Binding of CXCR4 Transmembrane Peptides to the Bacterial Chaperonin GroEL

The Escherichia coli chaperonin GroEL represents the paradigmatic molecular machine of protein folding. Most of our knowledge on GroEL function is derived from studies with denatured water-soluble proteins or short peptide mimetics. In our earlier studies, we observed that newly translated membrane protein CXCR4 gained significant folding enhancement upon interacting with GroEL in the presence of ATP and GroES. This highlights the mechanistic flexibility and substrate diversity of the chaperonin.

This work extends our previous observation to the study of binding of GroEL with CXCR4 transmembrane peptides.

The model peptide corresponding to the first transmembrane α-helix in the native tertiary structure of CXCR4 was commercially synthesized. A fluorescent tag was attached to the Nterminus of the peptide for ease of fluorescence characterization. Binding of CXCR4 peptide to GroEL was investigated by fluorescence anisotropy as well as isothermal titration calorimetry (ITC). Through model fitting to the anisotropy and ITC data, important thermodynamic parameters were obtained for the binding. In identifying the binding site of GroEL apical domains for the CXCR4 peptide, competitive binding of the peptide and a model "strongly binding peptide" (SBP) was performed by ITC. Furthermore, the kinetics of the CXCR4 peptide binding to GroEL was also studied by anisotropy.

Through anisotropic measurement of binding of CXCR4 peptide to GroEL, a dissociation constant and binding stoichiometry of 0.10µM and 7.00 was obtained. Thermodynamic parameters of the binding were also determined by ITC. By fitting the ITC data to one set of sites model, the values of the thermodynamic parameters were acquired directly as follows: ΔH=-627.10kcal/mol; ΔS=-186.23cal/mol; KD=0.23µM; N=5.96. The binding site of the CXCR4 peptide in GroEL was also probed through competitive binding with the model peptide SBP, pointing to the groove between paired α helices H and I in the apical domain. In addition, the binding kinetics suggests a slow dissociation of the peptide-GroEL complex.

The CXCR4 model peptide has been shown to bind to GroEL with high affinity. The binding stoichiometry was estimated to be 6 or 7 depending on the analysis method. Furthermore, the two thermodynamic parameters ΔH and ΔS that define the binding affinity have also been measured by ITC. The binding site of the CXCR4 peptide in the GroEL apical domain was investigated through competitive binding studies with the model peptide SBP. Meanwhile, the kinetic studies indicate a slow dissociation of the peptide-GroEL complex. These results obtained by the reduced approach of employing CXCR4 transmembrane peptides would be beneficial to understanding the GroEL-CXCR4 interaction in the assisted folding.