A Structure-based Mutational Analysis of Cyclophilin 40 Identifies Key Residues in the Core Tetratricopeptide Repeat Domain That Mediate Binding to Hsp90

Abstract

Cyclophilin 40 (CyP40) is a tetratricopeptide repeat (TPR)-containing immunophilin and a modulator of steroid receptor function through its binding to heat shock protein 90 (Hsp90). Critical to this binding are the carboxyl-terminal MEEVD motif of Hsp90 and the TPR domain of CyP40. Two different models of the CyP40-MEEVD peptide interaction were used as the basis for a comprehensive mutational analysis of the Hsp90-interacting domain of CyP40. Using a carboxyl-terminal CyP40 construct as template, 24 amino acids from the TPR and flanking acidic and basic domains were individually mutated by site-directed mutagenesis, and the mutants were coexpressed in yeast with a carboxyl-terminal Hsp90β construct and qualitatively assessed for binding using a β-galactosidase filter assay. For quantitative assessment, mutants were expressed as glutathione S-transferase fusion proteins and assayed for binding to carboxyl-terminal Hsp90β using conventional pulldown and enzyme-linked immunosorbent assay microtiter plate assays. Collectively, the models predict that the following TPR residues help define a binding groove for the MEEVD peptide: Lys-227, Asn-231, Phe-234, Ser-274, Asn-278, Lys-308, and Arg-312. Mutational analysis identified five of these residues (Lys-227, Asn-231, Asn-278, Lys-308, and Arg-312) as essential for Hsp90 binding. The other two residues (Phe-234 and Ser-274) and another three TPR domain residues not definitively associated with the binding groove (Leu-284, Lys-285, and Asp-329) are required for efficient Hsp90 binding. These data confirm the critical importance of the MEEVD binding groove in CyP40 for Hsp90 recognition and reveal that additional charged and hydrophobic residues within the CyP40 TPR domain are required for Hsp90 binding. Cyclophilin 40 (CyP40) is a tetratricopeptide repeat (TPR)-containing immunophilin and a modulator of steroid receptor function through its binding to heat shock protein 90 (Hsp90). Critical to this binding are the carboxyl-terminal MEEVD motif of Hsp90 and the TPR domain of CyP40. Two different models of the CyP40-MEEVD peptide interaction were used as the basis for a comprehensive mutational analysis of the Hsp90-interacting domain of CyP40. Using a carboxyl-terminal CyP40 construct as template, 24 amino acids from the TPR and flanking acidic and basic domains were individually mutated by site-directed mutagenesis, and the mutants were coexpressed in yeast with a carboxyl-terminal Hsp90β construct and qualitatively assessed for binding using a β-galactosidase filter assay. For quantitative assessment, mutants were expressed as glutathione S-transferase fusion proteins and assayed for binding to carboxyl-terminal Hsp90β using conventional pulldown and enzyme-linked immunosorbent assay microtiter plate assays. Collectively, the models predict that the following TPR residues help define a binding groove for the MEEVD peptide: Lys-227, Asn-231, Phe-234, Ser-274, Asn-278, Lys-308, and Arg-312. Mutational analysis identified five of these residues (Lys-227, Asn-231, Asn-278, Lys-308, and Arg-312) as essential for Hsp90 binding. The other two residues (Phe-234 and Ser-274) and another three TPR domain residues not definitively associated with the binding groove (Leu-284, Lys-285, and Asp-329) are required for efficient Hsp90 binding. These data confirm the critical importance of the MEEVD binding groove in CyP40 for Hsp90 recognition and reveal that additional charged and hydrophobic residues within the CyP40 TPR domain are required for Hsp90 binding. In the absence of hormone, estrogen receptor α is found predominantly in the nucleus as part of a multicomponent complex with Hsp90 1The abbreviations used are: Hspn , heat shock protein of n kDa; BSA, bovine serum albumin; CyP40, 40-kDa cyclophilin; ELISA, enzyme-linked immunosorbent assay; FKBP(s), FK506-binding protein(s); GST, glutathione S-transferase; hCyP40, human CyP40; hHsp90β, human Hsp90β; Hip, hsc70-interacting protein; Hop, Hsp90- and Hsp70-organizing protein; PP5, serine/threonine protein phosphatase type 5; TPR, tetratricopeptide repeat; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside. (1Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Google