Role of dimerization in the AAA+ adaptor SspB

AAA+ atp酶在所有生物王国中都参与了一系列重要的细胞事件，并且经常依赖于接头蛋白来调节底物特异性。CIpX是一种AAA+ atp酶，可展开蛋白质底物并将其转运到CIpP的蛋白水解腔中进行降解。递送蛋白SspB显著增强了具有特定c端肽序列（称为ssra -标签）的蛋白质的CIpXP降解。SspB是一种同型二聚体，它结合两个ssra标记的底物，并将它们呈现给CIpXP降解。我们目前的观点是，SspB通过以CIpX结合（XB）序列结束的柔性c端连接体松散地连接到CIpX上，允许它按顺序递送两种底物分子进行降解。然而，目前尚不清楚SspB二聚体是否在递送过程中分离，是否实际上递送了两种底物，二聚体中的两个XB区域是否对与CIpX的相互作用至关重要，或者SspB是否可以作为单体发挥作用。我建议通过使用在特定特性上有缺陷的突变蛋白，以及生化和生物物理分析来测试和完善我们的模型，以了解这个迷人的生物系统是如何工作的，从而回答这些问题。本文提出的研究将明确二聚化在SspB功能中的作用。除了CIpX， AAA+ atp酶的许多其他成员与低聚适配蛋白相互作用，包括SNARE/NSF。了解SspB二聚化在其与CIpXP的功能中的作用可能会广泛影响我们对其他低聚适配器与AAA+ ATPases相互作用的理解。

AAA+ ATPases are involved in a wide array of critical cellular events in all kingdoms of life and frequently rely on adaptor proteins to modulate substrate specificity. CIpX is an AAA+ ATPase that unfolds protein substrates and translocates them into the proteolytic chamber of CIpP for degradation. The delivery protein SspB dramatically enhances the CIpXP degradation of proteins with a specific C-terminal peptide sequence called the ssrA-tag. SspB is a homodimer that binds two ssrA-tagged substrates and presents them to CIpXP for degradation. Our current view is that SspB is loosely tethered to CIpX by flexible C-terminal linkers that end with a CIpX binding (XB) sequence, allowing it to sequentially deliver both substrate molecules for degradation. However, it is not known whether the SspB dimer comes apart during the delivery process, whether both substrates are in fact delivered, whether both XB regions in the dimer are critical for interactions with CIpX, or whether SspB can function as a monomer. I propose to answer these questions by using mutant proteins defective in specific properties together with biochemical and biophysical assays to test and refine our model for how this fascinating biological system works. The studies proposed herein will define the role of dimerization in SspB function. In addition to CIpX, many other members of the AAA+ ATPases interact with oligomeric adapter proteins including SNARE/NSF. Understanding the role of SspB dimerization in its function with CIpXP could broadly impact our understanding of the interactions of other oligomeric adapters with AAA+ ATPases.