OLIGOMERIZATION OF THE AMIDE SENSOR PROTEIN AMIC BY X-RAY AND NEUTRON-SCATTERING AND MOLECULAR MODELING

AmiC is the negative regulator of the amidase operon which is involved in amide metabolism in the cytosol of Pseudomonas aeruginosa. Crystal structures show that AmiC contains two large domains that are very si milar to the periplasmic leucine-isoleucine-valine binding protein (Li vJ) of Escherichia coli. Synchrotron X-ray and neutron (in 100% (H2O)- H-2 buffer) scattering data were obtained for AmiC in the presence of its substrate acetamide and its anti-inducer butyramide which binds mo re weakly to AmiC than acetamide. Guinier analyses to obtain radius of gyration R-G and molecular weight M-r values showed that AmiC formed trimers whose formation was favored in the presence of acetamide and w hich exhibited concentration-dependent properties at concentrations be tween 0.4 and 2 mg/mL. Above 2 mg/mL, where trimers predominated, the R-G data were identical within 0.05 nm for AmiC - acetamide and AmiC - butyramide with mean X-ray and neutron R-G values of 3.35 and 3.28 nm , respectively. Scattering curve fits constrained by the crystal struc ture of AmiC-acetamide were evaluated in order to describe a model for trimeric AmiC. A translational search of parallel alignments of three monomers to form a symmetric AmiC homotrimer gave a good X-ray curve fit. Combinations of calculated curves for monomeric, dimeric, trimeri c, and tetrameric AmiC as seen in the crystal structure of AmiC gave r easonable but weaker X-ray curve fits which did not favor the existenc e of tetrameric AmiC. It is concluded that AmiC exhibits novel ligand- dependent oligomerization properties in solution when these are compar ed to other members of the periplasmic binding protein superfamily, wh ere AmiC exists in monomeric and trimeric forms, the proportions of wh ich depend on the presence of acetamide or butyramide.