Scholar). This abundant molecular chaperone plays an essential role in maintaining steroid receptors in a hormone-activable state, and the association of receptor with Hsp90 is a prerequisite for efficient signal transduction (1Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Google Scholar). In addition to dimeric Hsp90 and the receptor subunit, mature steroid receptor complexes contain p23 and one of a group of modulating proteins that include the immunophilins cyclophilin 40 (CyP40), FKBP51, and FKBP52 (1Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Google Scholar). CyP40 was first isolated as a component of estrogen receptor α heterocomplexes (2Ratajczak T. Hlaing Scholar). This cyclophilin the and with its and immunophilins of the FK506-binding (1Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Google Scholar). The of these immunophilins is by an domain that is by and a function and a carboxyl-terminal protein interaction domain of the tetratricopeptide repeat motif T. Scholar). These immunophilins in steroid receptor function through association with steroid receptor complexes the immunophilins for a binding in the carboxyl-terminal of Hsp90 T. Scholar). of interaction the of PP5, a serine/threonine protein complexes W.B. 1997; Scholar). This recognition Hsp90 T. T. and chaperone 1997; T. T. and for steroid receptor interaction Toft D.O. Toft Scholar). Hsp90 as a for the to steroid receptors and to receptor to In that of the immunophilins and steroid receptor complexes an in the (1Pratt W.B. Toft D.O. Endocr. Rev. 1997; 18: 306-360Google Scholar). state, the of receptors as mature the interaction of receptor with and of the chaperone Toft D.O. Scholar). This is by of and Toft D.O. with TPR domains that are to in steroid immunophilins T. 1997; Scholar). The TPR domain in interaction with the domain of 1997; Scholar). TPR domains the and carboxyl-terminal of as recognition for and T. 1997; Scholar). In to Hip, the carboxyl-terminal of the binding 1997; 18: Scholar). is essential for the of Hsp90 to steroid receptors and with the TPR by immunophilins T. Scholar). The TPR domains in and Hop, to different for protein For Hip, part of its interaction for is by charged flanking the TPR domain 1997; Scholar). to the TPR domains of CyP40, FKBP51, and FKBP52 to Hsp90 recognition T. Scholar). The interaction of these immunophilins with is not and is that steroid receptor complexes in different for receptor W.B. Scholar). The the of a binding motif that is essential for interaction with and T. Toft D.O. Scholar). the TPR of a a of and T. Scholar). The of the TPR domain in this basic and that TPR in an a recognition for protein interaction Scholar). the TPR basic residues that are immunophilins this binding groove and to for Hsp90 interaction Scholar). for complexes and Hsp90 carboxyl-terminal and TPR domains in that to the within the binding groove and that hydrophobic by residues of this heat shock protein Scholar). a for CyP40, the first of a immunophilin T. Scholar). of the MEEVD the of the CyP40 TPR domain the peptide to a groove amino residues from the of three TPR T. Scholar). used the for TPR proteins to a comprehensive mutational analysis of the carboxyl-terminal Hsp90 domain of CyP40. identified residues that to a binding within the TPR domain to for the of the Hsp90 carboxyl-terminal MEEVD peptide interaction with CyP40 were from the Scholar). of the MEEVD peptide the of CyP40 was using the for Scholar). were using the from the of Scholar). The carboxyl-terminal of human CyP40 residues the TPR domain and flanking acidic and basic was the of a for with the yeast Scholar). This the the of CyP40 to the binding domain of residues The used for of the CyP40 were as and of the CyP40 was using For in the yeast β-galactosidase filter a carboxyl-terminal of human Hsp90β residues was the of from in with the and These a the to the of the a using a was of with the in the was for in the assay. 