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

Abstract

Plasmodium falciparum FK506-binding protein 35 (PfFKBP35) that binds to FK506 contains a conserved tetratricopeptide repeat (TPR) domain. Several known TPR domains such as Hop, PPP5, CHIP, and FKBP52 are structurally conserved and are able to interact with molecular chaperones such as Hsp70/Hsp90. Here, we present the crystal structure of PfFKBP35-TPR and demonstrate its interaction with Hsp90 C-terminal pentapeptide (MEEVD) by surface plasmon resonance and nuclear magnetic resonance spectroscopy-based binding studies. Our sequence and structural analyses reveal that PfFKBP35 is similar to Hop and PPP5 in possessing all the conserved residues which are important for carboxylate clamping with Hsp90. Mutational studies were carried out on positively charged clamp residues that are crucial for binding to carboxylate groups of aspartate, showing that all the mutated residues are important for Hsp90 binding. Molecular docking and electrostatic calculations demonstrated that the MEEVD peptide of Hsp90 can form aspartate clamp unlike FKBP52. Our results provide insightful information and structural basis about the molecular interaction between PfFKBP35-TPR and Hsp90.

Figure 1

Structural representation of PfFKBP35-TPR domain. (A) The dimer form of PfFKBP35-TPR crystal structure (2FBN), each monomer highlighted with different colors and all the seven helices are numbered. (B) The structural arrangement of PfFKBP35-TPR which consists three TPR domains and extra alpha helix (α7) are shown. The ribbon was generated by Pymol software. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 2

Structural and sequence comparison. (A) Structural alignment with human FKBP52. (B) Structural comparison of PfFKBP35-TPR (green) with FKBP52 (yellow) and Hop-T2A (blue). The 16 residues extra loop in FKBPTPR domains (between α2 and α3) and six residues insertion in Hop-T2A (between α4 and α5) highlighted with arrows. (C) The sequence alignment of TPR domains for Plasmodium falciparum FKBP35, FKBP52, FKBP51, FKBP38, Hop-T2A (Hsp organizing protein TPR2A), Hop-T1 (Hsp organizing protein TPR1), PPP5 (serine/threonine-protein phosphatase 5), and CHIP (C-terminus of Hsp70 interacting protein). The positively charged residues are highlighted with asterisk (*), identical residues are highlighted with red color, and conserved residues are highlighted with yellow color. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 3

(A) Binding mode of Hsp90 pentapeptide (MEEVD) with PfFKBP35-TPR, (B) Hop-T2A, (C) FKBP52. The Hsp90 pentapeptide, positively charged clamp residues, and the crucial hydrophobic residue (Tyr/Phe) are highlighted with sticks. Hydrogen bonds between TPR domain and pentapeptides are represented with dashed lines and hydrophobic interactions are highlighted with semicircles. (D) Superimposed structures of PfFKBP35-TPR, Hop-T2A, and FKBP52 with Hsp90 pentapetide. Terminal aspartate residue highlighted as sticks to show the binding mode difference. The Hsp90 pentapeptide binding conformation for PfFKBP35-TPR was achieved by GOLD molecular docking. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 4

Binding analysis of PfFKBP35 and the Hsp90 pentapeptide on BIAcore3000. Response of the wild-type PfFKBP35 at different protein concentrations ranging from 0.90 to 120 μM (from bottom to top) in response units (RU). All mutants showed almost no binding. A zoomed in view of one of the mutants K148A.

Figure 5

Chemical shift perturbations of ¹⁵N labeled PfFKBP35-TPR (0.1 mM) was monitored on 2D ¹H-¹⁵N HSQC spectrum upon the addition of Hsp90 pentapeptide (0.2 mM). (A) The spectrum of PfFKBP35-TPR in the absence of ligand has shown in blue while the spectrum in the presence of ligand is shown red. (B, C) Sections of ¹H-¹⁵N HSQC spectrum showing chemical shift changes of the residues involved in the ligand binding upon the addition of Hsp90 pentapeptide. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 6

(A) Electrostatic potential surface calculation results for PfFKBP35-TPR, (B) Hop-T2A, (C) FKBP52, and (D) FKBP51. Key differences like asparagine, lysine residues in binding pocket were highlighted with sticks. The Hsp90 pentapeptide structures are also shown in the binding pocket to highlight the binding orientation differences. The best docking pose of Hsp90 pentapeptide was shown with PfFKBP35-TPR. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]