Novobiocin induces a distinct conformation of Hsp90 and alters Hsp90-cochaperone-client interactions

Biochemistry. 2004 Jun 29;43(25):8217-29. doi: 10.1021/bi0497998.

Abstract

Hsp90 functions to facilitate the folding of newly synthesized and denatured proteins. Hsp90 function is modulated through its interactions with cochaperones and the binding and hydrolysis of ATP. Recently, novobiocin has been shown to bind to a second nucleotide binding site located within the C-terminal domain of Hsp90. In this report, we have examined the effect of novobiocin on Hsp90 function in reticulocyte lysate. Novobiocin specifically inhibited the maturation of the heme-regulated eIF2alpha kinase (HRI) in a concentration-dependent manner. Novobiocin induced the dissociation of Hsp90 and Cdc37 from immature HRI, while the Hsp90 cochaperones p23, FKBP52, and protein phosphatase 5 remained associated with immature HRI. Proteolytic fingerprinting of Hsp90 indicated that novobiocin had a distinct effect on the conformation of Hsp90, and molybdate lowered the concentration of novobiocin required to alter Hsp90's conformation by 10-fold. The recombinant C-terminal domain of Hsp90 adopted a proteolytic resistant conformation in the presence of novobiocin, indicating that alteration of Hsp90/cochaperone interactions was not the cause of the novobiocin-induced protease resistance within Hsp90's C-terminal domain. The concentration dependence of this novobiocin-induced conformation change correlated with the dissociation of Hsp90 and Cdc37 from immature HRI and novobiocin-induced inhibition of Hsp90/Cdc37-dependent activation of HRI's autokinase activity. The data suggest that binding of novobiocin to the C-terminal nucleotide binding site of Hsp90 induces a change in Hsp90's conformation leading to the dissociation of bound kinase. The unique structure and properties of novobocin-bound Hsp90 suggest that it may represent the "client-release" conformation of the Hsp90 machine.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Autoradiography
  • Benzoquinones
  • Enzyme Inhibitors / pharmacology
  • HSC70 Heat-Shock Proteins
  • HSP70 Heat-Shock Proteins / metabolism
  • HSP90 Heat-Shock Proteins / antagonists & inhibitors*
  • HSP90 Heat-Shock Proteins / chemistry*
  • HSP90 Heat-Shock Proteins / metabolism
  • Heme / deficiency
  • Heme / metabolism
  • Humans
  • Lactams, Macrocyclic
  • Molecular Chaperones / metabolism*
  • Molybdenum / pharmacology
  • Novobiocin / pharmacology*
  • Peptide Mapping / methods
  • Protein Binding
  • Protein Conformation
  • Protein Folding
  • Quinones / pharmacology
  • Rabbits
  • Reticulocytes / drug effects
  • Reticulocytes / metabolism
  • Tacrolimus Binding Proteins / metabolism
  • eIF-2 Kinase / antagonists & inhibitors
  • eIF-2 Kinase / metabolism

Substances

  • Benzoquinones
  • Enzyme Inhibitors
  • HSC70 Heat-Shock Proteins
  • HSP70 Heat-Shock Proteins
  • HSP90 Heat-Shock Proteins
  • HSPA8 protein, human
  • Lactams, Macrocyclic
  • Molecular Chaperones
  • Quinones
  • Novobiocin
  • Heme
  • Molybdenum
  • sodium molybdate(VI)
  • eIF-2 Kinase
  • Tacrolimus Binding Proteins
  • geldanamycin