| Abstract: | The 70-kDa heat shock protein (Hsp70) family of chaperones bind cognate substrates to perform a variety of different processes that are integral to cellular homeostasis. Although detailed structural information is available on the chaperone, the structural features of folding competent substrates in the bound form have not been well characterized. Here we use paramagnetic relaxation enhancement (PRE) NMR spectroscopy to probe the existence of long-range interactions in one such folding competent substrate, human telomere repeat binding factor (hTRF1), which is bound to DnaK in a globally unfolded conformation. We show that DnaK binding modifies the energy landscape of the substrate by removing long-range interactions that are otherwise present in the unbound, unfolded conformation of hTRF1. Because the unfolded state of hTRF1 is only marginally populated and transiently formed, it is inaccessible to standard NMR approaches. We therefore developed a 1H-based CEST experiment that allows measurement of PREs in sparse states, reporting on transiently sampled conformations. Our results suggest that DnaK binding can significantly bias the folding pathway of client substrates such that secondary structure forms first, followed by the development of longer-range contacts between more distal parts of the protein.The 70-kDa heat shock protein (Hsp70) chaperone system is an important component of the cellular proteostasis machinery, serving as a central hub to channel client proteins along folding, refolding, maturation, disaggregation, and proteolytic pathways in cooperation with other chaperone assemblies such as Hsp90, Hsp104, and GroEL/ES (1–3). Central to Hsp70 function is its ATP-dependent interaction with client proteins, facilitated by Hsp40 cochaperones and nucleotide exchange factors (NEFs) (4). Hsp70 is a weak ATPase that recognizes and binds substrates at sites containing large aliphatic hydrophobic residues, Ile, Leu, and Val, flanked by positively charged amino acids such as Arg and Lys (5). Initial binding of substrate to the ATP-form of Hsp70 can occur directly, or via Hsp40, with rapid on/off kinetics that give rise to a weak overall affinity for the interaction. Subsequent ATP hydrolysis, stimulated by interactions with Hsp40 and substrate, leads to a large conformational change in the chaperone, locking the substrate in the Hsp70 bound state. The resulting complex is of high affinity with slow substrate on/off rates (2).Escherichia coli DnaK is the best studied of Hsp70 chaperones. It is a 70-kDa protein comprised of an N-terminal ATPase and a C-terminal substrate binding domain that communicate allosterically to couple ATP hydrolysis with substrate binding (3). High-resolution structures of ADP- (6) and ATP-DnaK (7, 8) establish that these two domains dock on to one another in the ATP-DnaK state, but become detached from each other in the ADP-bound form. 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 (9–13). 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. For example, it has been shown that DnaK, in concert with cochaperones DnaJ (Hsp40) and GrpE (NEF), converts misfolded luciferase to a globally unfolded, yet folding-competent, conformation (14). Furthermore, the DnaK chaperone system is thought to “loosen” aggregated proteins for subsequent disaggregation by ClpB or proteolysis by ClpXP (15). Aggregated and misfolded proteins are stabilized by native and nonnative tertiary contacts and characterizing the extent to which these interactions either persist or are modified upon DnaK binding will provide insights into the mechanism by which DnaK carries out its myriad of important functions.Here we probe the existence of (transient) long-range tertiary interactions in a folding competent substrate, the human telomere repeat binding factor (hTRF1), which is globally unfolded when bound to DnaK (13), using paramagnetic relaxation enhancement (PRE) NMR spectroscopy. To evaluate whether DnaK binding modifies the energy landscape of the substrate in a way that affects long-range interactions in the unfolded state, we have recorded PREs in the unbound, unfolded conformation of hTRF1 under identical conditions to those used for studies of DnaK-bound hTRF1. The unfolded state of hTRF1 in water (referred to in what follows as uw-hTRF1) is only sparsely populated and transiently formed, rendering it invisible to standard NMR studies. However, the signal from the invisible state can be amplified using chemical exchange saturation transfer (CEST) and read out through the visible, folded hTRF1 state. We thus developed a 1H-based CEST experiment that facilitates measurement of PREs in uw-hTRF1. A comparison of PREs in this state with those in the hTRF1-DnaK bound conformation establishes that the large PREs in uw-hTRF1 are significantly reduced on DnaK binding, and the extent of residual long-range interactions in the DnaK-bound form is similar to hTRF1 denatured in 4 M urea. Taken together, our results suggest that DnaK may be able to modify the folding pathways of protein substrates by significantly influencing their tertiary structural tendencies in the bound conformation. |
