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 t he complete extracellular domain of the epidermal growth factor recept or (EGFR-ED) was examined by nuclear magnetic resonance (NMR) spectros copy. The H-1 MMR resonances of the methyl groups of TGF-alpha were us ed as probes of the interaction of TGF-alpha with the EGF receptor to determine the binding kinetics and the differential mobility within th e bound TGF-alpha. The methyl resonances were studied because there ar e 14 methyl containing residues well dispersed throughout the structur e of TGF-alpha and the relaxation properties of methyl groups are well understood. Changes in the longitudinal and transverse H-1 NMR relaxa tion rates of the methyl resonances of TGF-alpha caused by binding to the 85-kDa EGFR-ED were studied. From these measurements it was determ ined that the interaction was in the NMR fast exchange Limit. A bindin g mechanism to rationalize the different rates determined by NMR and s urface plasmon resonance techniques [Zhou, M., et al. (1993) Biochemis try 32, 8193-8198] is proposed. The transverse relaxation rate (R(2)) enhancements of the various methyl resonances displayed a regional dep endence within the bound TGF-alpha molecule. Resonances from the C-ter minus of TGF-alpha, which were flexible in the unbound molecule, revea led dramatic increases in their R(2) upon binding to the EGFR-ED along with resonances from the interior of TGF-alpha. However, upon binding , the R(2) enhancements of the methyl resonances from the N-terminus o f TGF-alpha, which were also flexible in the unbound TGF-alpha, were s light; indicating a retention of mobility of this region for bound TGF -alpha. The implications of these data with respect to the mechanism o f receptor activation and the design of antagonists are discussed.