Folding and Domain Interactions of Three Orthologs of Hsp90 Studied by Single-Molecule Force Spectroscopy

Abstract

The heat-shock protein 90 (Hsp90) molecular chaperones are highly conserved across species. However, their dynamic properties can vary significantly from organism to organism. Here we used high-precision optical tweezers to analyze the mechanical properties and folding of different Hsp90 orthologs, namely bacterial Hsp90 (HtpG) and Hsp90 from the endoplasmic reticulum (ER) (Grp94), as well as from the cytosol of the eukaryotic cell (Hsp82). We find that the folding rates of Hsp82 and HtpG are similar, while the folding of Grp94 is slowed down by misfolding of the N-terminal domain. Furthermore, the domain interactions mediated by the charged linker, involved in the conformational cycles of all three orthologs, are much stronger for Grp94 than for Hsp82, keeping the N-terminal domain and the middle domain in close proximity. Thus, the ER resident Hsp90 ortholog differs from the cytosolic counterparts in basic functionally relevant structural properties.

Hsp90 and Hsp70 chaperones: Collaborators in protein remodeling

“…One feature is a long, flexible, charged linker that connects the NTD and the MD in cytosolic eukaryotic Hsp90 as well as endoplasmic reticulum Hsp90, Grp94 (15,29,30), which is absent in bacterial Hsp90 and mitochondrial Hsp90, TRAP1 (15,31,32). This charged linker has been proposed to function as a modulator of Hsp90 conformational changes and co-chaperone binding (33)(34)(35)(36)(37). The second distinctive feature is a C-terminal extension containing the MEEVD motif that is present in cytosolic eukaryotic Hsp90 proteins and allows binding of many co-chaperones containing tetratricopeptide repeat (TPR) domains, including Hop, PP5, CHIP, Tpr2, Sgt1, and Tom70 ( Fig.…”

The hierarchical emergence of worm-like chain behaviour from globular domain polymer chains

“…For general SMFS experiments on polyproteins, it is often the case that the WLC model is used simply as a fitting model to validate the measurement of single protein properties, such as the peak forces and the peak-to-peak distances of the SMFS unfolding curves. Nevertheless, many SMFS experiments observe persistence lengths of around the length of a single amino acid: in titin 27,28,32,33 , general polyproteins [34][35][36] , and other structural motifs [37][38][39][40] , and this magnitude of persistence length corresponds well to that of individual, flexible amino acid chains 41,42 . Yet we have seen from the work of Li et al that in the absence of applied tensile force, the persistence length can be significantly larger.…”

Glucose-Regulated Protein 94 (GRP94): A Novel Regulator of Insulin-Like Growth Factor Production

“…It has the domain structure typical of the heat shock 90 (HSP90) family of proteins, including a C-terminal domain that mediates the constitutive dimerization of GRP94 ( Figure 1). Like all family members, the N-terminal domain of GRP94 is a typical ATP-binding domain [41] that affects the dimerization of GRP94 and its action cycle [42]. The N-terminal domain also mediates binding of antigenic peptides [43] through which GRP94 activates T cells, the basis for the immunological function of GRP94 [43,44].…”

“…GRP94 differs from the cytosolic HSP90 orthologs in inherent, functionally-relevant structural properties [42] such as the nucleotide-dependent conformational changes of the N-terminal domain [63] as well as the interactions mediated by the charged linker domain. These differences lead to a different action cycle of this protein [68,69] and probably also to its ability to chaperone folding of client proteins without the many co-chaperones that are required for activity of the cytosolic HSP90 orthologs [70].…”