NMR studies of the extracellular loop of the beta subunit of the high affinity IgE receptor.

Abstract

The high affiity IgE receptor, FcsRI, has been proposed to posses a tetrameric structure, aPy2 [I]. In this structure, the p subunit contains four hydrophobic membrane-spanning segments, and three connecting loops. The loops connecting transmembrane helices in the receptors could act structurally and biologically as protein domains. The synthesized peptide with sequences of the extracellular loop of the high affinity IgE receptors have been studied by NMR in the organic solvents. A synthetic 17-AA peptide with the sequence of the connecting loop 1-2 of the beta subunit (STLQTSDFDDEVLLLYR) was studied by ID and 2D-NMR spectroscopy in organic solvents. A set of ID and 2D proton NMR spectra was obtained on the Bruker 500 and 600 MHZ spectrometers in methanol and DMSO at 25°C. NMR-based molecular modelling was carried out by XPLOR 3. I program, using simulated annealing protocols with ambigous constraints [2], using 165 intraresidual, 9 sequential, 8 medium, and 36 ambigous distance constraints for thepeptide in the methanol and 59 intraresidual, 34 sequential, 9 medium, and 72 ambigous distance constraints for the peptide in the DMSO derived from 2D NOESY and ROESY experiments. The I 0 lowest energy structures were chosen from 20 generated structures for both solvents. The previous CD studies of the cytoplasmic 17-residue connecting peptide in TFE, indicated a conformational equilibrium in which a major component or moiety was an alpha helix structure (31. The addition of SDS at concentrations above critical micellar concentration, increased the alpha helical content in H,O. This indicated that this peptide could adopt a defined conformation dependent upon solvent polarity, and as such it could be a suitable subject for the NMR analysis. The spin system assignment of individual residues of the proton NMR spectra iii methanol and DMSO were achieved by TOCSY spectra, and the sequential assignments using the fingerprint regions of TOCSY and NOESY spectra. Proton chemical shifts values and 'JNH coupling constants were shown in the Table I . and Table 2 . for the methanol and DMSO solutions, respectively. The structural information from NOESY spectra (21 8 distance constraints) and coupling constants (17 torsional angle constraints) for the peptide conformation in methanol solution were used to calculate the model of the 17-residue bridge peptide of the high affinity IgE receptor. The ten lowest energy structures of the connecting peptide in methanol contained an alpha helical structure, confirming the result obtained by CD study that peptide could adopt an alpha helical conformation in the organic solvents. The lowest energy structure shown in the Figure 1 , indicated the location of the helical moiety in the middle of the peptide chain, between residues SER6 and GLUl I . The RMS values were 3.36 A and 0.46 A for the backbone atoms of the whole chain and of the alpha helical moiety, respectively. This indicated that peptide was flexible, especially at the ends of the chain, in agreement with the absence of the long range NOE distance constraints. These NMR based calculated structures of the 17-residue connecting peptide were in the good agreement with the structure of the sequence that connects two TM helices in the loop 1-2 of the beta subunitof FceRl in methanol at 25°C '