Preferential Substrate Binding Orientation by the Molecular Chaperone HscA

HscA, a specialized bacterial hsp70-class chaperone, interacts with the iron-sulfur cluster assembly protein IscU by recognizing a conserved LPPVK sequence motif at positions 99–103. We have used a site-directed fluorescence labeling and quenching strategy to determine whether HscA binds to IscU in a preferred orientation. HscA was selectively labeled on opposite sides of the substrate binding domain with the fluorescent probe bimane, and the ability of LPPVK-containing peptides having tryptophan at the N or C terminus to quench bimane fluorescence was measured. Quenching was highly dependent on the position of tryptophan in the peptide and the location of bimane on HscA implying a strong directional preference for peptide binding. Similar experiments showed that full-length IscU binds in the same orientation as IscU-derived peptides and that binding orientation is unaffected by the co-chaperone HscB. The preferred orientation of the HscA-IscU complex is the reverse of that previously described for peptide complexes of Escherichia coli DnaK and rat Hsc70 substrate binding domain fragments establishing that hsp70 isoforms can bind peptide/polypeptide substrates in different orientations. HscA, a specialized bacterial hsp70-class chaperone, interacts with the iron-sulfur cluster assembly protein IscU by recognizing a conserved LPPVK sequence motif at positions 99–103. We have used a site-directed fluorescence labeling and quenching strategy to determine whether HscA binds to IscU in a preferred orientation. HscA was selectively labeled on opposite sides of the substrate binding domain with the fluorescent probe bimane, and the ability of LPPVK-containing peptides having tryptophan at the N or C terminus to quench bimane fluorescence was measured. Quenching was highly dependent on the position of tryptophan in the peptide and the location of bimane on HscA implying a strong directional preference for peptide binding. Similar experiments showed that full-length IscU binds in the same orientation as IscU-derived peptides and that binding orientation is unaffected by the co-chaperone HscB. The preferred orientation of the HscA-IscU complex is the reverse of that previously described for peptide complexes of Escherichia coli DnaK and rat Hsc70 substrate binding domain fragments establishing that hsp70 isoforms can bind peptide/polypeptide substrates in different orientations. HscA is an hsp70 class molecular chaperone (Mr 66,000; also designated Hsc66) that is constitutively expressed in Escherichia coli (1Seaton B.L. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2066-2070Crossref PubMed Scopus (83) Google Scholar, 2Kawula T.H. Lelivelt M.J. J. Bacteriol. 1994; 176: 610-619Crossref PubMed Google Scholar, 3Vickery L.E. Silberg J.J. Ta D.T. Protein Sci. 1997; 6: 1047-1056Crossref PubMed Scopus (88) Google Scholar). Genetic studies in bacteria and eukaryotes indicate that HscA participates in the biogenesis of iron-sulfur proteins (4Strain J. Lorenz C.R. Bode J. Garland S. Smolen G.A. Ta D.T. Vickery L.E. Culotta V.C. J. Biol. Chem. 1998; 273: 31138-31144Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 5Zheng L. Cash V.L. Flint D.H. Dean D.R. J. Biol. Chem. 1998; 273: 13264-13272Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar, 6Takahashi Y. Nakamura M. J. Biochem. (Tokyo). 1999; 126: 917-926Crossref PubMed Scopus (228) Google Scholar, 7Tokumoto U. Takahashi Y. J. Biochem. (Tokyo). 2001; 130: 63-71Crossref PubMed Scopus (215) Google Scholar). The exact role of HscA is not known, but HscA binds IscU (8Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Crossref PubMed Scopus (200) Google Scholar, 9Silberg J.J. Hoff K.G. Tapley T.L. Vickery L.E. J. Biol. Chem. 2001; 276: 1696-1700Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), a protein proposed to serve as a template for iron-sulfur cluster formation (10Agar J.N. Krebs C. Frazzon J. Huynh B.H. Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (386) Google Scholar, 11Agar J.N. Zheng L. Cash V.C. Dean D.R. Johnson M.K. J. Am. Chem. Soc. 2000; 122: 2136-2137Crossref Scopus (116) Google Scholar), and may function to regulate cluster formation on IscU or transfer of iron-sulfur clusters from IscU to acceptor proteins. The interaction between HscA and IscU is facilitated by HscB (also designated Hsc20), a J-type co-chaperone protein that binds both IscU and HscA and targets IscU to HscA (3Vickery L.E. Silberg J.J. Ta D.T. Protein Sci. 1997; 6: 1047-1056Crossref PubMed Scopus (88) Google Scholar, 8Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Crossref PubMed Scopus (200) Google Scholar, 9Silberg J.J. Hoff K.G. Tapley T.L. Vickery L.E. J. Biol. Chem. 2001; 276: 1696-1700Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Recently, we found that HscA specifically recognizes a conserved LPPVK sequence motif found at positions 99–103 of IscU (12Hoff K.G. Ta D.T. Tapley T.L. Silberg J.J. Vickery L.E. J. Biol. Chem. 2002; 277: 27353-27359Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). A synthetic peptide corresponding to residues 98–106 (ELPPVKIHC) was found to bind to HscA and to stimulate HscA ATPase activity in a manner similar to IscU, suggesting that the LPPVK region plays a key role in binding to and regulation of HscA. Amino acid replacement studies on IscU and the Glu98–Cys106 peptide 11 The abbreviations used are: Glu98–Cys106 peptide, ELPPVKIHC peptide corresponding to IscU residues 98–106; HscA(Q421C)-bimane, HscA(D422C)-bimane, and HscA(A455C)-bimane, bimane-labeled HscA triple mutants derived from HscA(C315S/C448S); W-97 peptide, WELPPVKI; W-105 peptide, ELPPVKIW. revealed that Pro101 is the most critical position with Val102 and Lys103 also contributing to high affinity binding and regulation (13Hoff K.G. Cupp-Vickery J. Vickery L.E. J. Biol. Chem. 2003; 278: 37582-37589Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). To better understand the interaction of IscU with HscA we used the crystal structure of DnaK bound to the synthetic peptide NRLLLTG (14Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1064) Google Scholar) to construct models of HscA-peptide complexes (13Hoff K.G. Cupp-Vickery J. Vickery L.E. J. Biol. Chem. 2003; 278: 37582-37589Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). These models suggested that the LPPVK region of IscU could be accommodated in the peptide binding cleft of the substrate binding domain of HscA with the peptide backbone in an extended conformation, and the importance of Pro101 could be rationalized by its projection into a hydrophobic pocket in the central position of the cleft. It was not possible, however, to determine the orientation of the peptide based on model analysis, in HscA or peptide structure in models in the peptide was in opposite to the substrate binding domain (13Hoff K.G. Cupp-Vickery J. Vickery L.E. J. Biol. Chem. 2003; 278: 37582-37589Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). of HscA with of the IscU protein or with the co-chaperone HscB could on binding and orientation of the peptide in the the studies described we have site-directed fluorescence labeling and quenching to determine whether HscA binds to peptides and the full-length IscU protein in a orientation. The that HscA a strong directional preference for substrate binding and that the preferred orientation is opposite that previously for peptide complexes of hsp70 proteins. Protein and mutants of HscA and IscU the with from by The was from (3Vickery L.E. Silberg J.J. Ta D.T. Protein Sci. 1997; 6: 1047-1056Crossref PubMed Scopus (88) Google Scholar) by of for residues and to was used as a template to construct the and mutants that have a (8Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Crossref PubMed Scopus (200) Google Scholar) was used as a template for the and and of HscA and IscU mutants as previously described for the proteins (3Vickery L.E. Silberg J.J. Ta D.T. Protein Sci. 1997; 6: 1047-1056Crossref PubMed Scopus (88) Google Scholar, 8Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Crossref PubMed Scopus (200) Google Scholar). and W-105 by the Protein and at by high and was by of Glu98–Cys106 Biochem. PubMed Scopus Google Scholar), and of W-97 and W-105 for tryptophan Biochem. PubMed Scopus Google Scholar, C.R. Biochem. PubMed Scopus Google Scholar, L. Protein Sci. PubMed Scopus Google Scholar). and with a of in for at and at and was was the HscA (3Vickery L.E. Silberg J.J. Ta D.T. Protein Sci. 1997; 6: 1047-1056Crossref PubMed Scopus (88) Google bimane 1999; PubMed Scopus Google Scholar). The a labeling a of The and mutants labeling in different with labeling of the a fluorescence at a bimane-labeled HscA in experiments in the of to of of experiments in the of the of was to the for from to at an of and a of to for and found to by from or in IscU by of in experiments in the of of bimane-labeled HscA in the of and the of a of HscB and IscU in by the of from to with