Structural insights into GrpEL1-mediated nucleotide and substrate release of human mitochondrial Hsp70

Abstract

Maintenance of protein homeostasis is necessary for cell viability and depends on a complex network of chaperones and co-chaperones, including the heat-shock protein 70 (Hsp70) system. In human mitochondria, mitochondrial Hsp70 (mortalin) and the nucleotide exchange factor (GrpEL1) work synergistically to stabilize proteins, assemble protein complexes, and facilitate protein import. However, our understanding of the molecular mechanisms guiding these processes is hampered by limited structural information. To elucidate these mechanistic details, we used cryoEM to determine structures of full-length human mortalin-GrpEL1 complexes in previously unobserved states. Our structures and molecular dynamics simulations allow us to delineate specific roles for mortalin-GrpEL1 interfaces and to identify steps in GrpEL1-mediated nucleotide and substrate release by mortalin. Subsequent analyses reveal conserved mechanisms across bacteria and mammals and facilitate a complete understanding of sequential nucleotide and substrate release for the Hsp70 chaperone system.

Fig. 1. Structural determination of the Hsmortalin_R126W-GrpEL1 complex.

A Hsp70 proteins collaborate with co-chaperones to perform substrate capture and release. Mitochondrial Hsp70 (mortalin) interacts with a J-protein partner to capture substrates and hydrolyze ATP. Once substrate-bound, mortalin interacts with GrpE-like NEFs to facilitate substrate release via an unknown mechanism. NEF: nucleotide exchange factor. B Domain topology of Hsmortalin and HsGrpEL1. MTS: mitochondrial targeting sequence. IDL: interdomain linker. C Representative 2-D classes of the Hsmortalin_R126W-GrpEL1 complex. Colored arrows correspond to the densities observed in (D). D DeepEMhancer-sharpened map of the Hsmortalin_R126W-GrpEL1 complex colored by subunits and subdomains. The structure represents the exchange intermediate depicted in (A).

Fig. 2. The Hsmortalin_R126W NBD is fully expanded upon interaction with GrpEL1.

A Interface mapping between the mortalin_R126W IIB-NBD lobe and the GrpEL1-B short α-helix, and the mortalin_R126W IB-NBD lobe and the GrpEL1-A β-wing domain. The lack of EM density within the NBD suggests an apo-nucleotide NBD. B Structural comparison between the NBDs of Hsmortalin_R126W-GrpEL1, MtDnaK-GrpE (PDB: 8GB3), and ADP-PO₄-bound MgDnaK (PDB: 5OBW). Compared to ADP-PO₄-bound MgDnaK, the IIB-NBD lobe of Hsmortalin_R126W expands ~ 15° upon interaction with GrpEL1. C–E Comparison of the ATP binding residues in the IIB-NBD lobe. ATP-stabilizing interactions in the IIB-NBD lobe of Hsmortalin_R126W-GrpEL1 are removed by the movement of the IIB-NBD lobe. ADP-PO₄ is modeled from MgDnaK-ADP-PO₄. The semi-transparent model in panels (D and E) represents the MgDnaK-ADP-PO₄ structure (PDB: 5OBW).

Fig. 3. The mortalin interdomain linker is stabilized by interaction with the GrpEL1 long α-helix.

A Interaction mapping between the Hsmortalin_R126W linker and the GrpEL1 long α-helix. B Multiple sequence alignment between Hsp70 and GrpE-like species. The interdomain linker of Hsp70 is highly conserved, whereas the interacting GrpE-like region is variable. Hs: homo sapiens, Mg: mycoplasma genitalium, Mt: mycobacterium tuberculosis, Gk: geobacillus kaustophilus, Ec: eschericia coli. C Superposition of GrpE-like species from our Hsmortalin_R126W-GrpEL1 structure, MtDnaK-GrpE (PDB: 8GB3), and the AlphaFold2 prediction of HsGrpEL1.

Fig. 4. Hsmortalin_R126W in complex with GrpEL1 is substrate-bound to a mortalin truncation product.

