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 K_diss, 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 K_diss′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.

Keywords:

• zinc finger
• DNA–protein interaction
• TGEK linker
• Aspergillus nidulans

Acknowledgments

We thank Joseph Strauss for helpful discussion. This work was supported by the CNRS and the Institut Universitaire de France at Orsay, by CSIC, PEDECIBA Química, Master of Bioinformatics PEDECIBA-UdelaR, and ECOS-SUD program.