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The MEEVD peptide is in a with in and the in the MEEVD peptide a groove with residues from the CyP40 TPR domain and T. that of the MEEVD peptide in the for the two and of the interaction the of CyP40 the The and of the MEEVD peptide are as with the carboxyl-terminal and residues The residues identified in this as required for efficient interaction of CyP40 residues in Asn-231, Asn-278, Lys-308, and Arg-312) Hsp90 and in Ser-274, Lys-285, and Asp-329) binding with CyP40. and to are that and and a in the in of the MEEVD peptide in the binding and fusion proteins were expressed and as T. Scholar). were as the were by in of and for a was with for a of were and in and were and and for and were to of and In and protein was was the and were different for and were not were by and the by for in a fusion proteins in the were by with The was in and and fusion proteins were from the with by to the were using were by the assay Scholar). 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Scholar). were for and were for β-galactosidase using the filter assay to the were filter in and to the a in and The for to was for and the of interaction for was assessed qualitatively by the of to fusion proteins and protein were in of binding with for a of was and in by and the was for a 40 of were as a of and in and in an of this were by and three in of binding and in of binding with to the by the addition of the the was to of was in of For a binding was as as a with of protein to for binding of the protein to the were and by The of that to and construct was by using with for and the to for construct were three These were in microtiter 40 in was in to for and for an of bovine serum was to in as a for were with of in for an additional five with to of in was to the and to for proteins were by the five with in was as to and for the was by the five with to was in and as to and for the was by with of the was to the and the plate for with The was by the addition of of to the a that was using a with for and were three The assay was using 40 protein in of 40 protein with other the For the conventional pulldown and microtiter plate data were using the for for of and an in the of binding. The was to with for Hsp90 binding were to The carboxyl-terminal MEEVD motif of Hsp90 to critical for the binding of Hsp90 to proteins the immunophilins CyP40, FKBP51, and the serine/threonine phosphatase PP5, and T. Toft D.O. Scholar). of the CyP40 protein T. a in the with the five carboxyl-terminal residues a groove in the by the of the three TPR of an the of the CyP40 this T. a used the of the carboxyl-terminal residues as a to the interaction of the Hsp90 carboxyl-terminal MEEVD with CyP40 and The MEEVD peptide was the binding the carboxyl-terminal CyP40 with of the of the TPR The of CyP40 was the a that of the of the MEEVD peptide were with the of with Lys-227, Lys-308, and These three residues help define the binding groove the MEEVD peptide In the that three other Phe-234, Ser-274, and Asn-278, define the This the of the to the hydrophobic by The of the and domains of with two of different an for the of the of the three of the and CyP40 TPR domains in MEEVD binding a of The carboxyl-terminal the complex in the that the CyP40 the groove by the CyP40 TPR and and the and found in the by the found in the of the of the domain of in complex with the MEEVD motif of Hsp90 the importance of with the following residues of and residues Lys-227, Asn-231, Asn-278, Lys-308, and and a of residues from the domain were found to with acidic residues in the MEEVD In the a with of the MEEVD motif and to with and residues and a that the that of the MEEVD motif is and as by The is in by with the of and residues Lys-227, Asn-231, and Asn-278, and and in with The Arg-312) with of the MEEVD the residues and residues Phe-234, Lys-285, and with In of hydrophobic and were found in the and from the and CyP40 CyP40-MEEVD The that of the MEEVD peptide in hydrophobic with of the residues and residues Phe-234, and Lys-285, and that of the MEEVD motif hydrophobic with residues that include and residues Asn-231, Asn-278, and In the CyP40-MEEVD the of the Hsp90 required to the the hydrophobic by and to and of and Lys-285, The is the of a in CyP40 in of a in The in this in the hydrophobic by with the from the the of the as with in the CyP40 MEEVD interaction and hydrophobic with Asn-231, Asn-278, and as by the data Scholar). of the MEEVD peptide is not in the of the CyP40 MEEVD to hydrophobic with the amino residues of CyP40 as by the The TPR domain of to that of CyP40 and to the domain and its an the of is by the of amino acids of the first of TPR motif Scholar). Mutational analysis of the