similar and from experiments in of peptides to bimane-labeled HscA by fluorescence quenching from the in to to determine and for and not and the was of and on binding of tryptophan peptides to bimane-labeled HscA. of bimane-labeled HscA in the of of peptide and by of IscU tryptophan mutants on the fluorescence of bimane-labeled HscA. of bimane-labeled and in of IscU, or the for is to of of IscU tryptophan mutants to bimane-labeled HscA by fluorescence quenching from the in to to determine and was not of HscB on quenching of bimane-labeled HscA of bimane-labeled and in the of and of IscU or with HscB. IscU and HscB at a and to a to binding to and studies as described previously (3Vickery L.E. Silberg J.J. Ta D.T. Protein Sci. 1997; 6: 1047-1056Crossref PubMed Scopus (88) Google Scholar, J.J. Hoff K.G. Vickery L.E. J. Bacteriol. 1998; PubMed Google Scholar, J.J. Vickery L.E. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). ATPase in the of from for to for and of and was of ATPase activity of HscA mutants by peptides and IscU mutants with HscA at in in the of peptide or IscU, and The used and and the of of in a used site-directed labeling with the fluorescent probe bimane and quenching by tryptophan peptides to determine the orientation of substrates bound to HscA. of the quenching of bimane fluorescence by a that between the bimane and the of tryptophan M. Y. Chem. Scopus Google Scholar, PubMed Scopus Google Scholar). fluorescence with 1999; PubMed Scopus Google Scholar, A. A. J. Chem. Scopus Google Scholar), but its quenching by tryptophan can a of in proteins Biochemistry. 2002; PubMed Scopus (88) Google Scholar). The substrate binding domain of HscA was selectively labeled with and the ability of IscU-derived peptides having tryptophan at the N or C terminus and to quench bimane fluorescence was measured. of the full-length IscU protein having tryptophan in the corresponding positions and also for ability to quench bimane-labeled HscA. We have models of the substrate binding domain of HscA bound to the IscU peptide (13Hoff K.G. Cupp-Vickery J. Vickery L.E. J. Biol. Chem. 2003; 278: 37582-37589Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) based on the crystal structure of the DnaK substrate binding domain to the peptide NRLLLTG (14Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1064) Google Scholar). The of HscA sequence to that of suggesting that the peptide binding to have similar different of a model of the HscA substrate binding domain with The peptide is in a in the with Pro101 into a hydrophobic pocket in the central region of the cleft. model the peptide is with the N terminus on the of the substrate binding domain and the C terminus on the orientation is opposite that for the peptide complex (14Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1064) Google Scholar). We have designated the to orientation as the to the for the peptide To positions for bimane labeling we the model for residues to the position at the peptide the binding cleft. and the cleft on the and the cleft on the to labeling that bimane to selectively with or residues of bound of residues into HscA at positions or to bimane To labeling at the residues at positions and of HscA to by was used as a template to triple designated and labeling of the mutants to HscA suggesting that from the acid To whether the different of the was for ATPase and chaperone activity as as for with peptides and the ATPase activity of HscA, and in the and of the different peptides and of The an ATPase activity with HscA and a similar was for the mutants of the mutants peptide and ATPase activity of of ATPase activity with substrate binding. by IscU in the of HscB (8Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Crossref PubMed Scopus (200) Google Scholar) was also that the between substrate and co-chaperone was not The of the on the affinity for IscU by and of the mutants bound IscU with an affinity with that of HscA and mutants also chaperone activity similar to that of HscA J.J. Hoff K.G. Vickery L.E. J. Bacteriol. 