An NMR structural study on the dynamic interplay between chaperone factors involved in co-translational protein folding: Unveiling a modulating activity of TF in SecA sensing the nascent chain

Abstract

This research employs state-of-the-art methodologies in biochemistry and NMR structural biology to uncover the molecular complexities governing the actions of SecA and TF as they guide the NCs towards their 3-Dimensional fold. Employing the NMR SOFAST technique and using the Methyl TROSY approach, I have elucidated the dynamic interactions between SecA, TF, and diverse types and lengths of nascent chain ribosome complexes (RNCs), including those destined to the outer membrane (e.g., OmpA85, OmpA192, OmpA192ms), the cytosol (DHFR), or the inner membrane (PhoA).Our findings reveal that the conformation of SecA changes while interacting with the ribosome, significantly influenced by the presence of the NC, supporting a growing body of evidence (Huber et al., 2017; Wang et al., 2019; Z. Zhu, Wang, & Shan, 2022) that SecA participates in co-translational protein folding. Specifically, according to our results, SecA behaves differently depending on the size and type of the NC being synthesized, establishing stronger interactions with the longer RNCs. Notably, our observations also underscore the pivotal role of domains NBD-1 and NBD-2 in the SecA-RNC interactions, in agreement with the recent structural and biochemical studies (Huber et al., 2017; Knüpffer et al., 2019; Wang et al., 2019). Additional changes are observed in the PBD and HWD domains of SecA, explained by the well-documented domain rearrangements of the PBD, switching between the open and closed conformation (Vandenberk et al., 2019; Zimmer & Rapoport, 2009). We also demonstrate, for the first time, to the best of our knowledge, an interaction between the CTT of SecA and TF. Furthermore, the addition of TF to the SecA-RNC complex results in a concentration-dependent increase in SecA peak intensities, along with significant chemical shift perturbations (CSPs) primarily for the longer RNCs, indicating that TF can modulate the interaction between SecA and the RNC. Overall, these findings provide valuable insights into the conformational dynamics of the SecA co-translational protein targeting pathway in response to TF