Study of the molecular interaction between RssB Adaptor protein and IraP Anti‐Adaptor protein in Escherichia coli

Abstract

In exponential phase, the stationary phase sigma factor σS is maintained at low levels by proteolysis; RssB binds directly to σS and targets it to the ClpXP protease. Under stress conditions this process is inhibited, in part by sequestration of RssB by anti‐adaptor proteins. IraP is an anti‐adaptor made after phosphate starvation. We screened for RssB mutants resistant to IraP. The mutants identify a critical and conserved region between amino acids 214–221 in the C‐terminal domain of RssB. In vivo, cells carrying plasmids encoding RssBL214H or RssBA216T were able to degrade σS even in the presence of high levels of IraP. In a bacterial two‐hybrid assay, RssBL214H and RssBA216AT do not interact with IraP, while RssB+ does. In vitro, purified RssB mutant proteins were fully functional for RpoS degradation; RssBwt but not RssBL214H and RssBA216T were inhibited by IraP. Pull‐down analysis confirmed the loss of interaction of RssBL214H and RssBA216T with IraP. Thus, RssBL214H and RssBA216T have lost the interaction with IraP. Because other data suggests that IraP interacts with the N‐terminal domain of RssB, and these mutations are in the C‐terminal domain, we believe these mutations lead to an allosteric change in RssB, blocking the IraP interaction site. This allosteric change may normally be part of the activation cycle allowing RssB to deliver σS to the protease.