A structure-based mutational analysis of cyclophilin 40 identifies key residues in the core tetratricopeptide repeat domain that mediate binding to Hsp90.
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 Hsp90beta construct and qualitatively assessed for binding using a beta-galactosidase filter assay. For quantitative assessment, mutants were expressed as glutathione S-transferase fusion proteins and assayed for binding to carboxyl-terminal Hsp90beta 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.
Ward
Bryan K
BK
Western Australian Institute for Medical Research, Sir Charles Gairdner Hospital, Queen Elizabeth II Medical Centre, Nedlands, Western Australia 6009, Australia.
Allan
Rudi K
RK
Mok
Danny
D
Temple
Suzanna E
SE
Taylor
Paul
P
Dornan
Jacqueline
J
Mark
Peter J
PJ
Shaw
Daniel J
DJ
Kumar
Premlata
P
Walkinshaw
Malcolm D
MD
Ratajczak
Thomas
T
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000081406:Peptidyl-Prolyl Isomerase F; D021983:Cyclophilins; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D019696:Peptidylprolyl Isomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D020449:Repetitive Sequences, Amino Acid; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D000081406:Peptidyl-Prolyl Isomerase F; D017931:DNA Primers; D018841:HSP90 Heat-Shock Proteins; D010455:Peptides; D021983:Cyclophilins; D019696:Peptidylprolyl Isomerase