Novobiocin Induces a Distinct Conformation of Hsp90 and Alters Hsp90−Cochaperone−Client Interactions

Yun, Bo Geon; Huang, Wenjun; Leach, Natalie; Hartson, Steven D.; Matts, Robert L.

doi:10.1021/bi0497998

Cited by 100 publications

(123 citation statements)

References 79 publications

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“…3B), NB also diminished direct interactions of Hsp90 with the preprotein. Our results extend earlier observations that NB can disrupt Hsp90 complexes with steroid receptors and kinases (59,60) by measuring a reduction in actual intermolecular contact.…”

Section: Resultssupporting

confidence: 90%

“…For a mechanistic comparison with NB, the effect of GA on import was also studied. Both drugs have been demonstrated to impair Hsp90 function, although much lower concentrations of GA are typically required: 0.5-10 M GA in live cells or 10 -100 M GA in vitro (48,(53)(54)(55)(56)(57)(58)(59)(60)(61). Such concentrations agree with the ϳ2 M affinity of GA for Hsp90 measured in vitro (53).…”

Section: Resultssupporting

confidence: 55%

“…Also, it has been proposed that coumarin-derived antibiotics such as NB could affect the C-terminal dimerization of Hsp90 (60,61). To investigate these possibilities, the gel filtration behavior of Hsp90 itself was analyzed after different treatments.…”

Section: Resultsmentioning

confidence: 99%

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Hsp90 Functions in the Targeting and Outer Membrane Translocation Steps of Tom70-mediated Mitochondrial Import

Fan

Bhangoo

Young

2006

Journal of Biological Chemistry

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The Tom70 import receptor on the mitochondrial outer membrane specifically recognizes Hsp90 and Hsc70, a critical step for the import of mitochondrial preproteins, the targeting of which depends on these cytosolic chaperones. To analyze the role of Hsp90 in mitochondrial import, the effects of the Hsp90 inhibitors geldanamycin and novobiocin were compared. Geldanamycin occludes the N-terminal ATP-binding site of Hsp90, whereas novobiocin targets the C-terminal region of the chaperone. Here, novobiocin was found to inhibit preprotein import and, in particular, targeting to the purified cytosolic fragment of Tom70. Hsp90 cross-linking to preprotein and coprecipitation of Hsp90 with Tom70 were both impaired by novobiocin. Overall, novobiocin treatment increased preprotein aggregation, contributing to reduced import competence. In contrast, geldanamycin had no apparent effect on preprotein interactions with Hsp90, formation of preprotein-chaperone complexes, Hsp90 docking onto Tom70, or preprotein association with the outer membrane. Instead, geldanamycin impaired formation of preprotein import intermediates at the outer membrane. This suggests a novel active role for Hsp90 in import steps subsequent to Tom70 targeting. Our results outline the mechanisms of Hsp90 function in preprotein targeting and transport.The great majority of mitochondrial proteins are encoded in the nucleus, translated by cytosolic ribosomes, and imported into mitochondria by a translocation apparatus located in the mitochondrial outer and inner membranes (1). The TOM (translocase of the mitochondrial outer membrane) complex contains import receptors that mediate the targeting of preproteins to the membrane and a general import pore complex through which preproteins are translocated. Sorting of preproteins to their appropriate final compartment within the mitochondria is performed by the TIM (translocase of the inner membrane) machinery (2, 3).Before import, unfolded or hydrophobic mitochondrial preproteins are bound by cytosolic chaperones, notably Hsc70 (heat shock cognate protein of 70 kDa; the constitutive Hsp70 (heat shock protein of 70 kDa) form in mammals) and Hsp90. Such preproteins include members of the inner membrane metabolite carrier family. The integral membrane Tom70 import receptor contains a tetratricopeptide repeat (TPR) 3 domain similar to those found in several other cytosolic cochaperones of Hsp90 and Hsc70 and provides a specific docking site for the Hsp90 and Hsc70 chaperones. This interaction is thought to be an essential first step in Tom70-dependent preprotein targeting, followed by contact between the preprotein and Tom70 itself (4 -15). Additional cytosolic proteins, including co-chaperones of , might also interact with preproteins to further assist import. After targeting to Tom70, preproteins are translocated through the outer membrane via the Tom40 import pore complex. The Tom40 pore also translocates preproteins targeted through the major import receptor Tom20 (21-23). After translocation, preproteins of the met...

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Section: Resultssupporting

confidence: 90%

Section: Resultssupporting

confidence: 55%

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Hsp90 Functions in the Targeting and Outer Membrane Translocation Steps of Tom70-mediated Mitochondrial Import

Fan

Bhangoo

Young

2006

Journal of Biological Chemistry

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“…Novobiocin is a small molecule inhibitor that binds to the CTD of Hsp90 (Marcu et al, 2000a;Marcu et al, 2000b;Soti et al, 2002). Like GA and radicicol, novobiocin inhibits the formation of Hsp90 complexes with client or p23, but unlike GA it also reduces the affinity of Hsp90 for TPR-containing co-chaperones that bind to the CTD (Allan et al, 2006;Yun et al, 2004). The disruption of these Hsp90 complexes suggests that novobiocin causes structural rearrangements within Hsp90 that are distinct from the changes seen with GA. Epigallocatechin gallate (ECGG) the active ingredient found in green tea also directly binds to Hsp90 and acts as an AhR antagonist.…”

Section: Small Molecules Also Shift the Conformation Of Hsp90mentioning

confidence: 99%

Conformational dynamics of the molecular chaperone Hsp90

Krukenberg

Street

Lavery

et al. 2011

Quart. Rev. Biophys.

