Mapping the conformation of a client protein through the Hsp70 functional cycle

Abstract

The 70 kDa heat shock protein (Hsp70) chaperone system is ubiquitous, highly conserved, and involved in a myriad of diverse cellular processes. Its function relies on nucleotide-dependent interactions with client proteins, yet the structural features of folding-competent substrates in their Hsp70-bound state remain poorly understood. Here we use NMR spectroscopy to study the human telomere repeat binding factor 1 (hTRF1) in complex with Escherichia coli Hsp70 (DnaK). In the complex, hTRF1 is globally unfolded with up to 40% helical secondary structure in regions distal to the binding site. Very similar conformational ensembles are observed for hTRF1 bound to ATP-, ADP-and nucleotide-free DnaK. The patterns in substrate helicity mirror those found in the unfolded state in the absence of denaturants except near the site of chaperone binding, demonstrating that DnaKbound hTRF1 retains its intrinsic structural preferences. To our knowledge, our study presents the first atomic resolution structural characterization of a client protein bound to each of the three nucleotide states of DnaK and establishes that the large structural changes in DnaK and the associated energy that accompanies ATP binding and hydrolysis do not affect the overall conformation of the bound substrate protein.Hsp70 | protein folding | NMR | molecular chaperones | CEST T he Hsp70 chaperone system forms a central hub in the cellular proteostasis network, performing a large number of functions, all of which are predicated upon its relatively simple ATPdependent interaction with a client protein (1, 2). Hsp70 is a 70-kDa protein containing both nucleotide (NBD) and substrate (SBD) binding domains connected by a conserved linker (2). The beststudied Hsp70 is Escherichia coli DnaK, which recognizes an approximate seven-residue protein segment rich in large aliphatic hydrophobic residues flanked by positively charged amino acids (3). Client substrates enter the Hsp70 functional cycle by binding the ATP form of the chaperone, which has lower substrate affinity but faster binding and release rates compared with the ADP state (2). The crystal structure of ATP-DnaK (4, 5) shows that the two domains of Hsp70 are docked on each other, with the helical lid flanking the DnaK binding cleft in an open state. Subsequent ATP hydrolysis, stimulated by both Hsp40 and the bound substrate, locks the substrate in the binding pocket (2) and disengages the two domains of DnaK from each other, leading to major structural differences between the ATP-(4, 5) and ADP-bound (6) forms of the chaperone. Finally, ADP release, facilitated by nucleotide exchange factors (7), and rebinding of ATP free the bound substrate and reset the chaperone cycle.Although the structural transitions of DnaK and the role of allostery throughout the chaperone reaction cycle are becoming increasingly well understood (7), far less information is available on the substrate conformation in the DnaK-bound state. Our current view is derived primarily from studies on the isolated SBD of DnaK in conjunctio...

Hsp70 biases the folding pathways of client proteins

“…1A, blue) and binds ADP-DnaK with a K D of 32 μM (35°C) in a manner that is very similar to the well-characterized DnaK-binding peptide from the E. coli heat shock transcription factor σ 32 (13). Interestingly, DnaK-bound hTRF1 is globally unfolded but retains as much as 40% helicity in regions forming helices 1 and 3 in the native protein, which are distal to the DnaK binding site (13). hTRF1 is marginally stable and the folded conformer of the WT protein interconverts with an unfolded state with a fractional population of ∼4% and with a lifetime on the order of 3 ms (35°C), G !…”

“…Native hTRF1 has a predicted DnaK binding site (5) centered at Leu-30 in helix 2 (Fig. 1A, blue) and binds ADP-DnaK with a K D of 32 μM (35°C) in a manner that is very similar to the well-characterized DnaK-binding peptide from the E. coli heat shock transcription factor σ 32 (13). Interestingly, DnaK-bound hTRF1 is globally unfolded but retains as much as 40% helicity in regions forming helices 1 and 3 in the native protein, which are distal to the DnaK binding site (13).…”

“…These experiments have been developed for characterizing conformationally exchanging states that interconvert on the order of or less than several hundreds per second with excited state populations of ∼1% or higher (18). A comparison of the secondary structural propensities of DnaK-bound hTRF1 with those of the unfolded state revealed similar patterns of helicity in both states, showing that DnaK binding does not prevent the formation of secondary structure in the bound substrate protein (13).…”

“…In the latter case, the perturbation is propagated to proximal 1 H spins, resulting in dips in CEST profiles that are difficult to distinguish from chemical exchange (33). NOE dips are not a concern for applications that involve 15 N or 13 C CEST because the γ values of these spins are smaller than for 1 H, and hence the homonuclear dipolar effect that scales as γ 4 is, for all practical purposes, negligible. However, the effect is significant for 1 H CEST and requires a modification to the experimental approach.…”

“…In contrast to the detailed structural studies characterizing DnaK, little atomic resolution data are available on the conformation of folding-competent client proteins in the DnaK-bound state. It is known that DnaK binds substrates in a globally unfolded conformation with varying degrees of local residual native and nonnative secondary structure (9)(10)(11)(12)(13). However, whether stable or transient long-range interactions are present in DnaK-bound client proteins remains an open question that has relevance for understanding the function of DnaK in substrate refolding and disaggregation.…”