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

Iloro I, Narváez D, Guillén N, Camacho CM, Guillén L, Cora E, Pastrana-Ríos B - Biophys. J. (2008)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Protein Structure Function and Dynamics, University of Puerto Rico, Mayagüez Campus, Mayagüez, Puerto Rico.

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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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.
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fig1: 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.

Mentions: Amphiregulin (AR), whose structure has not yet been elucidated, and heparin binding-epidermal growth factor (HB-EGF) are ligands of the EGFR that are known to stimulate cell growth and proliferation and are often associated with oncogenesis (7–10). Amphiregulin, composed of 98 amino acids, and HB-EGF, composed of 86 residues (secondary structure composition is discussed herein), share a significant homology to all EGF family members containing an EGF-like domain (11,12) (Fig. 1) and share the same heparin-binding affinity (10,13). Recently discovered epiregulin (ER) (14), composed of 47 residues, is a ligand for EGFR and ErbB4, which also shares more than 41% amino acid sequence identity with EGF. ER exhibits the bifunctional regulatory property of inhibiting the growth of several epithelial cell lines while stimulating the growth of fibroblasts and various other cell types (8,15,16).


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

Iloro I, Narváez D, Guillén N, Camacho CM, Guillén L, Cora E, Pastrana-Ríos B - Biophys. J. (2008)

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.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2367206&req=5

fig1: 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.
Mentions: Amphiregulin (AR), whose structure has not yet been elucidated, and heparin binding-epidermal growth factor (HB-EGF) are ligands of the EGFR that are known to stimulate cell growth and proliferation and are often associated with oncogenesis (7–10). Amphiregulin, composed of 98 amino acids, and HB-EGF, composed of 86 residues (secondary structure composition is discussed herein), share a significant homology to all EGF family members containing an EGF-like domain (11,12) (Fig. 1) and share the same heparin-binding affinity (10,13). Recently discovered epiregulin (ER) (14), composed of 47 residues, is a ligand for EGFR and ErbB4, which also shares more than 41% amino acid sequence identity with EGF. ER exhibits the bifunctional regulatory property of inhibiting the growth of several epithelial cell lines while stimulating the growth of fibroblasts and various other cell types (8,15,16).

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Protein Structure Function and Dynamics, University of Puerto Rico, Mayagüez Campus, Mayagüez, Puerto Rico.

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.

Show MeSH
Related in: MedlinePlus