Wen Kuo-Kuang, Blake Milan S, Rubenstein Peter A
Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USA.
J Muscle Res Cell Motil. 2004;25(4-5):343-50. doi: 10.1007/s10974-004-6069-y.
Neisserial porins may play a role in the invasion of the host cell by the bacterium. The protein translocates to the host cell membrane and then to the cytosol during the invasive process, and we have shown it interacts with actin in vitro. Here, we have examined the nucleotide-dependence of the interaction of Neisseria porin, P.IB, with fluorescently labeled yeast G actin. Increasing free ATP between 0 to 0.5 mM retards complex formation between the two proteins. The ATP effect probably results from binding of the nucleotide to actin rather than to porin. Complex formation results in a biphasic release of bound nucleoside triphosphate from actin in the absence of free nucleotide at a rate slower than that of complex formation, but it does not induce hydrolysis of the actin-bound nucleotide. ATP prevents the porin-induced distortion of F-actin structure, and addition of ATP to the complex formed in the absence of free nucleotide induces actin polymerization indicating that P.IB stabilizes nucleotide-free G-actin. Our results suggest that P.IB causes an actin conformation change leading to the production of a polymerization-competent nucleotide-free protein.
奈瑟菌孔蛋白可能在细菌侵袭宿主细胞过程中发挥作用。在侵袭过程中,该蛋白转位至宿主细胞膜,然后进入细胞质,并且我们已证明它在体外与肌动蛋白相互作用。在此,我们研究了奈瑟菌孔蛋白P.IB与荧光标记的酵母G肌动蛋白相互作用的核苷酸依赖性。在0至0.5 mM之间增加游离ATP会延迟两种蛋白之间复合物的形成。ATP的作用可能是由于核苷酸与肌动蛋白结合而非与孔蛋白结合所致。在没有游离核苷酸的情况下,复合物的形成导致结合在肌动蛋白上的核苷三磷酸以比复合物形成速率慢的速度双相释放,但它不会诱导肌动蛋白结合核苷酸的水解。ATP可防止孔蛋白诱导的F -肌动蛋白结构变形,并且在没有游离核苷酸的情况下向形成的复合物中添加ATP会诱导肌动蛋白聚合,这表明P.IB可稳定无核苷酸的G -肌动蛋白。我们的结果表明,P.IB会导致肌动蛋白构象改变,从而产生具有聚合能力的无核苷酸蛋白。