A folding nucleus and minimal ATP binding domain of Hsp70 identified by single-molecule force spectroscopy

Abstract

The folding pathways of large proteins are complex, with many of them requiring the aid of chaperones and others folding spontaneously. Along the folding pathways, partially folded intermediates are frequently populated; their role in the driving of the folding process is unclear. The structures of these intermediates are generally not amenable to high-resolution structural techniques because of their transient nature. Here we employed single-molecule force measurements to scrutinize the hierarchy of intermediate structures along the folding pathway of the nucleotide binding domain (NBD) of Escherichia coli Hsp70 DnaK. DnaK-NBD is a member of the sugar kinase superfamily that includes Hsp70s and the cytoskeletal protein actin. Using optical tweezers, a stable nucleotide-binding competent en route folding intermediate comprising lobe II residues (183-383) was identified as a critical checkpoint for productive folding. We obtained a structural snapshot of this folding intermediate that shows native-like conformation. To assess the fundamental role of folded lobe II for efficient folding, we turned our attention to yeast mitochondrial NBD, which does not fold without a dedicated chaperone. After replacing the yeast lobe II residues with stable E. coli lobe II, the obtained chimeric protein showed native-like ATPase activity and robust folding into the native state, even in the absence of chaperone. In summary, lobe II is a stable nucleotide-binding competent folding nucleus that is the key to time-efficient folding and possibly resembles a common ancestor domain. Our findings provide a conceptual framework for the folding pathways of other members of this protein superfamily.

Keywords: elasticity; folding pathways; force; laser trapping; protein extension.

Fig. 1.

Refolding of NBD investigated by optical tweezers experiments. (A) Optical tweezers assay setup. EcNBD protein is tethered N and C terminally to dsDNA handles between two trapped beads. The mobile trap can be moved to exert a force on the molecule. (B) Series of measured stretching cycles: a fingerprint of the native protein (pink), and refolding intermediates (RFI1, red circle; RFI2, yellow star). Force-extension traces were recorded at a pulling speed of 200 nm/s and waiting time at zero force of 1 s. (C) The plot of natively refolded fraction dependent on the waiting time at zero force [EcNBD-nc (pink) and mtNBD (green)]. Fitting the data with a simple exponential equation gave a refolding rate for EcNBD-nc of 0.03 ± 0.02 s⁻¹; mtNBD does not refold in our experiments. (D) Contour lengths measured for the refolding intermediates RFI1 (red circle) and RFI2 (yellow star). Native unfolding (gray) shows, first, stretching of DNA (L_C = 0 nm), followed by the whole NBD unfolding (L_C = 134 nm). (E) Scatter plot of all unfolding events (n = 10 molecules). Unfolding of mature NBD (pink) is characterized by an unfolding force of 34 pN and a contour length change of ∆L_C = 134 nm (15). The refolding intermediates RFI1 (red circle) and RFI2 (yellow star) unfold at ∼5 and ∼7 pN, respectively. (F) Force-extension traces of the unfolding of isolated lobe II (aa 183–383). Unfolding of intact lobe II (pink, left), the next stretching shows the RFI1 and RFI2 unfolding (red circle and yellow star, right). (G) Scatter plot of the unfolding events in NBD 183–383. (H) Illustration of analyzed loop insertion variants Ins183 (K183-L20), Ins290 (A290-L20), and Ins364 (D364-L20). (I) Contour length transformation plots of EcNBD-nc, Ins290, Ins183 and Ins364 NBD variant. Ins183 and Ins364 show the same contour length for RFI1 and RFI2 as the EcNBD protein. The contour length differences for U-RFI1 in InsA290 are increased compared with EcNBD-nc (30.3 vs. 23.9 nm).

Fig. 2.

Refolding kinetics of EcNBD-nc and micromolar ATP binding capability of RFI2. (A) Refolding trajectories acquired at different force biases. (B) Chevron plots of all force dependent refolding transitions (circles) in apo with extrapolation to zero force rates (lines). Force-dependent probability plot with extrapolation to zero force. (C) Passive mode experiments at varying ATP concentrations at 4.0 pN show the binding of ATP to RFI2, which results in a longer RFI2 life-time. (D) Mg²⁺ATP interaction in the NBD structure using LigPlot software (38). (E) Chevron plot of the RFI1 ↔ RFI2 transition. Fits represent extrapolations of the force-dependent rates to zero force. Data points: force-dependent rates in the presence of 200 µM ATP and 5 mM MgCl₂ (RFI2 → RFI1, orange hexagons; RFI1 → RFI2, red hexagons; ATP-free, gray). The presence of ATP slows down the RFI2 unfolding rate (yellow hexagon). (F) Dependence of the transition rate (RFI2 → RFI1, yellow circles) on the ATP concentration at 4 pN. A fit to the data with the binding-unfolding-model (yellow line, fit, SI Appendix) yields a K_D of 33 µM, which is verified by simulations (brown circles).

Fig. 3.

Lobe II is an independent stable folding domain and able to bind nucleotide. (A) Design of Mini-NBD (aa 183–383). (B and C) The sequence of N-/C-terminal stretching cycles with a pulling speed of 200 nm/s and waiting time at zero force for 1 s in the absence of ATP (B) and the presence of 200 µM ATP and 5 mM MgCl₂ (C). (D and E) Passive mode experiments at 3.7 pN in the absence (D) and presence of 200 µM ATP and 5 mM MgCl₂ (E) RFI1 (red), RFI2 (orange) and RFI2f (green) are the populated states in this experiment with contour lengths of 36 nm (RFI1) and 0 nm (RFI2/RFI2f). (F) Crystal structure of AMP-PCP bound Mini-NBD at a resolution of 2.9 Å. (G) Superposition of the isolated lobe II domain with DnaK (aa 1–394) (PDB: 4B9Q) (rmsd = 0.7 Å). Flexible regions A, B, C, and D are indicated in circles. (H) LigPlot (38) analysis of AMP-PCP bound to Mini-NBD. The numbering is according to the EcNBD (PDB: 4B9Q). (I) Rmsd per residue plot. In gray, the unresolved structure is marked, and residues marked in bold (red ellipse) are involved in the nucleotide coordination. The overall rmsd of the structure alignment is 0.7 Å (25).

Fig. 4.

Insertion of lobe II of EcNBD can create a folding-competent mtNBD structure by acting as a folding nucleus. (A) Force-extension traces of mtNBD at a pulling velocity of 500 nm/s. Shown are only stretching cycles. First, natively folded mtNBD unfolds, followed by the unfolding of weakly formed structures that do not fold back to the native fold even after 10 min. (B) Scatter plot of force vs. contour length shows the broad scattering of refolding intermediates for mtNBD (green). To compare, the events for EcNBD are shown in red and orange (RFI1 and RFI2). (C) Design of the 50% mtNBD chimeric protein consisting of lobe II from EcNBD, lobe I, and C-terminal helix from mtNBD. (D) Force-extension stretching traces at a pulling speed of 200 nm/s show the characteristic refolding events of lobe II. After ∼230 s, the chimeric protein can refold completely to its stable native fold.