Nanomechanics of the substrate binding domain of Hsp70 determine its allosteric ATP-induced conformational change

Abstract

Owing to the cooperativity of protein structures, it is often almost impossible to identify independent subunits, flexible regions, or hinges simply by visual inspection of static snapshots. Here, we use single-molecule force experiments and simulations to apply tension across the substrate binding domain (SBD) of heat shock protein 70 (Hsp70) to pinpoint mechanical units and flexible hinges. The SBD consists of two nanomechanical units matching 3D structural parts, called the α-and β-subdomain. We identified a flexible region within the rigid β-subdomain that gives way under load, thus opening up the α/β interface. In exactly this region, structural changes occur in the ATP-induced opening of Hsp70 to allow substrate exchange. Our results show that the SBD's ability to undergo large conformational changes is already encoded by passive mechanics of the individual elements.laser trapping | parallel pathways | elasticity | force | protein extension W hen looking at protein structures at atomic resolution, it is often tempting to use macroscopic mechanical analogies to describe their function as molecular machines. However, such analogies are often misleading because boundaries between independently stable subdomains cannot often be determined from structures, owing to the high cooperativity of protein folding and structural transitions. Single-molecule protein nanomechanics have emerged as a tool to force biomolecules through their conformational space and, hence, identify hinges, breaking points, and mechanically stable subdomains (1-3).A prominent example of a protein machine undergoing large conformational change during its functional cycle is the ATP-regulated Hsp70 chaperone DnaK-a central molecular chaperone of the protein quality control network in a cell (4-6). Once ATP is bound to the nucleotide binding domain (NBD, blue-yellow; Fig. 1A) of DnaK, the initially closed substrate binding domain (SBD) opens its binding cleft by engaging the β-subdomain to the NBD. In doing so, it undergoes a dramatic ∼10 Å displacement of its lid subdomain ( Fig. 1A; refs. 7 and 8) to allow exchange of substrates (9). Several crystal structures of the isolated SBD (in which the NBD is absent) have been solved (10-15). In these structures, the absence or presence of peptide clients or nonnatural ligands induce no significant structural changes in the closed conformation. There is no indication in the crystal structures of the huge conformational change of the lid domain of the SBD, seen in the ATP form of the full-length two-domain DnaK. Therefore, the large conformational change of the SBD is only observed in the two-domain DnaK after ATP binding. Thus, although the crystal structures provide us with valuable insights into the 3D arrangement of individual atoms, the thermodynamic and mechanical stability of individual substructures are difficult to predict based on this information alone. Here, we ask how the large ATP-induced changes of the SBD, as seen in the two-domain DnaK, are mirrored in the subdomain integrity and nano...

Single-Molecule Mechanics in Ligand Concentration Gradient

“…on proteins. Although the small size of usual proteins may pose experimental challenge, large filamentous proteins (e.g., titin [50]) or proteins captured with DNA handles [51][52][53][54] may be investigated directly. Considering the emerging significance of single-molecule mechanics in understanding structural and functional detail, the addition of precisely adjusted ligand concentration gradients, as demonstrated here, may provide further access to understanding the exact mechanisms behind biomolecular phenomena.…”

Mechanical Properties of Chaperone BiP, the Master Regulator of the Endoplasmic Reticulum

“…However, by optical tweezers manipulation, we can specifically unfold the substrate without affecting BiP. Another study, by directly pulling DnaK using optical tweezers, the authors were able to study the mechanics and the order of events of unfolding of each domain of this Hsp70 [60,61]. This study shows that DnaK has more than two mechanical intermediates in each domain.…”

Activation of a Protein Kinase Via Asymmetric Allosteric Coupling of Structurally Conserved Signaling Modules

“…We attached DNA handles via thiol chemistry to two cysteines engineered at specific positions in the protein (see Methods) 13,14 . The handle position determines the direction and region of the protein subjected to the force applied through the optical tweezers (i.e., pulling geometry) [15][16][17] . We generated three PKA regulatory subunit constructs with unique pulling geometries to probe cAMP binding coupled to inter-domain interactions (Fig 1b, right).…”

“…Briefly, the purified target protein was concentrated to ~ 5 mg/mL in 10 mM DTT to reduce all cysteine residues. The 16 protein solution was run through 3 Micro Bio-Spin columns (Bio-Gel P6, Biorad) to remove the DTT before adding 10 mM 2,2'-dithiodipyriine (DTDP, Sigma) for 2 hours at room temperature.…”