Role of dimerization in the AAA+ adaptor SspB

AAA+ ATP酶参与所有生命领域中的一系列重要细胞事件，并且经常依赖于衔接蛋白来调节底物特异性。ClpX是AAA+ ATP酶，其将蛋白质底物展开并将其易位到ClpP的蛋白水解室中进行降解。递送蛋白SspB显著增强具有称为ssrA标签的特定C末端肽序列的蛋白质的ClpXP降解。SspB是一种同二聚体，结合两个ssrA标记的底物，并将其呈递给ClpXP进行降解。我们目前的观点是，SspB通过柔性C-末端接头与ClpX松散地连接，该接头以ClpX结合（XB）序列结束，使其能够顺序地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开，两种底物是否实际上都被递送，二聚体中的两个XB区域是否对与ClpX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质以及生物化学和生物物理测定来回答这些问题，以测试和完善我们的模型，了解这个迷人的生物系统是如何工作的。本文提出的研究将定义二聚化在SspB功能中的作用。除了ClpX之外，AAA+ ATP酶的许多其他成员与寡聚衔接蛋白（包括SNARE/NSF）相互作用。理解SspB二聚化在其与ClpXP的功能中的作用可以广泛地影响我们对其他寡聚衔接子与AAA+ ATP酶的相互作用的理解。

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.