Substrate protein folds while it is bound to the ATP-independent chaperone Spy

Abstract

Chaperones assist the folding of many proteins in the cell. While the most well studied chaperones use cycles of ATP binding and hydrolysis to assist protein folding, a number of chaperones have been identified that promote protein folding in the absence of high-energy cofactors. Precisely how ATP-independent chaperones accomplish this feat is unclear. Here we have characterized the kinetic mechanism of substrate folding by the small, ATP-independent chaperone, Spy. Spy rapidly associates with its substrate, Immunity protein 7 (Im7), eliminating its potential for aggregation. Remarkably, Spy then allows Im7 to fully fold into its native state while remaining bound to the surface of the chaperone. These results establish a potentially widespread mechanism whereby ATP-independent chaperones can assist in protein refolding. They also provide compelling evidence that substrate proteins can fold while continuously bound to a chaperone.

Chaperone-client interactions: Non-specificity engenders multifunctionality

“…Spy was very recently used as a simple chaperone folding system to identify the kinetic, thermodynamic, and structural properties that allow chaperones to promote client folding and to determine how they affect the folding landscape of client proteins (31)(32)(33). The "folding-friendly" amphiphilic and flexible nature of Spy's client-binding site was found to be critical to its chaperone activity (33).…”

“…The client-binding site, which encompasses a large part of the concave surface of Spy's cradleshaped structure, consists of four hydrophobic patches surrounded by positively charged hydrophilic residues and flexible N-terminal helix linkers between helix one and helix two. This combination of flexibility and amphiphilic binding surface allows Spy to dynamically bind the many conformational states that occur along the folding trajectory of its client proteins and hence mediate folding while remaining continuously but loosely bound to its clients (31,33). The attraction of the aggregation-prone unfolded client to the chaperone is driven by electrostatic forces, which are then complemented by hydrophobic interactions in the complex (32).…”

“…Although two separate groups have come to similar conclusions on the folding of Im7 in the presence of Spy (31,34), there is little evidence to suggest whether or not this mechanism also applies to other client proteins. As will be discussed in the cases below for Hsp60 and Hsp70, this caveat cannot be ignored.…”

Assembly pathway of a bacterial complex iron sulfur molybdoenzyme

“…An emerging functional feature of ATP binding chaperones is their highly dynamic behavior, and it seems that their massive domain movement is only weakly coupled to their nucleotide states. Rather, these ATPases are in a continual state of rapid flux (104). A mechanism for GroEL-assisted folding of large proteins involves their binding to the open (trans) ring, and subsequent folding in the bulk solution outside of the cavity (55).…”

“…Such an interaction was recently discovered between DmsD and GroEL, in that DmsD was shown to function as a connector to carry the DmsA substrate preprotein to GroEL for assisted folding (54). Although the numerous known folding chaperones have a fairly broad range of substrates, each chaperone family has a distinct mode of ATP binding (104). Currently, a major aim in the field is obtaining high-resolution structures of chaperone complexes acting on misfolded or unfolded proteins to better define the energetic regulation of protein quality control.…”