The accumulation-associated protein (Aap) from Staphylococcus epidermidis is a biofilm-related protein that was found to be a critical factor for infection using a rat catheter model. The B-repeat superdomain of Aap, composed of 5–17 B-repeats, each containing a Zn2+-binding G5 and a spacer subdomain, is responsible for Zn2+-dependent assembly leading to accumulation of bacteria during biofilm formation. We previously demonstrated that a minimal B-repeat construct (Brpt1.5) forms an antiparallel dimer in the presence of 2–3 Zn2+ ions. More recently, we have reported the presence of functional amyloid-like fibrils composed of Aap within S. epidermidis biofilms and demonstrated that a biologically relevant construct containing five and a half B-repeats (Brpt5.5) forms amyloid-like fibrils similar to those observed in the biofilm. In this study, we analyze the initial assembly events of the Brpt5.5 construct. Analytical ultracentrifugation was utilized to determine hydrodynamic parameters of reversibly associating species and to perform linked equilibrium studies. Linkage studies indicated a mechanism of Zn2+-induced dimerization similar to smaller constructs; however, Brpt5.5 dimers could then undergo further Zn2+-induced assembly into a previously uncharacterized tetramer. This led us to search for potential Zn2+-binding sites outside of the dimer interface. We developed a Brpt5.5 mutant that was unable to form the tetramer and was concordantly incapable of amyloidogenesis. CD and dynamic light scattering indicate that a conformational transition in the tetramer species is a critical step preceding amyloidogenesis. This mechanistic model for B-repeat assembly and amyloidogenesis provides new avenues for potential therapeutic targeting of staphylococcal biofilms.

中文翻译：

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葡萄球菌生物膜蛋白Aap形成四聚体物种，作为淀粉样蛋白形成之前的必要中间体。

表皮葡萄球菌的积累相关蛋白（Aap）是生物膜相关蛋白，使用大鼠导管模型发现它是感染的关键因素。Aap的B重复超域由5-17个B重复组成，每个重复域都包含一个Zn2 +结合G5和一个间隔子域，负责Zn2 +依赖的组装，导致细菌在生物膜形成过程中积累。我们以前曾证明，最小的B重复构建体（Brpt1.5）在存在2-3个Zn2 +离子的情况下会形成反平行二聚体。最近，我们已经报道了表皮葡萄球菌生物膜中由Aap组成的功能性淀粉样蛋白原纤维的存在，并证明了含有五个半B重复序列（Brpt5.5）的生物学相关构建物形成了类似于淀粉样蛋白原纤维的淀粉样蛋白原纤维。在生物膜中观察到。在这个研究中，我们分析了Brpt5.5结构的初始组装事件。分析超离心用于确定可逆结合物种的流体动力学参数，并进行链接的平衡研究。连锁研究表明，Zn2 +诱导的二聚化机制与较小的构建体相似。然而，Brpt5.5二聚体随后可能会进一步被Zn2 +诱导组装成以前未表征的四聚体。这导致我们在二聚体界面之外寻找潜在的Zn2 +结合位点。我们开发了一个Brpt5.5突变体，该突变体无法形成四聚体，并且始终不能产生淀粉样蛋白。CD和动态光散射表明四聚体物种中的构象转变是淀粉样蛋白生成之前的关键步骤。