1998; PubMed Google Scholar) as by of of the model substrate not that and HscA mutants in Quenching of HscA mutants labeled with bimane, and studies on with peptides in the of the of the peptide and the tryptophan W-97 and W-105 peptides on the bimane fluorescence of both and HscA. the the Glu98–Cys106 and W-97 peptides quenching the W-105 peptide bimane fluorescence The quenching by W-105 to W-97 that the bound peptide is with the C terminus on the of the substrate binding domain to The in quenching of W-97 and W-105 that of the peptide is bound in reverse to orientation. The of the peptides on the fluorescence of the also with binding orientation. was quenching of bimane fluorescence by the peptide, suggesting that peptide binding the of the probe at position a in quenching was with the The W-105 peptide quenching Glu98–Cys106 the W-97 peptide was These indicate a strong preference for peptide binding in the N terminus is on the of the substrate binding the same reverse orientation as with the is that the of peptides to quench fluorescence could from of the tryptophan the of the with and in of a preferred orientation. The binding affinity of the peptides for the was by of the in at of peptide bimane-labeled the of quenching by Glu98–Cys106 and W-97 was to with but the an to a binding corresponding to for the W-105 peptide a corresponding to bimane-labeled of the peptides a and bound with similar The of the of the W-97 and W-105 peptides to that of Glu98–Cys106 that the of tryptophan at the N or C terminus not binding The of a of the tryptophan residues on binding affinity is with the HscA-peptide model in residues and of the peptide binding cleft Similar fluorescence quenching experiments the with the Glu98–Cys106 and W-97 peptides quenching the W-105 peptide The that similar a different labeling position for the to binding orientation. on affinity of for substrates is L.E. Science. PubMed Scopus Google Scholar, D.R. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, D.R. L. PubMed Scopus Google Scholar, A. Science. 1994; PubMed Scopus Google Scholar, A. J. J. Biol. PubMed Scopus Google Scholar, L.E. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. Biochemistry. 1996; PubMed Scopus Google Scholar, L. J. J. Biol. 1996; PubMed Scopus Google Scholar), and and of HscA high substrate affinity to the (8Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Crossref PubMed Scopus (200) Google Scholar, 9Silberg J.J. Hoff K.G. Tapley T.L. Vickery L.E. J. Biol. Chem. 2001; 276: 1696-1700Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, J.J. Hoff K.G. Vickery L.E. J. Bacteriol. 1998; PubMed Google Scholar). To determine whether the bimane-labeled HscA mutants we quenching in the of or in binding of W-105 to bimane-labeled and W-97 to bimane-labeled is The affinity of for W-105 is in the of in the of and the affinity of for W-97 is in the of with the both the in the of similar to in the of These indicate that the in and or the of the bimane probe Quenching by IscU determine whether the binding orientation with the W-97 and W-105 peptides with the full-length IscU protein we the corresponding and proteins. mutants similar to IscU not suggesting that of tryptophan at positions not the structure of the revealed that IscU and as of a mutants in the ATPase activity of HscA as as of the mutants with interaction with the substrate binding domain in a manner similar to experiments bimane-labeled showed of and on the fluorescence of of to a of quenching the a in fluorescence The that quenching is with is with the reverse orientation from the peptide binding experiments described the to determine binding and to the with The structure of the IscU protein may of tryptophan to the bimane in and in HscA by IscU binding could fluorescence of the quenching studies the The of IscU, and on the fluorescence of and in The similar to found for the corresponding peptides in IscU and quenching HscA(A455C)-bimane, on the was IscU These indicate as found for the the IscU protein a strong preference for binding in the N terminus is on the and the C terminus is on the of the substrate binding the reverse to orientation. The in directional binding preference for the full-length IscU protein to that for the peptides that the the of the peptides not to binding of the binding of IscU, and for the bimane-labeled HscA mutants in The for IscU similar to found by for binding to the HscA mutants that bimane labeling not with binding. both and affinity to IscU and suggesting that the tryptophan have a on the interaction with labeled the affinity of could not be of the of binding of however, with affinity in the with and binding to bimane-labeled HscA in a similar manner as HscB on IscU of IscU with the complex of HscA is facilitated by the J-type co-chaperone HscB (8Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Crossref PubMed Scopus (200) Google Scholar). To determine whether HscB the orientation of IscU bound to we fluorescence