Structural analysis of substrate binding by the molecular chaperone DnaK

Abstract

DnaK and other members of the 70-kilodalton heat-shock protein (hsp70) family promote protein folding, interaction, and translocation, both constitutively and in response to stress, by binding to unfolded polypeptide segments. These proteins have two functional units: a substrate-binding portion binds the polypeptide, and an adenosine triphosphatase portion facilitates substrate exchange. The crystal structure of a peptide complex with the substrate-binding unit of DnaK has now been determined at 2.0 angstroms resolution. The structure consists of a beta-sandwich subdomain followed by alpha-helical segments. The peptide is bound to DnaK in an extended conformation through a channel defined by loops from the beta sandwich. An alpha-helical domain stabilizes the complex, but does not contact the peptide directly. This domain is rotated in the molecules of a second crystal lattice, which suggests a model of conformation-dependent substrate binding that features a latch mechanism for maintaining long lifetime complexes.

Fig. 1

Electron density map. (A) The experimental MAD-phased electron density map at 2.3 Å resolution in the separate regions of the substrate peptide and β strand 3 in the protein, contoured at 1σ. Superposed is the refined atomic model. Only the electron density within 5 Å sphere range of the model is shown [with the use of the “mapcover” feature in program O (32)]. The peptide is shown in full length and residues are numbered 1 to 7. Residues 422 to 428 of DnaK in β strand 3 are also labeled. (B) The corresponding 2F_o−F_c electron density map at 2.0 Å resolution, contoured at 1σ, produced by program O (32).

Fig. 2

Overall structure, (A) Stereo view of a Cα trace of the structure. Every tenth residue is marked with a filled circle and labeled. The alternative conformation of the NH₂-terminus from residues 389 to 394 is shown with the dashed line. N and C are the NH₂- and COOH-termini of the polypeptide chain, respectively. (B) Ribbon diagram of the structure in the same orientation as in (A), The α helices and β strands are shown in dark blue, the loops in gold yellow, the substrate peptide in red. The alternative conformation of the NH₂-terminus is shown in cyan. The bops between the β strands that interact with the helix αB are labeled. (C) A view of the overall structure rotated 90° around the vertical axis from the view in (B). (D) Schematic representation of the topology of the structure. (E) Buried residues and conserved surface residues in the substrate-binding unit of DnaK. The side chains of those residues with less than 10% solvent accessibility are colored yellow. Also, colored in red are the residues that have more than 40% solvent accessible surface and are, as well, identical in E. coli DnaK, bovine hsc70, and hamster BiP (see Fig. 3). Glycines are colored in red for their backbones. [Part (A) produced by MOLSCRIPT (55), (B) and (C) by SETOR (55), and (E) by GRASP (55)]

Fig. 3

Structure-based sequence alignment of the C-terminal substrate binding unit of E. coli DnaK, bovine hsc70, and hamster BiP. The secondary structure assignments, based on main-chain hydrogen bonding pattern, are shown as arrows and cylinders. Solvent accessibility is indicated for each residue by an open circle if the fractional solvent accessibility is greater than 0.4, a half-closed circle if 0.1 to 0.4, and a closed circle if less than 0.1 (30). Residues changed in binding-defective genetic mutations are identified by asterisks (51).

Fig. 4

Peptide binding to DnaK (A) Stereo view of the interactions of the three leucines in the peptide with the β subdomain. The protein and the peptide backbones are shown in brown and blue, respectively. The DnaK residues interacting with the side chain of Leu⁴ of the peptide are colored purple and those interacting with Leu³ and Leu⁵ of the peptide are colored yellow. (B) Hydrogen bonds formed between the peptide and the β-sandwich subdomain of DnaK. The hydrogen bonds are represented by black dashed lines. The residues of DnaK involved in forming the hydrogen bonds are highlighted white. The backbone amide and carbonyl groups interacting with the peptide are labeled. (C) Surface curvature of the p subdomain (residues 394 to 501) bound to the substrate peptide. The most convex parts of the molecular surface are coded green while the most concave and planar are coded gray and white, respectively. This view is as in (A) and (B) but rotated by 90° so that the NH₂-terminal end of the peptide is below. (D) Surface electrostatic potential of the substrate-binding fragment of DnaK complex with the peptide. The orientation of the structure is the same as in Fig. 2B; thus, this side is at the NH₂-terminal end of the peptide. The surface is colored according to the local electrostatic potential, ranging from dark blue (most positive region) to deep red (most negative). [Parts (A) through (D) produced by GRASP (55)]

Fig. 5

(A) Superposition of the Cα trace of the helices in two different crystal forms, based on the Cα positions in the β-sandwich subdomain (396–501). The type 2 (P2₁2₁2) structure is shown in brown and the type 1 (I222) is shown in green. (B) Residues at the interface between the β-sandwich subdomain and helix αB. The orientation of the structure is similar to that in Fig, 2B, The peptide is colored blue. The carbon atoms of the residues from αB are colored white and the residues from the β domain are colored yellow. Nitrogen and oxygen atoms are colored blue and red, respectively. (C) Schematic representation of the mechanistic function of DnaK. [Parts (A) and (B) were produced by GRASP (55)]