Nuclear Magnetic Resonance Solution Structure of the Pheromone E r-10 from the Ciliated Protozoan Euplotes raikovi

Abstract

The three-dimensional structure in solution of the pheromone E r-10 from the ciliated protozoan Euplotes raikovi has been determined by nuclear magnetic resonance spectroscopy. The structure of this 38-residue protein was obtained from 384 nuclear Overhauser enhancement distance constraints and 78 dihedral angle constraints using the distance geometry program DIANA for the structure calculation and the program AMBER for energy minimization. For a group of 20 conformers used to characterize the solution conformation, the average root-mean-square distance calculated for the backbone heavy atoms relative to the mean structure was 0·33 Å. The structure includes three short helices of residues 2 to 8, 12 to 19 and 24 to 33, and turn in the carboxy-terminal region of residues 34 to 38. These structural elements are held together by three disulfide bridges. The structure is quite stable relative to heat denaturation, since at both pH 4·6 and pH 6·0 only minor changes in the circular dichroism and nuclear magnetic resonance spectra were observed over the temperature range 20 to 80°C. The surface of the E r-10 structure shows an asymmetric charge distribution that results in a predominantly apolar surface on one side of the molecule. There is also a deep cleft in the structure with an asymmetric distribution of charged and apolar residues on the two walls. These surface features may be important for the homologous (autocrine) and heterologous binding of the pheromone to receptors.