Role of dimerization in the AAA+ adaptor SspB

AAA+ATPase参与所有生命王国中广泛的关键细胞事件，并经常依赖接头蛋白来调节底物专一性。CIpX是一种AAA+ATPase，它能打开蛋白质底物，并将它们转移到CIPP的蛋白水解室中进行降解。传递蛋白SspB极大地促进了具有特定C-末端多肽序列的蛋白质的CIpXP降解，该序列称为ssrA-Tag。SspB是一种同源二聚体，它结合两个标记了ssrA的底物，并将它们呈现给CIpXP进行降解。我们目前的观点是，SspB通过以CIpX结合(XB)序列结尾的灵活的C-末端连接子松散地与CIpX捆绑在一起，使其能够顺序地递送两个底物分子进行降解。然而，目前尚不清楚SspB二聚体是否在递送过程中分离，两个底物是否确实被递送，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性质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。本文提出的研究将确定二聚化在SspB功能中的作用。除CIpX外，AAA+ATPase的许多其他成员还与包括SNARE/NSF在内的寡聚体接头蛋白相互作用。了解SspB二聚化在其与CIpXP功能中的作用可能会广泛影响我们对其他寡聚适配器与AAA+ATPase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.