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. 2008 May 15;94(10):4041-55.
doi: 10.1529/biophysj.107.125856. Epub 2008 Jan 16.

The kinetics of the hydrogen/deuterium exchange of epidermal growth factor receptor ligands

Ibon Iloro  1 , Daniel Narváez, Nancy Guillén, Carlos M Camacho, Lalisse Guillén, Elsa Cora, Belinda Pastrana-Ríos
Affiliations

The kinetics of the hydrogen/deuterium exchange of epidermal growth factor receptor ligands

Ibon Iloro et al. Biophys J. .

Abstract

Five highly homologous epidermal growth factor receptor ligands were studied by mass spectral analysis, hydrogen/deuterium (H/D) exchange via attenuated total reflectance Fourier transform-infrared spectroscopy, and two-dimensional correlation analysis. These studies were performed to determine the order of events during the exchange process, the extent of H/D exchange, and associated kinetics of exchange for a comparative analysis of these ligands. Furthermore, the secondary structure composition of amphiregulin (AR) and heparin-binding-epidermal growth factor (HB-EGF) was determined. All ligands were found to have similar contributions of 3(10)-helix and random coil with varying contributions of beta-sheets and beta-turns. The extent of exchange was 40%, 65%, 55%, 65%, and 98% for EGF, transforming growth factor-alpha (TGF-alpha), AR, HB-EGF, and epiregulin (ER), respectively. The rate constants were determined and classified as fast, intermediate, and slow: for EGF the 0.20 min(-1) (Tyr), 0.09 min(-1) (Arg, beta-turns), and 1.88 x 10(-3) min(-1) (beta-sheets and 3(10)-helix); and for TGF-alpha 0.91 min(-1) (Tyr), 0.27 min(-1) (Arg, beta-turns), and 1.41 x 10(-4) min(-1) (beta-sheets). The time constants for AR 0.47 min(-1) (Tyr), 0.04 min(-1) (Arg), and 1.00 x 10(-4) min(-1) (buried 3(10)-helix, beta-turns, and beta-sheets); for HB-EGF 0.89 min(-1) (Tyr), 0.14 min(-1) (Arg and 3(10)-helix), and 1.00 x 10(-3) min(-1) (buried 3(10)-helix, beta-sheets, and beta-turns); and for epiregulin 0.16 min(-1) (Tyr), 0.03 min(-1) (Arg), and 1.00 x 10(-4) min(-1) (3(10)-helix and beta-sheets). These results provide essential information toward understanding secondary structure, H/D exchange kinetics, and solvation of these epidermal growth factor receptor ligands in their unbound state.