of residues and for efficient binding to Hsp90 residues Lys-227, Ser-274, Lys-285, Lys-308, and Scholar). the of of Hsp90 binding residues CyP40, Hop, and that these proteins to the MEEVD motif through using to the CyP40 and are a of amino residues to of the models the in CyP40 with the MEEVD the required to a of the MEEVD peptide in the CyP40 the CyP40 the models a binding groove to binding in the to the MEEVD motif the of the MEEVD peptide in the binding are an of the complex to define the of the This the CyP40-MEEVD this to for mutational analysis models for amino acids of CyP40 are critical for Hsp90 analysis of bovine CyP40 that a of TPR residues of importance to the of the TPR domain and for Hsp90 binding. Two and in the acidic the to with residues in and for and for T. Scholar). analysis an residues in and in and that in a with in T. In the of the the cyclophilin and TPR domains and is to in an of the amino of a of proteins reveal residues and that are not by to interaction with Hsp90 to of that to Hsp90 is that the TPR domain is a for Hsp90 binding to CyP40 and other proteins T. 1997; T. in that flanking acidic and carboxyl-terminal basic domains are required for efficient Hsp90 binding to CyP40 T. Scholar). of the acidic domain as in the construct CyP40 Hsp90 binding TPR and basic domains T. Scholar). was that of the acidic residues in this domain as and Hsp90 that are immunophilins and to the of CyP40, as and T. in the of binding to in the carboxyl-terminal acids of CyP40 was binding to the TPR domain was that a of residues in this the basic residues and the residues and Hsp90 binding. that part of the basic part of an binding the residues and to was that these residues a role in efficient Hsp90 by the models of CyP40-MEEVD a comprehensive mutational analysis of the TPR domain and flanking acidic and basic domains of CyP40 to amino acids that are critical for Hsp90 binding. was used to amino to to a of 24 residues of CyP40 within this carboxyl-terminal were using the a of the of Hsp90 binding using the yeast β-galactosidase filter assay in with the This that with to the for of yeast to of the and as another and as and the mutants as as as that of the CyP40 construct confirm and these in binding the and of the 24 CyP40 the expressed and as fusion assessed for Hsp90 interaction using a pulldown assay and an microtiter plate of the human carboxyl-terminal Hsp90 was the of the pulldown assay and the microtiter plate assay In the following Hsp90 and the following of binding of this was that of the and were two the in Hsp90 binding from assayed by the pulldown binding with assayed by the microtiter plate In the was a assessed by the conventional pulldown binding was assayed by the microtiter plate The other from the acidic and five from the basic and and from the TPR and in Hsp90 binding was The pulldown and microtiter plate with the using the yeast β-galactosidase filter that two of the mutants that a binding using this and in binding to Hsp90 with the conventional pulldown assay the microtiter plate assay. was assessed as a in the filter was to a in the pulldown and microtiter plate and analysis that with the the construct was expressed in yeast a in the filter assay of for this not of the of CyP40 Hsp90 binding of the three used is in of and to using a conventional pulldown assay in protein was with protein and to proteins the were and by from the of the of to for three assays. using an microtiter plate in was with to the of a microtiter plate and the of CyP40 by with by to and as the for three assay in For a from of to data the binding of mutants to as by a yeast assay and by pulldown and microtiter plate are in a The data the binding of mutants to as by a yeast assay and by pulldown and microtiter plate are that the binding of to the carboxyl-terminal domain of Hsp90 is by in the of Hsp90 of the and carboxyl-terminal domains of Hsp90 D.O. Scholar). the binding of CyP40 and other proteins by the microtiter plate assay using in of the carboxyl-terminal protein for analysis of and fusion protein binding. for Hsp90β were in with for the carboxyl-terminal Hsp90β protein not in the of Hsp90β not the binding of CyP40 to the carboxyl-terminal domain of to different by the binding of to Hsp90 D.O. to the of Hsp90β CyP40 binding to Hsp90β in a The five mutants that Hsp90 binding and were