Resolving hot spots in the C-terminal dimerization domain that determine the stability of the molecular chaperone Hsp90.
Human heat shock protein of 90 kDa (hHsp90) is a homodimer that has an essential role in facilitating malignant transformation at the molecular level. Inhibiting hHsp90 function is a validated approach for treating different types of tumors. Inhibiting the dimerization of hHsp90 via its C-terminal domain (CTD) should provide a novel way to therapeutically interfere with hHsp90 function. Here, we predicted hot spot residues that cluster in the CTD dimerization interface by a structural decomposition of the effective energy of binding computed by the MM-GBSA approach and confirmed these predictions using in silico alanine scanning with DrugScore(PPI). Mutation of these residues to alanine caused a significant decrease in the melting temperature according to differential scanning fluorimetry experiments, indicating a reduced stability of the mutant hHsp90 complexes. Size exclusion chromatography and multi-angle light scattering studies demonstrate that the reduced stability of the mutant hHsp90 correlates with a lower complex stoichiometry due to the disruption of the dimerization interface. These results suggest that the identified hot spot residues can be used as a pharmacophoric template for identifying and designing small-molecule inhibitors of hHsp90 dimerization.
Institute for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University, Düsseldorf, Germany.
Emanuele
E
Ciglia
Institute for Biochemical Plant Physiology, Heinrich-Heine-University, Düsseldorf, Germany.
Janina
J
Vergin
Institute of Biochemistry, Heinrich-Heine-University, Düsseldorf, Germany.
Sven
S
Reimann
Institute of Biochemistry, Heinrich-Heine-University, Düsseldorf, Germany.
Sander H J
SH
Smits
Institute of Biochemistry, Heinrich-Heine-University, Düsseldorf, Germany.
Lutz
L
Schmitt
Institute for Biochemical Plant Physiology, Heinrich-Heine-University, Düsseldorf, Germany.
Georg
G
Groth
Institute for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University, Düsseldorf, Germany.
Holger
H
Gohlke
D000409:Alanine / Q000235:genetics; D020816:Amino Acid Motifs; D018841:HSP90 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D056004:Molecular Dynamics Simulation; D009154:Mutation; D055503:Protein Multimerization; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D040681:Structural Homology, Protein; D044366:Transition Temperature
D018841:HSP90 Heat-Shock Proteins; D029701:Saccharomyces cerevisiae Proteins; D000409:Alanine