Interaction of transforming growth factor alpha with the epidermal growth factor receptor: binding kinetics and differential mobility within the bound TGF-alpha.
The interaction of transforming growth factor alpha (TGF-alpha) with the complete extracellular domain of the epidermal growth factor receptor (EGFR-ED) was examined by nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR resonances of the methyl groups of TGF-alpha were used as probes of the interaction of TGF-alpha with the EGF receptor to determine the binding kinetics and the differential mobility within the bound TGF-alpha. The methyl resonances were studied because there are 14 methyl containing residues well dispersed throughout the structure of TGF-alpha and the relaxation properties of methyl groups are well understood. Changes in the longitudinal and transverse 1H NMR relaxation rates of the methyl resonances of TGF-alpha caused by binding to the 85-kDa EGFR-ED were studied. From these measurements it was determined that the interaction was in the NMR fast exchange limit. A binding mechanism to rationalize the different rates determined by NMR and surface plasmon resonance techniques [Zhou, M., et al. (1993) Biochemistry 32, 8193-8198] is proposed. The transverse relaxation rate (R2) enhancements of the various methyl resonances displayed a regional dependence within the bound TGF-alpha molecule. Resonances from the C-terminus of TGF-alpha, which were flexible in the unbound molecule, revealed dramatic increases in their R2 upon binding to the EGFR-ED along with resonances from the interior of TGF-alpha. However, upon binding, the R2 enhancements of the methyl resonances from the N-terminus of TGF-alpha, which were also flexible in the unbound TGF-alpha, were slight; indicating a retention of mobility of this region for bound TGF-alpha. The implications of these data with respect to the mechanism of receptor activation and the design of antagonists are discussed.
Hoyt
D W
DW
Protein Engineering Network of Centres of Excellence, University of Alberta, Edmonton, Canada.
Harkins
R N
RN
Debanne
M T
MT
O'Connor-McCourt
M
M
Sykes
B D
BD
D000595:Amino Acid Sequence; D066246:ErbB Receptors / Q000737:chemistry*; D066298:In Vitro Techniques; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009038:Motion; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins; D012996:Solutions; D016211:Transforming Growth Factor alpha / Q000737:chemistry*
D011994:Recombinant Proteins; D012996:Solutions; D016211:Transforming Growth Factor alpha; D066246:ErbB Receptors