associated with residues that were from to the groove the MEEVD motif of Hsp90 are to with this motif and Scholar). These residues are to of the domain and in complex with the MEEVD motif are for MEEVD the Scholar). of these residues and were individually mutated to binding to Hsp90 Scholar). In the importance of the residues in is from the of the TPR domain of and a of of these and to residues Lys-227, Lys-308, and of CyP40, Hsp90 binding Scholar). of two other and Ser-274, help define the groove are not to in with the MEEVD motif a Hsp90 binding. the to the in CyP40, not binding of as is the for the CyP40 In the of the CyP40 CyP40-MEEVD a with Lys-227, and a with In the of a with Lys-308, and the of a with In the of the CyP40-MEEVD the of a with Lys-227, and the of a with In with and the of the MEEVD peptide in the binding the is in the Lys-227, Lys-308, and residues of CyP40 are in with the MEEVD analysis of the complex and CyP40-MEEVD this analysis that and hydrophobic with residues of this motif are of importance and that TPR domain residues the binding groove to for efficient Hsp90 interaction and in hydrophobic and a of Hsp90 binding with the and this was with the CyP40 was that with CyP40, using the pulldown and microtiter plate In the is in hydrophobic in with the and to the of the MEEVD motif Scholar). CyP40 residues that are to and are and and of these residues with in Hsp90 binding with that for CyP40 protein and to the CyP40-MEEVD The Hsp90 binding This is to an with a in In Hop, the is to a with of the MEEVD peptide in with residues and residues and a hydrophobic of the Scholar). In CyP40, of the to Hsp90 binding and In the CyP40 and models of CyP40-MEEVD interaction that the in a hydrophobic by of this to binding to Hsp90 these that and hydrophobic to Hsp90 interaction with the TPR domain of three CyP40, PP5, and of CyP40 mutational analysis data with that for with the of Hsp90 binding to PP5, in that the of residues and residues Lys-227, Lys-308, and and residues and Ser-274, and the was the are and CyP40 in the amino acids by found by mutational analysis to of importance in Hsp90 are CyP40-MEEVD the CyP40 that the of CyP40 part of the binding groove for the MEEVD and of this to CyP40 binding to the in not to of importance for MEEVD interaction and are amino In analysis that the CyP40 a with of this was not to in the Scholar). of the Hsp90 binding from analysis of CyP40. In Hop, the of to CyP40 one of the residues of the MEEVD motif that this is to for Hsp90 the mutated to the not Hsp90 binding is in proteins mutated to an in binding to Hsp90 the microtiter plate the was not different from using the conventional pulldown The was as an by the yeast assay is not to this that is a critical for Hsp90 binding. in that flanking acidic and carboxyl-terminal basic domains are required for efficient Hsp90 binding to CyP40 T. Scholar). analysis with bovine CyP40 that acidic residues and with residues in T. Scholar). mutated to of these acidic residues a in Hsp90 binding with is that one of the in bovine CyP40 is as a in human CyP40 that are in human CyP40 with the bovine CyP40 acidic is to an and was found to proteins mutated to of these residues Hsp90 binding is that is these residues in to Hsp90 interaction and that are to Hsp90 is that other acidic residues in this domain were not by mutational as and are residues for Hsp90 of help to define the residues in the acidic flanking are for Hsp90 binding. In is residues in the critical basic domain of CyP40 in T. are essential for Hsp90 binding of the two basic and the two and in this were to a Hsp90 binding The an additional one to part of an binding for from that these three basic are the Hsp90 binding not for Hsp90 binding. in the for basic residues within this for Hsp90 and mutational analysis of two basic residues in the critical basic domain help to reveal residues that are required for efficient Hsp90 This include basic residues as and not Mutational analysis of residues in the TPR and flanking acidic and basic domains of CyP40 a of critical residues in the TPR domain as are for recognition of the MEEVD motif of The binding groove for the MEEVD motif and the residues critical for interaction with to in CyP40, PP5, and In PP5, three residues that define the MEEVD binding groove and were found to essential for Hsp90 binding and the residues