荧光滴定表明GP(5)肽与profilin结合(K-D为84muM)，并且在没有profilin或存在profilin时，GP(5)肽弱抑制肌动蛋白结合核苷酸的交换。显微注射合成的GPPPPP三联体到感染李斯特氏菌的PtK2细胞中，当细胞内浓度为10mM时，细胞运动迅速停止，当等摩尔浓度的profilin和GP(5)同时微量注射时，这种抑制作用被完全中和。对[His-133-Ser]-Profilin的荧光研究表明，当该[His-133-Ser]-Profilin的等摩尔浓度的该[His-133-Ser]-Profilin突变体与GP(5)共注射时，很少或没有证据显示可饱和的GP(5)结合，多肽的抑制作用完全不受影响，表明GP(5)与野生型Profilin的结合代表了基于肌动蛋白的病原体运动的关键步骤。我们还提出了一个模型，该模型显示了VASP与其GPPPPP寄存器的焦点结合如何大大增加了细菌/火箭尾部界面上的Profilin和/或Profilin-Actin-ATP复合体的局部浓度。
Intracellular actin-based motility of Listeria monocytogenes requires protein-protein interactions involving two different proline-rich sequences: first, the tightly bound bacterial surface protein ActA uses its multiple oligoproline registers [consensus sequence = FE(D)FPPPPTD(E)E(D)] to tether vasodilator-stimulated phosphoprotein (VASP) to the bacterial surface; and second, VASP then deploys its own multiple GPPPPP (or GP(5)) registers to localize the actin-regulatory protein profilin to promote actin polymerization, We now report that fluorescence titration showed that GP(5)GP(5)GP(5) peptide binds to profilin (K-D of 84 mu M), and the peptide weakly inhibits exchange of actin-bound nucleotide in the absence or presence of profilin. Microinjection of synthetic GPPPPP triplet into Listeria-infected PtK2 cells promptly arrested motility at an intracellular concentration of 10 mu M. This inhibition was completely neutralized when equimolar concentrations of profilin and GP(5)GP(5)GP(5) were simultaneously microinjected. Fluorescence studies with [His-133-Ser]-profilin, a site-directed mutant previously shown to be defective in binding poly-L-proline [Bjorkegren, C., Rozycki, M., Schutt, C. E., Lindberg, U., & Karlsson, R. (1993) FEES Lett, 333, 123-126], exhibits little or no evidence of saturable GP(5)GP(5)GP(5) binding, When an equimolar concentration of this [His-133-Ser]-profilin mutant was co-injected with GP(5)GP(5)GP(5), the peptide's inhibitory action remained completely unaffected, indicating that GP(5)GP(5)GP(5) binding to wild-type profilin represents a key step in actin-based pathogen motility. We also present a model that shows how the focal binding of VASP with its GPPPPP registers can greatly increase the local concentration of profilin and/or profilin-actin-ATP complex at the bacteria/rocket-tail interface.