Table 1.
Data Collection and Refinement Statistics.
Figure 1.
Overall structure of the human peptide-bound HSP70 SBD.
A. Cartoon representation of NRLLLTG-bound HSP70 SBD Molecule A. The SBD and NRLLLTG peptide are shown in magenta and blue, respectively, and all domains and secondary structural elements are labeled. B. Superposition of molecules A and B in the asymmetric unit cell. Molecules A and B are color-coded magenta and green, respectively. C and D. Hinge region between the α and β subdomains. Molecules A and B are shown in panels C and D, respectively. Interacting (MolA) or potential interacting (MolB) residues are indicated in stick and CPK coloring (oxygen in red and nitrogen in blue). The electron density maps corresponding to these residues are contoured at 1.5 (MolA) and 1.0 (MolB) sigma. Hydrogen bonds are indicated with dotted lines.
Figure 2.
Sequence alignment of eukaryotic and bacterial HSP70 proteins.
Human HSP70 (HSPA1A or HSP72), HSPA2, HSPA5 (GRP78 or BiP), HSC70 (HSPA8 or HSP73), HSPA9 (GRP75, Mortalin-2 or MTHSP70) and E.coli DnaK (ecDnaK), E. coli HscA, Geobacillus kaustophilus HTA426 DnaK (gkDnaK) sequences are used for the alignment. Secondary structure elements and residue numbering for HSP70 is indicated above the protein sequence. The hinge region (also known as loop Lα,β) is highlighted in a red rectangular box, the arch residues are indicated with red asterisks, residues involved in NRLLLTG Leu5p substrate binding are indicated with green asterisks, and the juncture region residue Asn540 is indicated with a blue asterisk. The conserved hydrophobic residues in the helical bundle region are indicated with red triangles.
Figure 3.
Comparison of the substrate-bound HSP70 SBD to other DnaK and HSC70 SBD structures.
A. Comparison of the NRLLLTG-bound HSP70 SBD to the NRLLLTG-bound DnaK SBD (PDB code 1DKZ). HSP70 and DnaK are shown in magenta and blue cartoons, respectively. The corresponding peptide substrates are shown as stick models. B. A close-up view of the hydrophobic interactions within the helical bundle region of the α subdomain. Hydrophobic residues are shown as stick models. C. Comparison of the SBDα subdomain of the human HSP70-NRLLLTG structure with the isolated three-helix bundle region of the same protein in X-ray structure (PDB code 3LOF, blue) and NMR solution structure (PDB code 2LMG, green). D, Structural alignment of the SBDα subdomain of the HSP70-NRLLLTG structure with the three-helix bundle region of C. elegans Hsp70 (PDB code 2P32, cyan) and DnaK (PDB code 1DKZ, blue). E. Superposition of the lid subdomains of human HSP70 and rat HSC70. HSP70 (MolA) and HSC70 are colored in magenta and cyan, respectively. F. A close-up view of the interaction between the SBDα and SBDβ subdomains of HSP70. Interacting residues are highlighted as sticks, and hydrogen bonds are indicated with dotted lines.
Figure 4.
NRLLLTG peptide substrate binding by HSP70.
A. Omit electron density of the peptide substrate bound to the β subdomain of molecule A is contoured at 3.0 sigma. The NRLLLTG peptide is shown as a stick model in CPK coloring. B. Peptide binding site highlighting the flanking L1,2 and L3,4, and supporting L5,6 and L4,5 loops. The NRLLLTG peptide and arch residues are shown as stick models. C. Detailed view of the NRLLLTG-binding pocket in HSP70-NRLLLTG highlighting a network of van der Waals contacts with Leu5p of the NRLLLTG peptide. D. A close-up view of the interactions between Leu4p-Leu5p-Thr6p of the NRLLLTG peptide and the surrounding residues in HSP70-NRLLLTG. All interacting residues are shown as sticks and hydrogen bonds are shown as dotted lines.
Figure 5.
Putative druggable sites in the HSP70 SBD domain.
An overview of three hydrophobic pockets within the SBD domain have been circled and marked as A, B and C. Close-up of the surface area of these three sites are illustrated in CPK coloring mode, with carbon in green, oxygen in red and nitrogen in blue. Secondary structural elements within the area are labeled. The NRLLLTG substrate is removed from this figure (pocket A) to better illustrate the pocket A for comparison with the other pockets (B and C).