High-resolution solution structure of the EGF-like domain of heregulin-alpha

The solution structure of the 63-residue heregulin-alpha (HRC-alpha) epidermal growth factor (EGF)-like domain, corresponding to residues 177-239 of HRG-alpha, has been determined to high resolution using data from two-dimensional and three-dimensional homo- and heteronuclear NMR spectroscopy. The structure is based on a total of 887 internuclear distance and dihedral restraints derived from data obtained using unlabeled and uniformly N-15-labeled protein samples, at pH 4.5, 20 degrees C. A total of 20 structures were calculated using a hybrid distance geometry-simulated annealing approach with the program DGII, followed by restrained molecular dynamics using the program DISCOVER. The average maximum violations are 0.12 +/- 0.01 Angstrom and 1.4 +/- 0.3 degrees for distance and dihedral restraints, respectively. The backbone (N, C-alpha, C) atomic rms distribution about the mean coordinates for residues 3-23 and 31-49 is 0.29 +/- 0.07 Angstrom. The N- and C-terminal residues (1-2 and 50-63) and the Omega-loop comprising residues 24-30 are disordered. Comparison of the HRG-alpha EGF-like domain structure with the previously determined structure of human EGF [Hommel et al. (1992) J. Mol. Biol. 227, 271-282] reveals a high degree of structural similarity; excluding the N-terminal region (residues 1-13), the disordered Omega-loop region (residues 24-30) that contains a three-residue insertion in HRG-alpha relative to hEGF, and the disordered C-terminal region (residues 50-63), the C-alpha alignment between the HRG-alpha and hEGF minimized mean structures has a rms difference of similar to 1 Angstrom. in HRG-alpha the N-terminal residues 2-6 form a well-defined beta-strand rather than being disordered as found for hEGF. This structural difference correlates with functional data which suggest that the N-terminal region of the HRG-alpha EGF-like domain is responsible for the observed receptor specificity differences between HRG-alpha and EGF.