The Fe/S Assembly Protein IscU Behaves as a Substrate for the Molecular Chaperone Hsc66 from Escherichia coli *

IscU, a NifU-like Fe/S-escort protein, binds to and stimulates the ATPase activity of Hsc66, a hsp70-type molecular chaperone. We present evidence that stimulation arises from interactions of IscU with the substrate-binding site of Hsc66. IscU inhibited the ability of Hsc66 to suppress the aggregation of the denatured model substrate proteins rhodanese and citrate synthase, and calorimetric and surface plasmon resonance measurements showed that ATP destabilizes Hsc66·IscU complexes in a manner expected for hsp70-substrate complexes. Studies on the interaction of IscU with Hsc66 truncation mutants further showed that IscU does not bind the isolated ATPase domain of Hsc66 but does bind and stimulate a mutant containing the ATPase domain and substrate binding β-sandwich subdomain. These results support a role for IscU as a substrate for Hsc66 and suggest a specialized function for Hsc66 in the assembly, stabilization, or transfer of Fe/S clusters formed on IscU.

[1]  Shawn Y. Stevens,et al.  Structural insights into substrate binding by the molecular chaperone DnaK , 2000, Nature Structural Biology.

[2]  J. Silberg,et al.  The Hsc66-Hsc20 Chaperone System inEscherichia coli: Chaperone Activity and Interactions with the DnaK-DnaJ-GrpE System , 1998, Journal of bacteriology.

[3]  K. Flaherty,et al.  Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein , 1990, Nature.

[4]  J. Rothman,et al.  The ATPase core of a clathrin uncoating protein. , 1987, The Journal of biological chemistry.

[5]  Bernd Bukau,et al.  The Hsp70 and Hsp60 Chaperone Machines , 1998, Cell.

[6]  J. Reinstein,et al.  The role of ATP in the functional cycle of the DnaK chaperone system. , 1995, Journal of molecular biology.

[7]  J. Agar,et al.  Modular organization and identification of a mononuclear iron-binding site within the NifU protein , 2000, JBIC Journal of Biological Inorganic Chemistry.

[8]  Bernd Bukau,et al.  Substrate specificity of the DnaK chaperone determined by screening cellulose‐bound peptide libraries , 1997, The EMBO journal.

[9]  D. Dean,et al.  Assembly of Iron-Sulfur Clusters , 1998, The Journal of Biological Chemistry.

[10]  J. Silberg,et al.  Interaction of the iron-sulfur cluster assembly protein IscU with the Hsc66/Hsc20 molecular chaperone system of Escherichia coli. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[11]  E. Eisenberg,et al.  Effect of Nucleotide on the Binding of Peptides to 70-kDa Heat Shock Protein (*) , 1995, The Journal of Biological Chemistry.

[12]  William J. Welch,et al.  ATP-induced protein Hsp70 complex dissociation requires K+ but not ATP hydrolysis , 1993, Nature.

[13]  T. Kawula,et al.  Mutations in a gene encoding a new Hsp70 suppress rapid DNA inversion and bgl activation, but not proU derepression, in hns-1 mutant Escherichia coli , 1994, Journal of bacteriology.

[14]  C. Krebs,et al.  IscU as a scaffold for iron-sulfur cluster biosynthesis: sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU. , 2000, Biochemistry.

[15]  J. Reinstein,et al.  The second step of ATP binding to DnaK induces peptide release. , 1996, Journal of molecular biology.

[16]  Logan S. Ahlstrom,et al.  Chaperone-assisted protein folding. , 1997, Current opinion in structural biology.

[17]  R. McMacken,et al.  DnaJ dramatically stimulates ATP hydrolysis by DnaK: insight into targeting of Hsp70 proteins to polypeptide substrates. , 1999, Biochemistry.

[18]  D. Mckay,et al.  Kinetics of peptide binding to the bovine 70 kDa heat shock cognate protein, a molecular chaperone. , 1996, Biochemistry.

[19]  K. Johnson.  [61] Rapid kinetic analysis of mechanochemical adenosinetriphosphatases , 1986 .

[20]  B. Seaton,et al.  A gene encoding a DnaK/hsp70 homolog in Escherichia coli. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[21]  W. Burkholder,et al.  Specificity of DnaK-peptide binding. , 1994, Journal of molecular biology.

[22]  R. Mooney,et al.  Activation of phosphatidylinositol-3-kinase by platelet-derived growth factor and insulin-like growth factor-1 is inhibited by a transmembrane phosphotyrosine phosphatase. , 1993, The Journal of biological chemistry.

[23]  S. Rüdiger,et al.  Interaction of Hsp70 chaperones with substrates , 1997, Nature Structural Biology.

[24]  P. Christen,et al.  Kinetics of molecular chaperone action. , 1994, Science.

[25]  S. Garland,et al.  Suppressors of Superoxide Dismutase (SOD1) Deficiency in Saccharomyces cerevisiae , 1998, The Journal of Biological Chemistry.

[26]  J. Silberg,et al.  Kinetic Characterization of the ATPase Cycle of the Molecular Chaperone Hsc66 from Escherichia coli * , 2000, The Journal of Biological Chemistry.

[27]  Craig M. Ogata,et al.  Structural Analysis of Substrate Binding by the Molecular Chaperone DnaK , 1996, Science.

[28]  J. Silberg,et al.  Hsc66 and Hsc20, a new heat shock cognate molecular chaperone system from Escherichia coli , 1997, Protein science : a publication of the Protein Society.

[29]  R. Jordan,et al.  Modulation of the ATPase Activity of the Molecular Chaperone DnaK by Peptides and the DnaJ and GrpE Heat Shock Proteins (*) , 1995, The Journal of Biological Chemistry.