A proposed architecture for the central domain of the bacterial enhancer-binding proteins based on secondary structure prediction and fold recognition

The expression of genes transcribed by the RNA polymerase with the alternative sigma factor <r54 (Ecr54) is absolutely dependent on activator proteins that bind to enhancer-like sites, located far upstream from the promoter. These unique prokaryotic proteins, known as enhancer-binding proteins (EBP), mediate open promoter complex formation in a reaction dependent on NTP hydrolysis. The best characterized proteins of this family of regulators are NtrC and Nif A, which activate genes required for ammonia assimilation and nitrogen fixation, respectively. In a recent IRBM course (“Frontiers of protein structure prediction,” IRBM, Pomezia, Italy, 1995; see web site http://www.mrc-cpe.cam.uk/ irbm-course95/), one of us (J.O.) participated in the elaboration of the proposal that the Central domain of the EBPs might adopt the classical mononucleotide-binding fold. This suggestion was based on the results of a new protein fold recognition algorithm (Map) and in the mapping of correlated mutations calculated for the sequence family on the same mononucleotide-binding fold topology. In this work, we present new data that support the previous conclusion. The results from a number of different secondary structure prediction programs suggest that the Central domain could adopt an alfi topology. The fold recognition programs ProFIT 0.9, 3D PROFILE combined with secondary structure prediction, and 123D suggest a mononucleotide-binding fold topology for the Central domain amino acid sequence. Finally, and most importantly, three of five reported residue alterations that impair the Central domain ATPase activity of the Eo-54 activators are mapped to polypeptide regions that might be playing equivalent roles as those involved in nucleotide-binding in the mononucleotide-binding proteins. Furthermore, the known residue substitutions that alter the function of the Ecr54 activators, leaving intact the Central domain ATPase activity, are mapped on a region proposed to play an equivalent role as the effector region of the GTPase superfamily.