Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, MA 01003.
Molecular and Cellular Biology Program, University of Massachusetts Amherst, Amherst, MA 01003.
Proc Natl Acad Sci U S A. 2021 Apr 27;118(17). doi: 10.1073/pnas.2010523118.
Bacterial protein degradation is a regulated process aided by protease adaptors that alter specificity of energy-dependent proteases. In , cell cycle-dependent protein degradation depends on a hierarchy of adaptors, such as the dimeric RcdA adaptor, which binds multiple cargo and delivers substrates to the ClpXP protease. RcdA itself is degraded in the absence of cargo, and how RcdA recognizes its targets is unknown. Here, we show that RcdA dimerization and cargo binding compete for a common interface. Cargo binding separates RcdA dimers, and a monomeric variant of RcdA fails to be degraded, suggesting that RcdA degradation is a result of self-delivery. Based on HDX-MS studies showing that different cargo rely on different regions of the dimerization interface, we generate RcdA variants that are selective for specific cargo and show cellular defects consistent with changes in selectivity. Finally, we show that masking of cargo binding by dimerization also limits substrate delivery to restrain overly prolific degradation. Using the same interface for dimerization and cargo binding offers an ability to limit excess protease adaptors by self-degradation while providing a capacity for binding a range of substrates.
细菌蛋白降解是一个受调控的过程，需要蛋白酶衔接物的辅助，以改变能量依赖型蛋白酶的特异性。在 ，细胞周期依赖性蛋白降解依赖于衔接子的层次结构，如二聚体 RcdA 衔接子，它可以结合多个货物并将底物递送到 ClpXP 蛋白酶。在没有货物的情况下，RcdA 自身会被降解，而 RcdA 如何识别其靶标尚不清楚。在这里，我们表明 RcdA 二聚化和货物结合竞争一个共同的界面。货物结合会分离 RcdA 二聚体，而 RcdA 的单体变体不能被降解，这表明 RcdA 的降解是自我递呈的结果。基于 HDX-MS 研究表明，不同的货物依赖于二聚化界面的不同区域，我们生成了对特定货物具有选择性的 RcdA 变体，并显示出与选择性变化一致的细胞缺陷。最后，我们表明通过二聚化掩盖货物结合也限制了底物的递呈，以限制过度丰富的降解。将二聚化和货物结合使用相同的界面提供了一种通过自我降解限制过多蛋白酶衔接物的能力，同时提供了结合一系列底物的能力。