A Model of the Hsmortalin_R126W substrate binding domain (SBD) bound to a substrate fit into the DeepEMhancer-sharpened cryoEM map. B Rigid-body docking of the mortalin SBD into the posterior substrate EM density (low-pass filtered to 5 Å). C Residue mapping of the substrate within the mortalin substrate binding site. The density of the substrate is shown as a gray contour. D Comparison of the modeled Hsmortalin substrate with the crystal structure of EcDnaK bound to the NRLLLTG peptide (PDB: 4EZW).

Fig. 5. GrpEL1 forms unique interactions with the Hsmortalin_R126W NBD and SBD.

A Structural mapping of the Hsmortalin_R126W SBD with the GrpEL1-B long α-helix and β-wing domain. B Residue mapping of the interactions between the GrpEL1-B long α-helix and Hsmortalin_R126W SBDβ cleft. C Residue mapping of the interactions between the GrpEL1-B β-wing domain and SBDα helical domain. D Multiple sequence alignments of the GrpEL1-B β-wing-SBDα interacting regions across GrpE-like and Hsp70 species. E Designation of the GrpEL1 β-wing face that interacts with the Hsmortalin_R126W NBD (β-wing face-N) and the face that interacts with the SBD (β-wing face-S).

Fig. 6. Mutation of Y173A in GrpEL1 results in a shift of the Hsmortalin SBD.

A DeepEMhancer-sharpened map of mortalin_R126W-GrpEL1_Y173A. Shadow represents the silhouette of the mortalin_R126W-GrpEL1_WT SBD position. B Superposition of mortalin_R126W-GrpEL1_WT and mortalin_R126W-GrpEL1_Y173A. In the mortalin_R126W-GrpEL1_Y173A structure, the SBD is translated ~ 6 Å away from GrpEL1-B compared to WT. C DeepEMhancer-sharpened map of mortalin_R126W-GrpEL1_Y173A−lid. Shadow represents the silhouette of the mortalin_R126W-GrpEL1_WT SBD position.

Fig. 7. Flexibility analysis of mortalin_R126W-GrpEL1_WT and mortalin_R126W-GrpEL1_Y173A.

A Anisotropic network modeling (ANM) mode 1 of mortalin_R126W-GrpEL1_WT and mortalin_R126W-GrpEL1_Y173A describes a lateral motion of the SBD. The SBD plane is colored in beige. B ANM mode 2 of mortalin_R126W-GrpEL1_Y173A describes a medial motion of the SBD. The SBD plane is colored in beige. Regions colored in blue describe low amounts of motion whereas regions in red describe high amounts of motion. C Analysis of lateral motion in replicate 1 of the mortalin_R126W-GrpEL1_WT all-atom simulation. The angular plot denotes the angle between vectors v₁ and v₃ (Supplementary Fig. 14) throughout a 150 ns replicate. The structural snapshot in dark blue represents a representative substate from 78 to 95.9°, in light blue a representative substate from 96 to 105°, and in seafoam green a representative substate from 105.1 to 134°. The percentage of trajectory in each region is annotated on the angular plot. The angle of the initial substate is denoted by a black line on the angular plot. D Analysis of medial motion in replicate 1 of the mortalin_R126W-GrpEL1_Y173A all-atom simulation. The angular plot denotes the angle between vectors v₁ and v₂ (Supplementary Fig. 14) throughout a 150 ns replicate. The structural snapshot in yellow represents a representative substate from 74 to 86.9°, in orange a representative substate from 87 to 96°, and in red a representative substate from 96.1 to 114°. The percentage of trajectory in each region is annotated on the angular plot. The angle of the initial substate is denoted by a black line on the angular plot.

Fig. 8. GrpEL1-mediated nucleotide and substrate release mechanism of human mortalin.

Upon interaction with a substrate and J-protein, mortalin hydrolyzes ATP and stably interacts with substrate. GrpEL1 interacts asymmetrically with mortalin and facilitates ADP release via interactions at the NBD. Flexibility within the SBD loosens the SBDα subdomain. Following ATP binding in a step-wise mechanism, the NBD dissociates first, which enables the opening of the SBD and subsequent substrate release. Alternatively, ATP binding induces concerted dissociation of mortalin whereby decoupling of SBDα and GrpEL1-B enables substrate release. The intermediate representing the mortalin_R126W-GrpEL1_WT structure is highlighted in orange.