in CyP40 were required for Hsp90 binding critical and identified from as in the interaction with the carboxyl-terminal of the MEEVD the were found in binding to essential for efficient binding to Hsp90 Scholar). that the residues in the CyP40 TPR Lys-227, Asn-278, Lys-308, and are required for Hsp90 binding that the interaction in the binding of CyP40 to the MEEVD to in the was not to mutational the in CyP40, Asn-231, was found to critical for Hsp90 binding The CyP40 mutational data that other TPR residues Ser-274, and from of the CyP40-MEEVD models to with the MEEVD are in required for efficient Hsp90 binding. The TPR residues identified by as for Hsp90 recognition are not a mutational analysis of the TPR domain was not to residues as an with Asn-231, and in hydrophobic with the of not a mutational analysis of the is that this is of with to Hsp90 binding is proteins and of the in the recognition Scholar). The is CyP40, PP5, and in these three its role in the of the hydrophobic for the of the MEEVD peptide of the CyP40 binding to Hsp90 that is to a with T. for Hsp90 interaction in the of maintaining the of CyP40. In other residues within the TPR other of CyP40 acidic and basic domains for critical for binding to other Hsp90 for binding Toft D.O. Scholar). The of mature steroid receptor complexes is a the of as Hip, Hop, and the of binding to Hsp90 the This for in the of by this is in the of and and are by immunophilin to the of the binding the binding and of 18: Scholar). In are a of other proteins not to steroid receptor complexes the to to Hsp90 Scholar). the to a for the interaction of Hsp90 and with one the for the TPR proteins that different with this by the protein through its domain and Hsp90 through its These are the residues required for the interaction are in TPR domains and in the carboxyl-terminal residues of and Hsp90 that to to in the and Hsp90 residues of the and MEEVD that the and domains of Hop, MEEVD peptide was to and was and a peptide was to and peptide was not Scholar). that the and MEEVD different hydrophobic in the and domains for binding Scholar). with to Hsp90 in the proteins with the to of different hydrophobic the of with this is that in the CyP40 the not in a to with the residues of of the in of the MEEVD peptide in the binding groove and in the the not the residues of in hydrophobic with the and are different in CyP40 is not in of residues part of a that binding of and CyP40 to In addition to the MEEVD a by amino acids within the domain of Hsp90β to of importance in the binding of proteins Toft D.O. Scholar). found that an that this is critical for CyP40 interaction in the of an MEEVD domain not is from these and that the interaction of immunophilins and other proteins with Hsp90 is not and that of Hsp90 from the MEEVD and domain are required for binding proteins Toft D.O. Scholar). in interaction to the domain of and for to carboxyl-terminal of Hsp90 by amino acids not to for CyP40, and interaction with Hsp90 Toft D.O. Scholar). with to a carboxyl-terminal construct of Hsp90 binding of PP5, and Hop, the a from FKBP52 and to Scholar). that additional are required as as the recognition for binding to Scholar). these that the of binding to Hsp90 by different proteins by Hsp90 interaction of the MEEVD motif and of domain is that Hsp90 from these domains different TPR proteins the TPR and that this the MEEVD the to receptor for of FKBP52 receptor complexes in binding of hormone, to Scholar). that Hsp90 a to immunophilins and to receptor of receptor by association with Hsp90 by binding to the in Hsp90 a in association with the the for the to is that FKBP52 to the receptor W.B. are to not CyP40 to the estrogen In the association of immunophilins to steroid receptors is to by in Hsp90 and the receptors W.B. Scholar). In this is that CyP40 and the two immunophilins that are found associated with the estrogen receptor α T. Hsp90 binding with and Toft D.O. Scholar). residues that for Hsp90 binding to Two the flanking acidic and basic are to essential for Hsp90 binding. within these domains Hsp90 for In this the TPR domain the residues for the recognition of the MEEVD motif and the flanking the residues required for interaction with other Hsp90 for critical for the of to the modulating that immunophilins Hsp90 for yeast two and used in this and for the and for help with the