Requirement for binding multiple ATPs to convert a GroEL ring to the folding-active state

Abstract

Production of the folding-active state of a GroEL ring involves initial cooperative binding of ATP, recruiting GroES, followed by large rigid body movements that are associated with ejection of bound substrate protein into the encapsulated hydrophilic chamber where folding commences. Here, we have addressed how many of the 7 subunits of a GroEL ring are required to bind ATP to drive these events, by using mixed rings with different numbers of wild-type and variant subunits, the latter bearing a substitution in the nucleotide pocket that allows specific block of ATP binding and turnover by a pyrazolol pyrimidine inhibitor. We observed that at least 2 wild-type subunits were required to bind GroES. By contrast, the triggering of polypeptide release and folding required a minimum of 4 wild-type subunits, with the greatest extent of refolding observed when all 7 subunits were wild type. This is consistent with the requirement for a ''power stroke'' of forceful apical movement to eject polypeptide into the chamber.chaperonin ͉ chemical inhibitor ͉ nucleotide ͉ protein folding ͉ pyrazolol pyrimidine

Reconciling theories of chaperonin accelerated folding with experimental evidence

“…After protein binding, the subsequent binding of ATP [52,82] can further unfold the substrate protein [10,11,44,52,64]. ATP binding also provides enough energy (roughly 46 kcal/mol per ring [83,84]) to allosterically free GroES [85], and possibly the substrate protein (yellow) from the opposite ring, less than 1 s later [52]. The subsequent binding of GroES to the top ring eventually frees the protein from the opening of GroEL and into the newly formed ''cis'' chamber, where it can continue folding [10,11,21,40,52,[64][65][66][67][68]86].…”

“…2a and 4, sideways arrows). ATP binding and consumption provides the energy for GroEL to repeatedly bind to proteins, denature them, and (sometimes) release them [40,44,[82][83][84][85]. Stringent GroEL-dependent proteins typically endure many such ATPase cycles before folding [13] (see also [7,16,80,91,102]).…”

Chaperonotherapy for Alzheimer’s Disease: Focusing on HSP60

“…One strategy aims at targeting HSP60 cysteine residues either as oxidizable sites [128] or for covalent binding, presumably through interaction with an electrophilic compound [129][130][131]. The other approach targets ATP binding and hydrolysis sites-functions, thus affecting those ATP-dependent conformational changes of HSP60 which are crucial for the protein folding function [132][133][134].…”

“…4.6, left) -can form a complex with HSP60 thus affecting its protein-folding activity [132,133]. A recent study further showed that mizoribine (6) slowed down the folding cycle by affecting ATP hydrolysis.…”

“…4.6, right), was reported to block ATP binding and hydrolysis thus affecting the protein-folding function of HSP60 [133]. Despite its activity, no update on the use of EC3016 (7) has appeared since the first report of its HSP60 inhibitory function.…”