Structural organization of the fibrinogen-binding region of the clumping factor B MSCRAMM of Staphylococcus aureus
Structural organization of the fibrinogen-binding region of the clumping factor B MSCRAMM of Staphylococcus aureus
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DOI：
10.1074/jbc.m106741200
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发表时间：
2001-11-30
影响因子：
4.8
通讯作者：
Höök, M
中科院分区：
文献类型：
--
作者：
Perkins, S;Walsh, EJ;Höök, M
金黄色葡萄球菌的凝集因子B（Clf B）是结合纤维蛋白原的表面蛋白（Ni Eidhin，D.，珀金斯，S.，Francois，P.，Vaudaux，P.，Hook，M.，和Foster，T. J.，1998 Mol. Microbiol. 30，245-257）。配体结合活性位于类似于500个残基的A区（残基44-542），其代表MSCRAMM蛋白的N-末端一半。我们现在假设ClfB A区由三个亚结构域组成，分别命名为N1、N2和N3。为了检验这一假设，我们表达了各个推定的亚结构域、串联基序N12和N23以及全长A区N123的重组形式。远紫外圆二色性光谱表明，每个亚结构域主要由β-折叠组成，具有很少或没有可辨别的α-螺旋。热诱导的个别子域的展开发生与一个单一的状态转换，是可逆的，这表明子域可以折叠为离散的单位。凝胶渗透色谱表明N2、N3和N23为球形。相比之下，结构域N1似乎是延长的，并在含有该亚结构域的区段（即N12或N123）上赋予稍微延长的结构。N123、N12和N23都与纤维蛋白原结合，但N23对纤维蛋白原的亲和力高于对全长A区、N123或N12观察到的亲和力。然而，N23的延长的N末端是配体结合所必需的。通过蛋白水解加工产生的并且缺乏N-末端延伸的N23的形式不能结合纤维蛋白原。单个亚结构域的重组形式不结合纤维蛋白原。添加重组N23有效地抑制ClfB介导的细菌粘附纤维蛋白原，和N123引起细菌附着的一些减少，而N12基本上是无活性的。针对A区的中央N2结构域的抗体在抑制细菌粘附到固定的纤维蛋白原上是最有效的，尽管抗N3或抗N1抗体也引起ClfB介导的粘附到纤维蛋白原上的一些减少。
The clumping factor B (ClfB) of Staphylococcus aureus is a surface protein that binds to fibrinogen (Ni Eidhin, D., Perkins, S., Francois, P., Vaudaux, P., Hook, M., and Foster, T. J., 1998 Mol. Microbiol. 30, 245-257). The ligand-binding activity is located in the similar to 500-residue A-region (residues 44-542), which represents the N-terminal half of the MSCRAMM protein. We now hypothesize that the ClfB A-region is composed of three subdomains, which we have named N1, N2, and N3, respectively. To examine this hypothesis, we expressed recombinant forms of the individual putative subdomains, the tandem motifs N12 and N23, and the full-length A-region N123. Far UV circular dichroism spectra showed that each subdomain is composed mainly of beta -sheets with little or no discernible alpha -helices. Heat-induced unfolding of individual subdomains occurred with a single state transition and was reversible, indicating that the subdomains can fold as discreet units. Gel permeation chromatography indicated that N2, N3, and N23 are globular. In contrast, domain Nl appeared to be elongated and conferred a somewhat elongated structure on segments containing this subdomain (i.e. N12 or N123). N123, N12, and N23 all bound to fibrinogen, but N23 had a higher affinity for fibrinogen than that observed for the full-length A-region; N123 or for N12. However, an extended N terminus of N23 was required for ligand binding. A form of N23 that was generated by proteolytic processing and lacked the N-terminal extension was unable to bind fibrinogen. Recombinant forms of individual subdomains did not bind fibrinogen. The addition of recombinant N23 effectively inhibited ClfB-mediated bacterial adherence to fibrinogen, and N123 caused some reduction in bacterial attachment, whereas N12 was essentially inactive. Antibodies raised against the central N2 domain of the A-region were the most effective at inhibiting bacterial adhesion to immobilized fibrinogen, although anti-N3 or anti-N1 antibodies also caused some reduction in ClfB-mediated adherence to fibrinogen.