GroEL/GroES cycling: ATP binds to an open ring before substrate protein favoring protein binding and production of the native state

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 106; no. 48; pp. 20264 - 20269
• Main Authors: Tyagi, Navneet K, Fenton, Wayne A, Horwich, Arthur L
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 01.12.2009; National Acad Sciences
• Subjects: Adenosine triphosphatase; Adenosine Triphosphate - metabolism; Aggregation; Binding sites; Biological Sciences; Carboxylic Acids; Chaperonin 10 - metabolism; Chaperonin 60 - metabolism; Chaperonins; Emission spectra; Encapsulating; Encapsulation; Fluorescence; Fluorescence Resonance Energy Transfer; Kinetics; Mathematical rings; Models, Molecular; Protein Binding; Protein Folding; Protein refolding; Proteins; Ribulose-Bisphosphate Carboxylase - metabolism
• DOI: 10.1073/pnas.0911556106
• ISSN: 0027-8424

The GroEL/GroES reaction cycle involves steps of ATP and polypeptide binding to an open GroEL ring before the GroES encapsulation step that triggers productive folding in a sequestered chamber. The physiological order of addition of ATP and nonnative polypeptide, typically to the open trans ring of an asymmetrical GroEL/GroES/ADP complex, has been unknown, although there have been assumptions that polypeptide binds first, allowing subsequent ATP-mediated movement of the GroEL apical domains to exert an action of forceful unfolding on the nonnative polypeptide. Here, using fluorescence measurements, we show that the physiological order of addition is the opposite, involving rapid binding of ATP, accompanied by nearly as rapid apical domain movements, followed by slower binding of nonnative polypeptide. In order-of-addition experiments, approximately twice as much Rubisco activity was recovered when nonnative substrate protein was added after ATP compared with it being added before ATP, associated with twice as much Rubisco protein recovered with the chaperonin. Furthermore, the rate of Rubisco binding to an ATP-exposed ring was twice that observed in the absence of nucleotide. Finally, when both ATP and Rubisco were added simultaneously to a GroEL ring, simulating the physiological situation, the rate of Rubisco binding corresponded to that observed when ATP had been added first. We conclude that the physiological order, ATP binding before polypeptide, enables more efficient capture of nonnative substrate proteins, and thus allows greater recovery of the native state for any given round of the chaperonin cycle.