Ligand-Driven Vectorial Folding of Ribosome-Bound Human CFTR NBD1

Abstract

SUMMARY The mechanism by which protein folding is coupled to biosynthesis is a critical, but poorly understood aspect of protein conformational diseases. Here we use FRET to characterize tertiary structural transitions of nascent polypeptides and show that the first nucleotide-binding domain (NBD1) of human CFTR, whose folding is defective in cystic fibrosis, folds via a cotranslational multi-step pathway as it is synthesized on the ribosome. Folding begins abruptly as NBD1 residues 389–500 emerge from the ribosome exit tunnel, thereby initiating compaction of a small, N-terminal α/β-subdomain. Real-time kinetics of synchronized nascent chains revealed that subdomain folding is rapid, occurs coincident with synthesis, and is facilitated by direct ATP binding to the nascent polypeptide. These findings localize the major CF defect late in the NBD1 folding pathway and establish a paradigm wherein a cellular ligand promotes vectorial domain folding by facilitating an energetically favored local peptide conformation.

Kinetic Analysis of Ribosome-bound Fluorescent Proteins Reveals an Early, Stable, Cotranslational Folding Intermediate

“…In vitro transcription was carried out (43) using SP6 polymerase at 40°C for 1 h and added directly (20% volume) to a rabbit reticulocyte lysate (RRL) in vitro translation reaction supplemented with [ 35 S]methionine and a synthetic suppressor aminoacyl-tRNA, [ 14 C]Lys-tRNA amb (0.8 M) (25,44,45). Translation was carried out at 24°C for 1 h as described previously (25). For fluorescence spectroscopy, [ 35 S]methionine was omitted and the translation reaction was supplemented with 40 M methionine.…”

“…In such a system, conformational status can be assessed by functional properties (17)(18)(19)(20), limited proteolysis (21,22), conformation specific antibodies (23,24), fluorescence resonance energy transfer (25,26), and recently, NMR spectroscopy (27,28). Although relatively few substrates have been examined to date, findings indicate that folding in the cell can increase kinetics and improve folding efficiency when compared with in vitro refolding studies (17,18,20,21).…”

In vivo translation rates can substantially delay the cotranslational folding of the Escherichia coli cytosolic proteome

“…Under this assumption, these domains are only capable of forming a stable, folded tertiary structure once all the residues comprising the domain have emerged from the exit tunnel and are sterically permitted to come into contact, which can include tertiary structure formation in the last 2 nm of the exit tunnel (24,32). Recent experimental results, however, call this assumption into question because the CATH-defined NBD1 domain of human CFTR protein was found to exhibit stable subdomain folding during synthesis on the ribosome (33). Thus, noncooperative folding can occur on the ribosome, with a stable native tertiary structure forming before synthesis of the entire domain is complete.…”

The ribosome regulates flavodoxin folding

“…Depending on nascent chain conformation, the exit tunnel can hold between 30 (extended) to 70 (α-helical) amino acid residues. Folding or misfolding of the nascent chain may already start in the exit tunnel (151)(152)(153)(154) and in most cases commences while the nascent chain gradually emerges from it (51)(52)(53)55,(155)(156)(157)(158)(159)(160)(161)(162)(163). As synthesis of the nascent chain is orders of magnitude slower than it takes a protein to fold (i.e., seconds versus micro-to milliseconds), nascent chains can already sample parts of their conformational space before the next amino acid residue is added.…”

“…Methodologies like epitope recognition, enzymatic activity, cofactor binding, NMR and fluorescence spectroscopy indicate structural ordering and acquisition of activity of polypeptides once they arrive outside the exit tunnel (51)(52)(53)55,(155)(156)(157)(158)(159)(160)(161)(162)(163). Cotranslational folding has been observed for all α (51), all β (53,55,159,162) and α/β proteins (54,178,179).…”