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Figures

FIGURE 1
FIGURE 1
Solid surface Connolly models were generated for these ligands using their corresponding PDBs. (A) EGF (PDB:1EPJ), (C) TGF-α (PDB:1YUG), (E) HB-EGF (modified from PDB:1XDT, HB-EGF peptide composed of 79 residues), and (G) epiregulin (PDB:1K37, ER peptide composed of 46 residues). In white are the hydrophobic regions of the ligand with IR active side chains: red, negatively charged (aspartates and glutamates); blue, positive charged (arginine); and green for the tyrosine side chains. (A) The Kabash and Sander (34) rendition of these ligands is also shown to highlight the secondary structure differences (B) EGF, (D) TGF-α, (F) HB-EGF, and (H) ER, although they all contain the EGF-like domain with the characteristic two β-strands and three loops. In blue are the amino and in red are the carboxy terminal ends. (I) Sequence alignment: based on National Center for Biotechnology Information database sequences corresponding to accession numbers AAS83395, NP_003227, BAA22146 or 1K37, NP_001936, AAA51773, for human EGF, TGF-α, ER, HB-EGF, and AR, respectively. Highlighted are the conserved and class-specific residues in light gray and orange, respectively. Conserved disulfide bridges are shown as solid gray lines. Conserved secondary structure motif β-strands are shown as arrows (same colors as β-sheets). (J) Dendogram for the EGF-like family of ligands.
FIGURE 2
FIGURE 2
MS analysis: TOF-ES MS spectra of (A) EGF, (B) TGF-α and MALDI, (C) AR, (D) HB-EGF, and (E) ER. EGF and HB-EGF are in dimer states, whereas TGF-α, AR, and ER are in their monomeric state.
FIGURE 3
FIGURE 3
Overlaid spectra of EGFR ligands corresponding to initial and final spectra collected during the H/D exchange experiment within the spectral region of 3600–1400 cm−1 for (A) EGF, (B) TGF-α, (C) AR, (D) HB-EGF, and (E) ER. The spectra demonstrate the extent of hydration of the protein film and the overall band shifts and intensity changes for all five ligands.
FIGURE 4
FIGURE 4
Overlaid difference spectra of the EGFR ligands. (A) EGF, (B) TGF-α, (C) AR, (D) HB- EGF, and (E) ER within the spectral region 1725–1400 cm−1. The difference spectra were generated by subtraction of the first spectrum from all subsequent spectra.
FIGURE 5
FIGURE 5
The kinetics of exchange for EGF is summarized in three plots. (A) Initial times of exchange. (B) Percent of unexchanged protein versus time. EGF was observed to exchange 40% after 80 min. A straight line shown at the bottom of each multiexponential decay plot (A and B) is the residual of the fit with the resulting standard deviation 0.039. (C) Evolution of the absorbance at selected wavenumbers versus time, which includes a summary of the kinetic components assigned to Tyr, Arg, and the different structural motifs.
FIGURE 6
FIGURE 6
The kinetics of H/D exchange for TGF-α is summarized in three plots. (A) Initial times of exchange. (B) Percent of unexchanged protein versus time. TGF-α was observed to exchange 65% after 15 min. A straight line shown at the bottom of each multiexponential decay plot (A and B) is the residual of the fit with the resulting standard deviation 0.071. (C) Evolution of the absorbance at selected wavenumbers versus time, which includes the kinetics assigned for Tyr, Arg, and the different structural motifs.
FIGURE 7
FIGURE 7
The kinetics of exchange for AR is summarized in three plots. (A) Initial times of exchange. (B) Percent of unexchanged protein versus time. AR was observed to exchange 55% after 80 min. A straight line shown at the bottom of each multiexponential decay plot (A and B) is the residual of the fit with the resulting standard deviation 0.083. (C) Evolution of the absorbance at selected wavenumbers versus time, which includes a summary of the kinetic components assigned to Tyr, Arg, and the different structural motifs.
FIGURE 8
FIGURE 8
The kinetics of exchange for HB-EGF is summarized in three plots. (A) initial times of exchange. (B) Percent of unexchanged protein versus time. HB-EGF was observed to exchange 65% after 20 min. A straight line shown at the bottom of each multiexponential decay plot (A and B) is the residual of the fit with the resulting standard deviation 0.127. (C) Evolution of the absorbance at selected wavenumbers versus time, which includes a summary of the kinetic components assigned to Tyr, Arg, and the different structural motifs.
FIGURE 9
FIGURE 9
The kinetics of exchange for ER is summarized in three plots. (A) Initial times of exchange. (B) Percent of unexchanged protein versus time. ER was observed to exchange 98% after 150 min. A straight line shown at the bottom of each multiexponential decay plot (A and B) is the residual of the fit with the resulting standard deviation 0.650. (C) Evolution of the absorbance at selected wavenumbers versus time, which includes a summary of the kinetic components assigned to Tyr, Arg, and the different structural motifs.
FIGURE 10
FIGURE 10
2DCOS obtained from the baseline-corrected spectra corresponding to EGF and TGF-α (A and C) synchronous and (B and D) asynchronous plots, respectively.
FIGURE 11
FIGURE 11
2DCOS corresponding to AR, HB-EGF, and ER obtained from the baseline corrected spectra (A, C, and E) synchronous plots and (B, D, and F) asynchronous plots, respectively.
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