The nucleotide-bound/substrate-bound conformation of the Mycoplasma genitalium DnaK chaperone

Abstract

Hsp70 chaperones keep protein homeostasis facilitating the response of organisms to changes in external and internal conditions. Hsp70s have two domains-nucleotide binding domain (NBD) and substrate binding domain (SBD)-connected by a conserved hydrophobic linker. Functioning of Hsp70s depend on tightly regulated cycles of ATP hydrolysis allosterically coupled, often together with cochaperones, to the binding/release of peptide substrates. Here we describe the crystal structure of the Mycoplasma genitalium DnaK (MgDnaK) protein, an Hsp70 homolog, in the noncompact, nucleotide-bound/substrate-bound conformation. The MgDnaK structure resembles the one from the thermophilic eubacteria DnaK trapped in the same state. However, in MgDnaK the NBD and SBD domains remain close to each other despite the lack of direct interaction between them and with the linker contacting the two subdomains of SBD. These observations suggest that the structures might represent an intermediate of the protein where the conserved linker binds to the SBD to favor the noncompact state of the protein by stabilizing the SBDβ-SBDα subdomains interaction, promoting the capacity of the protein to sample different conformations, which is critical for proper functioning of the molecular chaperone allosteric mechanism. Comparison of the solved structures indicates that the NBD remains essentially invariant in presence or absence of nucleotide.

Keywords: ATP hydrolysis; Hsp70 protein; Mycoplasma genitalium; allostery; chaperone cycle.

Figure 1

Overall structure of the M. genitalium DnaK protein. (A) Stereoview of the NDB and SBD domains represented as ribbons of MgDnaKΔCt connected by a highly conserved hydrophobic linker. The structure is depicted in rainbow coloring and the magnesium ion is represented as a red sphere. (B) View of the SBD domain 90° apart from the view on (A) with the C‐terminal tail shown in green. (C) Close‐up view of the substrate binding pocket where the construct C‐terminal tail is bound, mimicking a peptide substrate. The hydrophobic residues forming the substrate binding pocket are shown as sticks and follow the rainbow coloring on (A). The residues from the C‐terminal tail are shown as green atom‐colored sticks. (D) Stereoview of the linker region of MgDnaKΔCt:ADP·Pi (green ribbon) showing its interaction with residues from the SBD subdomains β and α. Residues from the linker and from subdomains β and α are depicted in stick mode and colored yellow, red and orange, respectively. http://imolecules3d.wiley.com/imolecules3d/review/vOYMnEAmHM3p6wxYgulJNC3xv0P4Zo7iUujMANFeHj0yq36fAFGzP4asF8kjFW9E825/1620

Figure 2

Structural comparison of MgDnaKΔCt (blue ribbon) with the ADP‐bound/substrate‐bound GkDnaKΔCt structure (green ribbon, PDB ID 2V7Y). Superposition of the NDB domains (A) and the SBD domains (B) of MgDnaKΔCt:ADP·Pi and GkDnaKΔCt:ADP·Pi. http://imolecules3d.wiley.com/imolecules3d/review/vOYMnEAmHM3p6wxYgulJNC3xv0P4Zo7iUujMANFeHj0yq36fAFGzP4asF8kjFW9E825/1621

Figure 3

Comparison of the nucleotide binding site from the MgDnaK‐NBD structures. (A) Two sulfate ions and a glycerol molecule are found in the nucleotide binding pocket of apoMgDnaK‐NBD. (B) An ADP molecule and an inorganic phosphate together with four water molecules (red spheres) are shown octahedrally coordinating a magnesium ion (green sphere) in MgDnaK‐NBD:ADP·Pi. The nucleotide, added as ATP, was fully hydrolyzed and the magnesium ion bridges the phosphate β and the inorganic phosphate. (C) An AMP‐PNP molecule was clearly defined in the MgDnaK‐NBD:AMPPNP structure. Phosphate β coordinates with a magnesium ion (green sphere), which presents only partial occupancy. In all three panels the 2Fo‐Fc electron density corresponding to the ligands is shown contoured at 1σ. Ligands are represented as sticks following an atom‐colored code, while blue is used to color the protein.