Nonnatural protein-protein interaction-pair design by key residues grafting

Abstract

Protein-protein interface design is one of the most exciting fields in protein science; however, designing nonnatural protein-protein interaction pairs remains difficult. In this article we report a de novo design of a nonnatural protein-protein interaction pair by scanning the Protein Data Bank for suitable scaffold proteins that can be used for grafting key interaction residues and can form stable complexes with the target protein after additional mutations. Using our design algorithm, an unrelated protein, rat PLCdelta(1)-PH (pleckstrin homology domain of phospholipase C-delta1), was successfully designed to bind the human erythropoietin receptor (EPOR) after grafting the key interaction residues of human erythropoietin binding to EPOR. The designed mutants of rat PLCdelta(1)-PH were expressed and purified to test their binding affinities with EPOR. A designed triple mutation of PLCdelta(1)-PH (ERPH1) was found to bind EPOR with high affinity (K(D) of 24 nM and an IC(50) of 5.7 microM) both in vitro and in a cell-based assay, respectively, although the WT PLCdelta(1)-PH did not show any detectable binding under the assay conditions. The in vitro binding affinities of the PLCdelta(1)-PH mutants correlate qualitatively to the computational binding affinities, validating the design and the protein-protein interaction model. The successful practice of finding a proper protein scaffold and making it bind with EPOR demonstrates a prospective application in protein engineering targeting protein-protein interfaces.

Fig. 1.

Comparison of the designed ERPH1–EPOR complex model with the EPO–EPOR complex structure. (A) The structure of EPO–EPOR complex structure (PDB ID code 1EER). Red, EPO; green and cyan, EPOR dimer. (B) The designed ERPH1–EPOR complex model. Red, EPO; green and cyan, EPOR dimer. Images were created by using PyMOL (48).

Fig. 2.

Key interactions at the designed ERPH1–EPOR interface compared with those at the EPO–EPOR interface. (A) Hydrogen-bond interactions at the ERPH1–EPOR interface. (B) Hydrogen-bond interactions at the EPO–EPOR interface. (C) The hydrophobic cluster at the designed ERPH1–EPOR interface. (D) The hydrophobic cluster at the EPO–EPOR interface. ERPH1 is shown in gray ribbons, and EPOR is shown in pale green ribbons. The residues at the interfaces are shown in colored sticks.

Fig. 3.

K_D determination of ERPH1 binding to hEPOsR from in vitro SPR study. (A) Sensograms of the binding of increasing concentrations of ERPH1 to hEPOsR. The concentrations of ERPH1 used were 0, 3, 9, 27, 81, 150, and 400 nM from the bottom up. (B) K_D determination of the binding of ERPH1 to hEPOsR. Data points represent the equilibrium average response shown in A. The solid line represents the theoretical curve that was globally calculated from the steady-state fit model provided by BIAevaluation 4.1 software (Biacore).

Fig. 4.

Effects of ERPH1 on the EPO–EPOR signal pathway. (A) Dose-dependent inhibitory effects of ERPH1 on EPO–EPOR signal pathway. Different amounts of ERPH1 cDNA with a secretory signal peptide sequence were transfected into 293T cells. A total of 1.5 units (1.5 nM) of hEPO (Roche) was added as the competitor. Data were normalized by transfection with a blank pcDNA3.1_SP/myc-His A vector and are expressed as the mean ± SD (n = 3). (B) The expression levels of ERPH1 protein detected by Western blotting.

Fig. 5.

Dose-dependent inhibitory effects of purified recombinant ERPH1 on EPO activity. (A) The relative luciferase activity when different concentrations of ERPH1 were added. (B) IC₅₀ plot of ERPH1. The IC₅₀ was determined to be 5.7 ± 1.2 μM (R² = 0.9695).