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 Hsp90␤ construct and qualitatively assessed for binding using a ␤-galactosidase filter assay. For quantitative assessment, mutants were expressed as glutathione S-transferase fusion proteins and assayed for binding to carboxyl-terminal Hsp90␤ 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 ␣ is found predominantly in the nucleus as part of a multicomponent complex with Hsp90 1 (1). 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 (1). 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 (1). CyP40 was first isolated as a component of estrogen receptor ␣ heterocomplexes (2). This novel cyclophilin binds the immunosuppressant cyclosporin A and shares structural similarity with its partner co-chaperones, FKBP51 and FKBP52, both immunophilins of the FK506-binding class (1). The structure of these large immunophilins is characterized by an amino-terminal immunophilin-like domain that is targeted by immunosuppressant drugs and overlaps a peptidylprolyl isomerase function and a carboxyl-terminal protein interaction domain incorporating 3 units of the tetratricopeptide repeat (TPR) motif (3). These immunophilins have been implicated in steroid receptor function through their association with Hsp...

On the role, ecology, phylogeny, and structure of dual-family immunophilins

“…The most conserved amino acid residues in the FCBP TPRs are indicated by all arrowheads. Residues that are known to interact with the MEEVD sequence of Hsp90, based on structural studies of CYN and FKBP TPRs (Cliff et al 2006;Scheufler et al 2000;Ward et al 2002;Wu et al 2004), are specifically indicated with closed arrowheads (detailed in Results); note that they are also among the most highly conserved, supporting the functional importance of a potential FCBP TPRHsp90 interaction…”

“…The binding exhibits various degrees of specificity between the two partners; Hsp90, for example, is preferred over Hsp70 to bind to the TPRs of the large FKBPs (Assimon et al 2015;Guy et al 2015). In particular, peptide-TPR co-crystal structure and mutational analysis have established that the invariant C-terminal pentapeptide, MEEVD, of Hsp90 (Gupta 1995) interacts with specific residues that are common to the TPRs of long FKBPs and CYNs (Assimon et al 2015;Cliff et al 2006;Scheufler et al 2000;Ward et al 2002;Wu et al 2004). The residues, found to be absolutely essential for MEEVD binding, are: K5, N9 in TPR1, N6 in TPR2, and K2, R6 in TPR3.…”

Independent Functions of hsp90 in Neurotransmitter Release and in the Continuous Synaptic Cycling of AMPA Receptors

“…hsp90 is also required for protein translocation into mitochondria (Young et al, 2003) and peroxisomes (Crookes and Olsen, 1998). The trafficking functions of hsp90 depend on cytoskeletal elements (Galigniana et al, 1998(Galigniana et al, , 2002Pratt et al, 1999) and involve specific interactions between C-terminal sequences of hsp90 and tetratricopeptide repeat (TPR) domains in several effector molecules (Chen et al, 1996;Young et al, 1998;Russell et al, 1999;Scheufler et al, 2000;Ward et al, 2002). TPR domains are involved in a variety of cellular functions, including protein transport and targeting (for review, see Blatch and Lassle, 1999).…”

Crystallographic structure of the tetratricopeptide repeat domain of Plasmodium falciparum FKBP35 and its molecular interaction with Hsp90 C‐terminal pentapeptide

“…These data suggest that the sequence and three-dimensional structures of TPR domain and TPR-recognition motif in Hsp90 are conserved in Plasmodium parasite and their molecular basis of the interaction between PfFKBP35-TPR and the Hsp90 pentapeptide are similar to the known TPR domain and Hsp90 interactions. 16,24,25 Despite the similarity, however, possible specificity of this interaction could also be understood as seen in Hsp70-Hop-Hsp90 complex. 16 Hop has two TPR domains TPR2A1 and TPR1 that interact exclusively with Hsp90 and Hsp70, respectively.…”

“…The MEEVD pentapeptide present at the C-terminus of Hsp90 (hereafter Hsp90 pentapeptide), which is conserved in all eukaryotes, serves as a TPR recognition motif. 16,[23][24][25] Hsp90 orthologue of P. falciparum (PfHsp90, PF07_0029) also contains the invariant MEEVD at the C-terminal. 26,27 It has been shown that the TPR domain of PfFKBP35 (hereafter PfFKBP35-TPR) associates with the PfHsp90.…”