Biochemical and structural studies on the high affinity of Hsp70 for ADP

Abstract

The molecular chaperone 70-kDa heat shock protein (Hsp70) is driven by ATP hydrolysis and ADP-ATP exchange. ADP dissociation from Hsp70 is reportedly slow in the presence of inorganic phosphate (P(i) ). In this study, we investigated the interaction of Hsp70 and its nucleotide-binding domain (NBD) with ADP in detail, by isothermal titration calorimetry measurements and found that Mg(2+) ion dramatically elevates the affinity of Hsp70 for ADP. On the other hand, P(i) increased the affinity in the presence of Mg(2+) ion, but not in its absence. Thus, P(i) enhances the effect of the Mg(2+) ion on the ADP binding. Next, we determined the crystal structures of the ADP-bound NBD with and without Mg(2+) ion. As compared with the Mg(2+) ion-free structure, the ADP- and Mg(2+) ion-bound NBD contains one Mg(2+) ion, which is coordinated with the β-phosphate group of ADP and associates with Asp10, Glu175, and Asp199, through four water molecules. The Mg(2+) ion is also coordinated with one P(i) molecule, which interacts with Lys71, Glu175, and Thr204. In fact, the mutations of Asp10 and Asp199 reduced the affinity of the NBD for ADP, in both the presence and the absence of P(i) . Therefore, the Mg(2+) ion-mediated network, including the P(i) and water molecules, increases the affinity of Hsp70 for ADP, and thus the dissociation of ADP is slow. In ADP-ATP exchange, the slow ADP dissociation might be rate-limiting. However, the nucleotide-exchange factors actually enhance ADP release by disrupting the Mg(2+) ion-mediated network.

Figure 1

ITC analysis of the interactions of the Hsp70 NBD (A–D) and the full-length Hsp70 (E–H) with ADP, in the presence and absence of Mg²⁺ ion and P_i. (A) The NBD in the P_i- and Mg²⁺ ion-containing buffer. (B) The NBD in the P_i-free and Mg²⁺ ion-containing buffer. (C) The NBD in the P_i-containing and Mg²⁺ ion-free buffer. (D) The NBD in the P_i- and Mg²⁺ ion-free buffer. (E) Hsp70 in the P_i- and Mg²⁺ ion-containing buffer. (F) Hsp70 in the P_i-free and Mg²⁺ ion-containing buffer. (G) Hsp70 in the P_i-containing and Mg²⁺ ion-free buffer. (H) Hsp70 in the P_i- and Mg²⁺ ion-free buffer.

Figure 2

Crystal structures of the ADP-bound NBD, with and without Mg²⁺ ion. (A) Crystal structure of the ADP- and Mg²⁺ ion-bound NBD (green). The stick models represent ADP and P_i, and the balls represent Mg²⁺ (magenta) and K⁺ (gray) ions. (B) Crystal structure of the ADP-bound and Mg²⁺ ion-free NBD (purple). The stick model represents ADP, and the gray ball represents the K⁺ ion. (C) The nucleotide-bound pocket of the ADP- and Mg²⁺ ion-bound NBD. The electron density indicates the F_o − F_c omit map (3σ level). (D) The nucleotide-bound pocket in the ADP-bound and Mg²⁺ ion-free NBD. The electron density indicates the F_o − F_c omit map (3σ level). (E) Anomalous difference Fourier map (4σ level) in the nucleotide-binding pocket of the ADP- and Mg²⁺ ion-bound NBD. (F) Anomalous difference Fourier map (4σ level) in the nucleotide-binding pocket of the ADP-bound and Mg²⁺ ion-free NBD. Molecular graphics were generated and rendered with the PyMOL program (DeLano Scientific, Palo Alto, CA).

Figure 3

Interactions between the NBD and ADP, in the presence and absence of Mg²⁺ ion. (A) The direct interactions in the presence of Mg²⁺ ion (stereo view). (B) The direct interactions in the absence of Mg⁺ ion (stereo view). (C) The indirect interactions in the presence of Mg²⁺ ion (stereo view). (D) The indirect interactions in the absence of Mg²⁺ ion (stereo view). The interacting residues are shown as stick models, with the translucent ribbon model of the NBD in green (A and C) and purple (B and D). ADP and P_i are also shown as stick models. The Mg²⁺ ion is colored magenta, and the K⁺ ion is represented by a gray ball. Water molecules are shown as red dots. Black and red dashed lines represent hydrogen bonds and coordinate bonds, respectively.

Figure 4

Interactions between the NBD and ADP, in the presence of Na⁺ ion. (A) ITC analysis of the interactions between the NBD and ADP, in buffer containing Pi-, Mg²⁺, and Na⁺ ions. (B) Crystal structure of the ADP- and Mg²⁺ ion-bound NBD containing Na⁺ ion. (C) The direct interactions between the NBD and ADP (stereo view). (D) The indirect interactions between the NBD and ADP (stereo view). The interacting residues are shown as stick models, with the ribbon model of the NBD in cyan. ADP and P_i are also shown as stick models. The Mg²⁺ ion is colored magenta, and the Na⁺ ion is represented by a gray ball. Water molecules are shown as red dots. Black and red dashed lines represent hydrogen bonds and coordinate bonds, respectively.

Figure 5

ITC analysis of the interactions of the NBD mutants with ADP. (A) The D10A mutant in the P_i-free and Mg²⁺ ion-containing buffer. (B) The D199A mutant in the P_i-free and Mg²⁺ ion-containing buffer. (C) The D10A mutant in the P_i- and Mg²⁺ ion-containing buffer. (D) The D199A mutant in the P_i- and Mg²⁺ ion-containing buffer.

Figure 6

Amino acid sequence alignment of the NBDs of five Hsp70-family proteins. Sequences are the NBDs of human (Homo sapiens) Hsp70 (NCBI Gene ID: 3303), Hsc70 (NCBI Gene ID: 3312), Bip/Grp78 (NCBI Gene ID: 3309), Grp75/mtHsp70 (NCBI Gene ID: 3313), and E. coli DnaK (NCBI Gene ID: 944750). Above the sequences, the secondary structures of human Hsp70 NBD are indicated. The residues involved in the Mg²⁺ ion-mediated network are indicated by green arrows.