283

262

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The molecular chaperone Hsp90 is an essential eukaryotic protein that makes up 1-2% of all cytosolic proteins. Hsp90 is vital for the maturation and maintenance of a wide variety of substrate proteins largely involved in signaling and regulatory processes. Many of these substrates have also been implicated in cancer and other diseases making Hsp90 an attractive target for therapeutics. Hsp90 is a highly dynamic and flexible molecule that can adapt its conformation to the wide variety of substrate proteins with which it acts. Large conformational rearrangements are also required for the activation of these client proteins. One driving force for these rearrangements is the intrinsic ATPase activity of Hsp90, as seen with other chaperones. However, unlike other chaperones, studies have shown that the ATPase cycle of Hsp90 is not conformationally deterministic. That is, rather than dictating the conformational state, ATP binding and hydrolysis shifts the equilibrium between a pre-existing set of conformational states in an organismdependent manner. In vivo Hsp90 functions as part of larger heterocomplexes. The binding partners of Hsp90, co-chaperones, assist in the recruitment and activation of substrates, and many co-chaperones further regulate the conformational dynamics of Hsp90 by shifting the conformational equilibrium towards a particular state. Studies have also suggested alternative mechanisms for the regulation of Hsp90's conformation. In this review, we discuss the structural and biochemical studies leading to our current understanding of the conformational dynamics of Hsp90 and the role that nucleotide, co-chaperones, post-translational modification and clients play in regulating Hsp90's conformation. We also discuss the effects of current Hsp90 inhibitors on conformation and the potential for developing small molecules that inhibit Hsp90 by disrupting the conformational dynamics.

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“…The N-terminal nucleotide binding domain of Hsp90 is the site of action for the Hsp90 inhibitor geldanamycin (26 -28), whereas its C-terminal domain contains a binding site for novobiocin, which also inhibits Hsp90 function (29 -32). Analyses with these compounds, with nucleotides and nucleotide analogs, and with site direct mutants that alter Hsp90 ATP binding and/or ATPase activity have revealed that Hsp90 function is regulated via the binding and hydrolysis of ATP, which modulates the switching of Hsp90 between at least three alternative conformations (1)(2)(3)(4)32). In the presence of geldanamycin, Hsp90 binds weakly to client kinases in a salt-labile fashion (20,24,33).…”

mentioning

confidence: 99%

Definition of Protein Kinase Sequence Motifs That Trigger High Affinity Binding of Hsp90 and Cdc37

Prince

Matts

2004

Journal of Biological Chemistry

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Hsp90 cooperates with its co-chaperone Cdc37 to provide obligatory support to numerous protein kinases involved in the regulation of cellular signal transduction pathways. In this report, the crystal structure of the Src family tyrosine kinase Lck was used to guide the creation of kinase constructs to determine features recognized by Hsp90 and its "kinase-specific" co-chaperone Cdc37. Two parameters were assayed: the ability and extent to which the constructs bound to Hsp90 and Cdc37, and the ability of the constructs to trigger saltresistant high affinity complexes with Hsp90 and Cdc37 independent of the presence of molybdate. Although Hsp90 interacted with both the N-terminal and C-terminal lobes (NL and CL, respectively) of the catalytic domains of the kinases, the lobes themselves were not sufficient to trigger the high affinity binding of Hsp90. Only constructs containing a complete N-or C-terminal lobe and part of the adjacent lobe bound to Hsp90 and Cdc37 in salt-stable complexes independent of molybdate. The two minimum constructs that bound Hsp90 and Cdc37 contained the ␣-C-helix and the ␤4-and ␤5-strands of the NL through to end of the CL and the NL through to the ␣-E-helix and the amino acids that cap the helix. Cdc37 interacted with only the NL and minimally required the ␣-C-helix and ␤4-and ␤5-strands of this lobe of Lck. The results indicate that the high affinity binding activity of Hsp90 is triggered through its interaction with adjacent subdomain structures of kinase catalytic domains. Furthermore, the ␣-C-helix and part of its adjoining loop connection to the ␤4-strand appear to be the primary determinants recognized by Cdc37.

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Novobiocin Induces a Distinct Conformation of Hsp90 and Alters Hsp90−Cochaperone−Client Interactions

Cited by 100 publications

References 79 publications

Hsp90 Functions in the Targeting and Outer Membrane Translocation Steps of Tom70-mediated Mitochondrial Import

Hsp90 Functions in the Targeting and Outer Membrane Translocation Steps of Tom70-mediated Mitochondrial Import

Conformational dynamics of the molecular chaperone Hsp90

Definition of Protein Kinase Sequence Motifs That Trigger High Affinity Binding of Hsp90 and Cdc37

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