How SecB maintains clients in a translocation competent state.

Abstract

Bacterial secretory proteins must remain soluble and non-folded until they reach the SecYEG translocase. Preprotein intrinsic features and chaperones can delay protein folding. The SecB chaperone is known to delay folding of some proteins, however the link between the folding state of a client and its interaction with SecB, until the client-transfer to the translocase remains elusive. This study unravels how a model client, maltose binding protein (MBP), is kept in a non-folded state by SecB. Using single-molecule FRET and hydrogen-deuterium exchange mass spectrometry, we describe in detail the folding pathway of the client and demonstrate that SecB acts first as an unfoldase, reverting partial folding and then as holdase, preventing folding. The presence of an SP delays the folding and stabilizes the client to SecB interaction. Single-point mutations that abolish formation of some foldons drastically increase the lifetime of the SecB-bound state. Towards delivery to the translocase, SecA interacts with the MBP:SecB complex forming a quaternary super-assembly thus, further stabilizing the disordered state of the client. Collectively, our study demonstrates the interplay between secretory chaperones and a model client, with SecB combining unfoldase and holdase activities to retain the client in a translocation-competent state while SecA secures this complex until later translocation.