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

• Published in: Molecular cell 2011-03, Vol.41 (6), p.682-692
• Main Authors: Khushoo, Amardeep, Yang, Zhongying, Johnson, Arthur E., Skach, William R.
• Format: Article
• Language: English
• Subjects: adenosine triphosphate; Adenosine Triphosphate - metabolism; ATP; Binding Sites; biosynthesis; Compaction; Cystic fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator - chemistry; Cystic Fibrosis Transmembrane Conductance Regulator - genetics; Cystic Fibrosis Transmembrane Conductance Regulator - metabolism; energy transfer; fluorescence; Fluorescence Resonance Energy Transfer; Humans; Kinetics; Ligands; Models, Molecular; polypeptides; Protein Binding; Protein Folding; Protein structure; Protein Structure, Tertiary; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Ribosomes; Ribosomes - metabolism; Tunnels
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2011.02.027

The mechanism by which protein folding is coupled to biosynthesis is a critical, but poorly understood, aspect of protein conformational diseases. Here we use fluorescence resonance energy transfer (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 multistep pathway as it is synthesized on the ribosome. Folding begins abruptly as NBD1 residues 389–500 emerge from the ribosome exit tunnel, 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. ► CFTR NBD1 folds cotranslationally on the ribosome via distinct intermediates ► NBD1 folding is initiated by compaction of an ATP-binding N-terminal subdomain ► N-terminal subdomain folding is rapid and occurs coincident with synthesis ► ATP binding stimulates de novo NBD1 folding