quenching studies in the of the HscB on the fluorescence of the the of the however, HscB binding in an in fluorescence by a in the not to from in the of the bimane probe from HscB binding. IscU and HscB stimulate the ATPase activity of HscA (8Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Crossref PubMed Scopus (200) Google Scholar), was not to as with IscU of the and of fluorescence and of a of HscB IscU to HscA in the of in of IscU with HscB to the complex a in fluorescence and in to The is that of HscB suggesting that IscU binding in quenching of as was in the of HscB and of with HscB to a similar fluorescence to that with IscU with the of on the opposite to the bimane on the of the HscA substrate binding of with in in quenching suggesting as in the of is on the of the substrate binding domain in the with also indicate a reverse to orientation for IscU to HscA in the of HscB. IscU and with HscB quenching for a similar to that in the of HscB and and with of residues of IscU on the of the HscA substrate binding of with on the in quenching with of the residues of IscU on the of the substrate binding These with for the indicate that interaction of HscB with IscU and not the orientation of the IscU sequence in the peptide binding cleft. The studies that HscA a strong directional preference for binding of and IscU-derived peptides the LPPVK the bimane fluorescent probe to of the of quenching of HscA labeled on opposite sides of the substrate binding domain by and tryptophan peptides that the reverse to binding orientation is the to orientation. The that peptides the LPPVK sequence bound with the same as the IscU protein and the of an of the co-chaperone HscB on binding orientation indicate that the LPPVK region with the substrate binding domain to binding orientation. These with previously proposed models in the LPPVK region binds to the substrate binding domain in an extended with Pro101 in the peptide binding cleft (13Hoff K.G. Cupp-Vickery J. Vickery L.E. J. Biol. Chem. 2003; 278: 37582-37589Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). for proposed to serve as a for the assembly of iron-sulfur clusters (10Agar J.N. Krebs C. Frazzon J. Huynh B.H. Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (386) Google Scholar, 11Agar J.N. Zheng L. Cash V.C. Dean D.R. Johnson M.K. J. Am. Chem. Soc. 2000; 122: 2136-2137Crossref Scopus (116) Google Scholar), and the interaction of HscA and IscU may an role in iron-sulfur cluster formation The binding orientation that we on the of different of the IscU to the Amino of IscU that to the LPPVK motif be on the of the HscA substrate binding be on the that the structure of the HscA substrate binding domain is similar to that of DnaK X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1064) Google Scholar, also K.G. Cupp-Vickery J. Vickery L.E. J. Biol. Chem. 2003; 278: 37582-37589Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), the of the the binding cleft and the the to between the and of may have for iron-sulfur cluster The structure of the iron-sulfur complex of IscU is not known, but residues have in cluster (10Agar J.N. Krebs C. Frazzon J. Huynh B.H. Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (386) Google Scholar, 11Agar J.N. Zheng L. Cash V.C. Dean D.R. Johnson M.K. J. Am. Chem. Soc. 2000; 122: 2136-2137Crossref Scopus (116) Google Scholar, J. J. Am. Chem. Soc. 2000; 122: Scopus (78) Google Scholar, J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus (83) Google Scholar, Biochemistry. 2002; PubMed Scopus Google Scholar). the HscA-IscU complex residues and of IscU on the of the HscA substrate binding is to the from with the same iron-sulfur cluster as and bound to HscA. of with from IscU could and studies indicate that IscU iron-sulfur complexes as a or (8Hoff K.G. Silberg J.J. Vickery L.E. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7790-7795Crossref PubMed Scopus (200) Google Scholar, J.N. Krebs C. Frazzon J. Huynh B.H. Dean D.R. Johnson M.K. Biochemistry. 2000; 39: 7856-7862Crossref PubMed Scopus (386) Google Scholar, 11Agar J.N. Zheng L. Cash V.C. Dean D.R. Johnson M.K. J. Am. Chem. Soc. 2000; 122: 2136-2137Crossref Scopus (116) Google Scholar, J. J. Am. Chem. Soc. 2000; 122: Scopus (78) Google Scholar, J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus (83) Google Scholar, Biochemistry. 