NMR-based modelling revealed an alpha helical structure for cytoplasmic domain of the alpha subunit of Fc epsilon RI, the high affinity IgE receptor.

Abstract

FceRl is a tebameric complex, apy,, of non-covalently attached subunits: one IgEbinding a subunit with the binding site in the extracellular part of the chain, one p subunit and a dimer of disulphide-linked y subunits [I] . The direct experimental structure determination of the intact FceRI receptor has not been reported. The understanding of IgE receptor structure should be medically important, since it plays a central role in mediating many of the pathophysiological phenomena associated with allergy [2]. However, division of this receptor by the membrane into domains led to the strategy of structure determination of synthesized domain peptides by CD, FTIR, NMR and molecular modelling studies [3-81. These 3D domain structures form a basis for determining the structure of whole subunits and receptors. In the present work, a synthetic 23-AA peptide with the sequence of the cytoplasmic domain of the alpha subunit (FSTHKQFESILKIQKTGKGKKKG) was studied by 2D-NMR and CD spectroscopy in aqueous, organic and mixed solutions. A set of ID and 2D proton NMR spectra was obtained on the Bruker 500 and 600 MHZ spectrometers for each solvent system. NMR-based molecular modelling was carried out by XPLOR 3.1 program [9], using distance geomeby and simulated annealing protocols using 126 intraresidual, 60 sequential and 48 medium distance constraints derived from 2D NOESY and ROESY experiments. The 10 lowest energy structures were chosen from 20 generated structures, and then subjected to the refinement against volume integrales from the 300 ms mixing time NOESY spectrum. Spin system assignments of the NMR spectra of the peptide in 90% methanol / 10% water were made from TOCSY spectm, and sequential assignments using the fmgerprint region of the phase sensitive NOESY and ROESY spectra. Assignments for the proton resonances of the peptide are summarised in Table 1. The coupling constants are presented in the Figure 1 . The low coupling constant values between HIS-4 and GLN-14 residues indicated a helical conformation in that region.