Filter binding assay for the geldanamycin-heat shock protein 90 interaction.

A filter binding assay to measure affinity of [3H-allyl]17-allylamino geldanamycin ([3H]AAG) for the ATP binding site of the N-terminal domain of human Hsp90alpha (hHsp90alpha9-236) was developed. Diethylaminoethyl cellulose or glass fiber filters impregnated with polyethyleneimine were used to capture the [3H]AAG-Hsp90 complex, and conditions which washed >98% of free [3H]AAG from the filters were developed. The complex formed at a rapid rate (k(on)=2.5 x 10(7)Lmol(-1) x s(-1)) and dissociated with a half-life of 2.3 min (k(off)=5 x 10(-3) x s(-1)). hHsp90alpha9-236 bound to [3H]AAG with a K(d) value of 0.4+/-0.1 microM. [3H]AAG had similar affinities for full-length hHsp90alpha and for hHsp90alpha9-236 variants containing biotinylated N-terminal biotinylation signal sequences and N- or C-terminal His(6) tags. Geldanamycin, ADP, ATP, and radicicol-all known to bind to the ATP domain of Hsp90-competed with [3H]AAG for binding to hHsp90alpha9-236, showing K(d) values in good agreement with reported values.

[1]  J. Reinstein,et al.  C-terminal regions of Hsp90 are important for trapping the nucleotide during the ATPase cycle. , 2000, Journal of molecular biology.

[2]  L. Weber,et al.  Sequence and regulation of a gene encoding a human 89-kilodalton heat shock protein , 1989, Molecular and cellular biology.

[3]  D. Santi,et al.  An FKBP12 binding assay based upon biotinylated FKBP12. , 2001, Analytical biochemistry.

[4]  L. Pearl,et al.  ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo , 1998, The EMBO journal.

[5]  I. H. Segel.  Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems , 1975 .

[6]  M. Yarus,et al.  On the properties and utility of a membrane filter assay in the study of isoleucyl-tRNA synthetase. , 1970, Analytical biochemistry.

[7]  L. Pearl,et al.  Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin. , 1999, Journal of medicinal chemistry.

[8]  Roger Fan,et al.  Dynamic activation of endothelial nitric oxide synthase by Hsp90 , 1998, Nature.

[9]  L. Pearl,et al.  Identification and Structural Characterization of the ATP/ADP-Binding Site in the Hsp90 Molecular Chaperone , 1997, Cell.

[10]  W. Pratt.  The hsp90-based Chaperone System: Involvement in Signal Transduction from a Variety of Hormone and Growth Factor Receptors , 1998, Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine.

[11]  C. Nicchitta,et al.  Ligand Interactions in the Adenosine Nucleotide-binding Domain of the Hsp90 Chaperone, GRP94 , 2000, The Journal of Biological Chemistry.

[12]  D. Cane,et al.  Dissecting and exploiting intermodular communication in polyketide synthases. , 1999, Science.

[13]  C. Nicchitta,et al.  Ligand Interactions in the Adenosine Nucleotide-binding Domain of the Hsp90 Chaperone, GRP94 , 2000, The Journal of Biological Chemistry.

[14]  L. Neckers,et al.  Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[15]  N. Rosen,et al.  Development of a purine-scaffold novel class of Hsp90 binders that inhibit the proliferation of cancer cells and induce the degradation of Her2 tyrosine kinase. , 2002, Bioorganic & medicinal chemistry.

[16]  Y. Yarden,et al.  Sensitivity of Mature ErbB2 to Geldanamycin Is Conferred by Its Kinase Domain and Is Mediated by the Chaperone Protein Hsp90* , 2001, The Journal of Biological Chemistry.

[17]  F. Hartl,et al.  In Vivo Function of Hsp90 Is Dependent on ATP Binding and ATP Hydrolysis , 1998, The Journal of cell biology.

[18]  S. Kimmins,et al.  Maturation of steroid receptors: an example of functional cooperation among molecular chaperones and their associated proteins , 2000, Cell stress & chaperones (Print).

[19]  J. Buchner,et al.  Coordinated ATP Hydrolysis by the Hsp90 Dimer* , 2001, The Journal of Biological Chemistry.

[20]  C. Diorio,et al.  Inhibition of the oncogene product p185erbB-2 in vitro and in vivo by geldanamycin and dihydrogeldanamycin derivatives. , 1995, Journal of medicinal chemistry.

[21]  L. Neckers,et al.  Geldanamycin as a Potential Anti-Cancer Agent: Its Molecular Target and Biochemical Activity , 2004, Investigational New Drugs.

[22]  Piper Pw.  The Hsp90 chaperone as a promising drug target. , 2001 .

[23]  Neal Rosen,et al.  Crystal Structure of an Hsp90–Geldanamycin Complex: Targeting of a Protein Chaperone by an Antitumor Agent , 1997, Cell.