2002; PubMed Scopus Google Scholar) that could for cluster binding. The of HscA binding on the structure of IscU not studies of from indicate that protein a structure in and that the region residues corresponding to the LPPVK motif and of coli IscU is C. C. J. Biol. 2003; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). studies of IscU from that the of IscU protein is in A. J. and M. A. Protein for IscU is to and and the LPPVK region is as a the structure on structure that binding of IscU to HscA of the structure of the IscU studies on the of HscA on the IscU structure and iron-sulfur cluster formation may into the role of the chaperone in cluster formation with hsp70 to orientation of and IscU-derived peptides to the substrate binding domain of HscA is opposite that described previously for hsp70 peptide The crystal structure of the substrate binding domain of coli DnaK showed that the synthetic peptide NRLLLTG was bound with its N terminus the residues to HscA residues and and its C terminus the the to HscA (14Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1064) Google Scholar), we have designated as the The central of the NRLLLTG peptide was bound in a hydrophobic pocket in the of the cleft with residues the by and the pocket (14Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1064) Google Scholar). studies of a complex of the NRLLLTG peptide and a of the DnaK substrate binding domain the showed binding in the same and orientation as was in the crystal structure S. M. Protein Sci. 2003; PubMed Scopus Google Scholar). studies on of DnaK and Hsc70 have also revealed of binding a DnaK substrate binding domain showed that a of the was in and was bound to the peptide binding cleft of the Y. Biochemistry. 1998; PubMed Scopus Google Scholar). found with the the DnaK peptide was in a with bound in the central pocket and and on the and of the Y. Biochemistry. 1998; PubMed Scopus Google Scholar). Similar also in studies of a of rat Hsc70 The C terminus of Hsc70 was and was bound in the central pocket with and to the in positions corresponding to the Y. J. Biol. 1999; PubMed Scopus Google Scholar). the to and HscA, peptide binding to with a preferred orientation. It is not known, however, whether directional binding is to complexes or is a of hsp70 It is also not whether the binding orientation with DnaK and Hsc70 or the reverse orientation with HscA most of the crystal structure of the complex that for is at the central in a hydrophobic pocket to (14Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1064) Google Scholar), and could be in the of binding. The that the peptide however, The NRLLLTG peptide is in a of and that could be in binding but binding in the reverse with of the interaction of DnaK with peptides S. J. J. 2001; PubMed Scopus Google Scholar) have that peptides having an sequence to in binding suggesting that on both peptide backbone and may to both binding and may also binding. The region the peptide binding cleft of DnaK a that peptides residues (14Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1064) Google Scholar, S. L. J. J. 1997; PubMed Scopus Google Scholar, S. A. Biol. 1997; PubMed Scopus Google Scholar). is most on the of the and the NRLLLTG peptide is bound with its N terminus on the and the C terminus on the (14Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1064) Google Scholar). that for peptide complex of DnaK the plays a role in binding on the of on binding of the NRLLLTG peptide to DnaK have an of binding J. Chem. 2003; Scopus Google Scholar), but based on binding of to DnaK that the to the of binding 2000; PubMed Scopus Google Scholar). The importance of and may for different the of HscA-IscU the preference for at the central position of the peptide of the and of the the of an in the peptide backbone at position the of with the NRLLLTG complex with of the substrate binding domain of HscA also an with residues the of the cleft and residues the (13Hoff K.G. Cupp-Vickery J. Vickery L.E. J. Biol. Chem. 2003; 278: 37582-37589Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), and could to binding studies suggested the that the reverse peptide binding orientation and Lys103 of IscU to in with HscA and that orientation K.G. Cupp-Vickery J. Vickery L.E. J. Biol. Chem. 2003; 278: 37582-37589Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 9Silberg J.J. Hoff K.G. Tapley T.L. Vickery L.E. J. Biol. Chem. 2001; 276: 1696-1700Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). of the structure of the HscA-IscU complex in peptide is bound in the reverse from that of the of substrate by class of molecular We Ta and for for and with fluorescence and Silberg for with