title: In vitro and in silico analysis of the Aspergillus nidulans DNA-CreA repressor interactions.
abstract: The CreA protein mediates carbon catabolite repression in the fungus Aspergillus nidulans. Its DNA-binding domain belongs to the Cys2-His2 class, binding specifically to a 5' SYGGRG 3' nucleotide sequence. EMSA experiments showed that the CreA(G27D) mutation resulted in a 30-fold increase of the Kdiss, and footprinting revealed a altered pattern of protein/DNA contacts. We modeled the CreA and the CreA(G27D) complexes in silico. A 15 ns molecular dynamics simulation of the solvated CreA(G27D) and CreA models was carried out using the MOE 2007.09 suite and the Amber99 force field. We have focused our analysis in residues Arg14, Glu16, His17, and Arg20 and Arg44, Asp46, and Arg50, previously, shown to be responsible for the specific contacts of the two Zn fingers. The electrostatic and the total potential energies showed the CreA(G27D) mutation to decrease the affinity of the complex, in agreement with the Kdiss's values. The in silico approach highlighted the role of the inter-finger linker. We identified several differential structural characteristics of the CreA and CreA(G27D)/DNA complexes and observed that the latter resulted in a lower dynamic flexibility of the complex.
authors.affiliation: a Biologia Molecular Departamento de Bioquímica Clínica, Facultad de Química , UdelaR , General Flores 2124, C.P. 1157, 11800 Montevideo , Uruguay .
authors.forename: Patricia
authors.initials: P
authors.lastname: Esperón
authors.forename: Claudio
authors.initials: C
authors.lastname: Scazzocchio
authors.forename: Margot
authors.initials: M
authors.lastname: Paulino
mesh_terms: D001233:Aspergillus nidulans / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003198:Computer Simulation; D004271:DNA, Fungal / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D056004:Molecular Dynamics Simulation; D009154:Mutation; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics; D016335:Zinc Fingers
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; D012097:Repressor Proteins; C071237:CreA protein, Aspergillus nidulans
keywords: Aspergillus nidulans; DNA–protein interaction; TGEK linker; zinc finger