Fig 1.
Constructs and domain boundaries.
FKBP8: green, glutamate rich domain; yellow, PPIase domain; red, TPR domain; orange, calmodulin binding region and cyan, transmembrane region. Hsp90: green, N-terminal domain; orange, charged linker region; yellow, middle domain; red, C-terminal domain and cyan, conserved MEEVD TPR-binding motif. Hsp72: green, N-terminal domain; yellow, substrate binding domain and red, lid region. Numbers represent the approximate domain boundaries.
Table 1.
Data collection and refinement statistics.
Fig 2.
Isothermal titration calorimetry interactions.
ITC interactions between FKBP1-380 and A), Hsp90β EDASRMEEVD peptide B); human Hsp70 GSGPTIEEVD peptide; C), full-length Hsp90β; D), full-length human Hsp70; E), between FKBP92-207 and full-length Hsp90β; F), between FKBP1-380 and Hsp90β295–724 or G), Hsp90β295–689; H), between FKBP92-380 and full-length Hsp90β; I), between FKBP1-380 and human Hsp90α464–732; J), between FKBP1-380 and yHsp90546-709; K), and between the FKBP1-380 K307E mutant and yHsp90546-709. L), Gel-filtration chromatography of yHsp90546-709 (black trace), FKBP1-380 (red trace) and yHsp90546-709—FKBP1-380 (blue trace), showing that yHsp90546-709 and FKBP1-380 co-migrate as a complex.
Fig 3.
PyMol Cartoon structure of the FKBP92-380-DSTMEEVD complex.
A), FKBP92-380 consists of a single PPIase domain (left domain) and a TPR domain (right helical domain) that acts as the binding site of the DSTMEEVD Hsp90β peptide (yellow stick representation). B), Superimposition of the MEEVD containing peptides found bound to the four TPR domains of FKBP92-380 of the unit cell of the crystal. The peptide sequences bound were DTEMEEVD, ATEMEEVD (where the side chain of the first aspartate residue was modeled as an alanine residue), EMEEVD and EMEE. For amino acid residues defined by visible density the conformation of the amino acid was very similar in all cases. C), Representation of the electron density shown as a mesh for the bound DTEMEEVD peptide.
Fig 4.
Superimposition with the NMR structure of the PPIase domain of FKBP38.
Superimposition of the PPIase domains from NMR (yellow) and crystallography (green) show that the structures are essentially the same. However, in the crystal structure, relative to that of the NMR structure, a loop is present (Red) that disrupts β-strand 3 into two shorter β-strands.
Fig 5.
Superimposition of the TPR domain of FKBP52 and FKBP92-380.
A), Superimposition of the TPR domains of two molecules of the unit cell of the FKBP52 structure with bound peptide (green colored molecule with gold peptide and cyan colored molecule with blue peptide) and that of FKBP92-380 (magenta colored molecule and peptide) showing that the TPR domains are essentially the same. However, the bound MEEVD containing peptides are in radically different conformations. The peptides from the FKBP52 structure do not superimpose and are bound in an opposite polarity to that seen for FKBP92-380. While the hydrophobic residues, methionine and valine, of the conserved MEEVD motif of Hsp90 are buried in hydrophobic pockets of FKBP92-380, the same residues are surprisingly exposed to solvent in the FKBP52 structure. B), Superimposition of FKBP92-380 (green molecule with yellow bound peptide) and the TPR domain of CHIP (cyan molecule and bound peptide) showing that the bound MEEVD containing peptide conformations are essentially the same.
Fig 6.
Illustrations of the bound DTEMEEVD peptide.
A), Pymol cartoon showing interactions between the DTEMEEVD peptide (cyan) and the FKBP92-380 protein (green). Hydrogen bonds and salt bridges are shown as dotted blue lines and water molecules as cyan spheres. B), Ligplot showing the interactions between the DTEMEEVD peptide and the FKBP92-380 protein. Hydrogen bonds and salt bridges are shown as dotted cyan colored lines and water molecules as cyan spheres. Red amino acid residues represent the peptide and those in black represent FKBP92-380.
Fig 7.
ATPase activity of yeast Hsp90.
Using a 5-molar excess of FKBP92-380, over Hsp90 did not substantially alter the ATPase activity of the chaperone. Geld, geldanamycin.