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High-resolution solution structure of the 18 kDa substrate-binding domain of the mammalian chaperone protein Hsc701

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Abstract

The three-dimensional structure for the substrate-binding domain of the mammalian chaperone protein Hsc70 of the 70 kDa heat shock class (HSP70) is presented. This domain includes residues 383–540 (18 kDa) and is necessary for the binding of the chaperone with substrate proteins and peptides. The high-resolution NMR solution structure is based on 4150 experimental distance constraints leading to an average root-mean-square precision of 0.38 Å for the backbone atoms and 0.76 Å for all atoms in the beta-sandwich sub-domain. The protein is observed to bind residue Leu539 in its hydrophobic substrate-binding groove by intramolecular interaction. The position of a helical latch differs dramatically from what is observed in the crystal and solution structures of the homologous prokaryotic chaperone DnaK. In the Hsc70 structure, the helix lies in a hydrophobic groove and is anchored by a buried salt-bridge. Residues involved in this salt-bridge appear to be important for the allosteric functioning of the protein. A mechanism for interdomain allosteric modulation of substrate-binding is proposed. It involves large-scale movements of the helical domain, redefining the location of the hinge area that enables such motions.

Introduction

The 70 kDa heat shock proteins (Hsp70 s) are a class of chaperone proteins that exist in nearly all organisms and organelles. Originally identified in response to stress or heat shock (Lindquist, 1987), the Hsp70s have since been recognized as playing essential roles in many cellular functions, including protein folding, protein translocation, oligomeric assembly, and protein degradation (reviewed by Beissinger and Buchner 1998, Bukau and Horwich 1998, Craig 1993, Craig et al 1993, Craig et al 1995, Hartl 1996, Haynes et al 1997, Schatz and Dobberstein 1996). Hsc70 (heatshock cognate) is the mammalian Hsp70 that is constitutively expressed to high levels and is thought to be the molecular chaperone involved in the in vivo folding and repair of proteins by binding to newly synthesized or partially unfolded polypeptides Hendrick and Hartl 1993, Craig 1993, Martin and Hartl 1997.

Hsp70 proteins consist of three domains. The N-terminal 44 kDa fragment, shown to have ATPase activity Chappell et al 1987, Flaherty et al 1990 is followed by an 18 kDa domain that binds substrate, which in turn is followed by a 10 kDa domain that may be involved in interaction with the co-chaperones of the DnaJ class (Cyr et al., 1994) and with the regulation of the substrate-binding kinetics and affinity Wawrzynow and Zylicz 1995, Wawrzynow et al 1995, Zhu et al 1996, Rudiger et al 1997b, Wang et al 1998. Deletion studies on full Hsc70 have shown that the fragment 1–540 of Hsc70, lacking the C-terminal 10 kDa domain, is sufficient for chaperone function in clathrin uncoating (Ungewickell et al., 1997). In addition, the fragment Hsc 1–554 shows allosteric conformational changes upon ATP binding, as observed by small-angle X-ray scattering (Wilbanks et al., 1995). Experiments using short peptides showed that the Hsp70s preferentially bind to peptides rich in internal hydrophobic residues, which mimic unfolded proteins. The optimal binding size was found to be about seven amino acid residues long Flynn et al 1989, Flynn et al 1991, Gragerov et al 1994, Rudiger et al 1997a, Rudiger et al 1997b. Isolated 18 kDa substrate-binding-domain (SBD) binds peptides with an affinity (8 μM) indistinguishable from that of the native Hsc70 (5–8 μM) for the S-peptide of RNase-A (Wang et al., 1993). Thus, all the substrate-binding determinants are located in an 18 kDa domain directly following the ATP-binding site. The full 27 kDa C-terminal fragment is not necessary for this function. The three-dimensional structure of the 44 kDa ATPase domain of Hsc70 has been determined by X-ray crystallography to a resolution of 2.2 Å (Flaherty et al., 1990).

The first experimental structural information on the substrate-binding domains of the Hsp70s originated from NMR spectroscopy (Morshauser et al., 1995). The study revealed that the secondary structure of the substrate-binding domain of Hsc70 has a beta-strand topology not seen before in other proteins. The topology was quite different from that predicted by molecular modeling (Rippmann et al., 1991). An identical topology was found for the substrate-binding domain of DnaK (Wang, 1995). These studies were followed by a high-resolution crystal structure of the substrate-binding and variable domain of homologous prokaryotic DnaK (Zhu et al., 1996). Very recently, we reported a solution structure of a 21 kDa substrate-binding domain of DnaK (Wang et al., 1998).

