Abstract
Trigger factor (TF) is a highly conserved multi-domain molecular chaperone that exerts its chaperone activity at the ribosomal tunnel exit from which newly synthesized nascent chains emerge. TF also displays promiscuous substrate binding for a large number of cytosolic proteins independent of ribosome binding. We asked how TF recognizes a variety of substrates while existing in a monomer-dimer equilibrium. Paramagnetic nuclear magnetic resonance (NMR) and electron spin resonance (ESR) spectroscopy were used to show that dimeric TF displays a high degree of structural polymorphism in solution. A series of peptides has been generated to quantify their TF binding affinities in relation with their sequence compositions. The results confirmed a previous predication that TF preferentially binds to peptide fragments that are rich in aromatic and positively charged amino acids. NMR paramagnetic relaxation enhancement analysis showed that TF utilizes multiple binding sites, located in the chaperone domain and part of the prolyl trans-cis isomerization domain, to interact with these peptides. Dimerization of TF effectively sequesters most of the substrate binding sites, which are expected to become accessible upon binding to the ribosome as a monomer. As TF lacks ATPase activity, which is commonly used to trigger conformational changes within molecular chaperones in action, the ribosome-binding-associated disassembly and conformational rearrangements may be the underlying regulatory mechanism of its chaperone activity.
Highlights
Molecular chaperones are pivotal in facilitating protein folding and maintaining proteostasis in vivo (Hartl, 2016; Hartl and Hayer-Hartl, 2002)
Isolated ribosome binding domain (RBD), substrate binding domain (SBD), and peptidyl trans–cis isomerization domain (PPI) exhibit well-resolved 2D 15N−1H backbone amide and 13C−1H side-chain methyl correlation spectra, whose chemical shifts assignments have been previously reported at a residue-specific level (Huang and Hsu, 2016; Yao et al, 2008)
These assignments serve as the basis to complete the backbone and side-chain methyl nuclear magnetic resonance (NMR) chemical shift assignments through a divide-and-conquer assignment strategy despite the apparent high-molecular weight of full-length trigger factor (TF) of approximately 100 kDa (Morgado et al, 2017; Saio et al, 2014, 2018)
Summary
Molecular chaperones are pivotal in facilitating protein folding and maintaining proteostasis in vivo (Hartl, 2016; Hartl and Hayer-Hartl, 2002). TF binds to the ribosomal protein L23 through the RBD in a 1 : 1 stoichiometry at the exit of the ribosomal tunnel from which newly synthesized nascent polypeptide chains emerge during translation (Ferbitz et al, 2004; Lakshmipathy et al, 2007; Merz et al, 2008; Rutkowska et al, 2008). TF forms a dragon-like cradle at the ribosomal tunnel exit to sequester emerging nascent chains and continues to hold onto its substrates after being released from the ribosome until folding is complete (Ferbitz et al, 2004; Hesterkamp and Bukau, 1996). MTSL into single-cysteine TF variants, i.e., 14C, 150C, 326C, and 376C, was achieved by incubating the protein samples with 10 mM DTT, which was removed using a desalting column (PD-10, GE Healthcare, USA). As previously described [2], the ESE signals were fitted to a stretched exponential function to extract T2 values from the ESE data using the MATLAB software
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