Backbone dynamics of the EGF-like domain of heregulin-alpha.
The backbone dynamics of the 63 residue epidermal growth factor (EGF)-like domain of heregulin-alpha (HRG-alpha) have been characterized by measurement of longitudinal relaxation rate constants (R1), transverse relaxation rate constants (R2), and steady-state ¿1H¿-15N nuclear Overhauser effects for the 15N nuclear spins using proton-detected heteronuclear NMR spectroscopy. Analysis of the R2/R1 ratios in conjunction with the known structure of the HRG-alpha EGF-like domain yields a rotational correlation time of approximately 8.4 ns, suggesting that the protein aggregates under the solution conditions used (3.8 mM protein, 50 mM sodium acetate, pH 4.5, 20 degreesC), and that it tumbles with an axially symmetric diffusion tensor (D parallel/D perpendicular=1.4). Sedimentation equilibrium experiments confirm that the EGF-like domain of HRG-alpha undergoes weak self-association under these conditions and are consistent with a simple monomer-dimer equilibrium with a dimer-dissociation constant Kd=1.6(+/-0.4) mM. The relaxation data were analyzed using a reduced spectral density mapping approach to avoid systematic effects of aggregation on the usual model-free formalism. The reduced spectral densities show that residues near the N terminus (residues 3 to 5 and 7 to 12), in the Omega-loop between beta-strands 2 and 3 (residues 24 to 31), and in particular the C-terminal 13 residues (residues 51 to 63), have significant mobility on a picosecond/nanosecond time-scale. In addition, conformational exchange on a microsecond time-scale was identified for residues 44 to 46 on the basis of observed differences in R2 at 11.7 and 14.1 T. The mobility identified near the N terminus and in the vicinity of residues 44 to 46 may be important in allowing the interactions of heregulin with multiple receptors.
Fairbrother
W J
WJ
Department of Protein Engineering, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
Liu
J
J
Pisacane
P I
PI
Sliwkowski
M X
MX
Palmer
A G
AG
D004815:Epidermal Growth Factor / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D008958:Models, Molecular; D020880:Neuregulins; D011487:Protein Conformation; D014461:Ultracentrifugation
D006023:Glycoproteins; D020880:Neuregulins; D004815:Epidermal Growth Factor