The process of allosteric communication between ATPase and substrate-binding domains that modulates the affinity of the chaperone towards substrate is poorly understood in the absence of a high-resolution structure of a complete Hsp70. Fluorescence and proteolytic degradation studies have demonstrated changes in both the ATPase and the substrate-binding domains upon nucleotide or polypeptide binding Buchberger et al 1995, Fung et al 1996.

Here, we present the first high-resolution structure of an eukaryotic Hsp70 substrate-binding domain, that of Hsc70, residues Ser383-Glu540. This construct includes the full substrate-binding area as defined by the studies of Wang et al. (1993) but lacks the C-terminal 10 kDa variable region, which was part of the construct used in the X-ray diffraction studies of DnaK. We reiterate that the last 10 kDa does not appear relevant for proper chaperone function Ungewickell et al 1997, Wilbanks et al 1995. The NMR structure is very similar to both the NMR and crystal structures of DnaK substrate-binding domain. Just as DnaK-SBD in solution, Hsc70-SBD in solution binds to its own C-terminal domain, placing a leucyl residue (Leu539) in its substrate-binding pocket. When compared with the crystal structure of DnaK substrate-binding domain, which binds intermolecularly to a hydrophobic peptide, it is clear that the helical domain, commonly accepted to be a modulator of the chaperone activity, is in a vastly different conformation. The helix position of Hsc70 observed in solution is defined by contacts in a hydrophobic groove and a salt-bridge. Our study suggests that large changes in helix position, hinged at the basis of the helical domain, are responsible for the modulation of substrate-binding kinetics and affinity of the chaperones in the Hsp70 class. Comparison with mutagenesis data suggests that the helix position observed in this structure is relevant for the allosteric functioning of the protein, and most likely represents the ADP (high-affinity) state.

Section snippets

Quality of the NMR structure

The various constraints used in structural calculations are summarized in Table 1. The first set of statistics pertains to an initial medium-resolution structure. The input for the final structure calculation includes a total of 4150 NOEs, of which 891 were intramolecular, 1020 were sequential, 653 were medium-range, and 1586 were long-range. Figure 1(a) shows the distribution of NOEs per residue for Hsc70 SBD. Numerous restraints were observed for nearly all residues except for the first ten

NMR structure

Here, we demonstrated a comprehensive NOE analysis in which the assignments are based almost exclusively on spectroscopic information. Supplementing the standard 3-D single- edited NOESYs (13C-resolved and 15N-resolved) with 3-D double-edited counterparts (the 13C,13C-resolved NOESY and the 13C,15N-resolved NOESY) and high-resolution versions of the 4-D experiments (13C,13C-resolved NOESY and the 13C,15N-resolved NOESY) led to the unambiguous assignment of an unparalleled quantity of NOEs. It

Sample preparation

The gene coding for rat Hsc70 substrate-binding domain (HscSBD Ser383-Glu540, see Figure 10) was under IPTG-inducible phage T7 promoter control in Escherichia coli strain JM109 (DE3). Up to 50 mg/l of fully 13C/15N-labeled pure protein was obtained from M9 medium cell cultures containing 1 g/l of 15N-labeled ammonium chloride and 2 g/l of 13C-labeled d -glucose (Cambridge Isotopes, Inc.). The peptide sequence (His)6 follows the native HscSBD to allow affinity purification with a Ni column

Acknowledgements

This work was supported by NIH grant RO1 GM52421. We are indebted to Dr C. Wang (Taipei, Taiwan) for plasmid pPDB70 encoding for the Hsc70 fragment studied. We are grateful to Dr Alexander V. Kurochkin for expert advise on protein purification and to Dr Maurizio Pellecchia for help with the molecular graphics. NMR experiments at 750 MHz were carried out at the National Magnetic Resonance Facility at Madison, USA, subsidized by the NIH (RR02301, RR02781 and RR08438), the NSF ( BIR-9214394 and

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    Present addresses: W. Hu, Memorial Sloan-Kettering Cancer Center, New York, NY 10029, USA; H. Wang, Pharmaceutical Discovery Division, Abbott Laboratories, Abbott Park, IL 60064, USA.

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