title: Reciprocal domain evolution within a transactivator in a restricted sequence space.
abstract: offhough the concept of domain merging and shuffling as a major force in protein evolution is well established, it has been difficult to demonstrate how domains coadapt. Here we show evidence of coevolution of the Sinorhizobium meliloti NifA (SmNifA) domains. We found that, because of the lack of a conserved glycine in its DNA-binding domain, this transactivator protein interacts weakly with the enhancers. This defect, however, was compensated by evolving a highly efficient activation domain that, contrasting to Bradyrhizobium japonicum NifA (BjNifA), can activate in trans. To explore paths that lead to this enhanced activity, we mutagenized BjNifA. After three cycles of mutagenesis and selection, a highly active derivative was obtained. Strikingly, all mutations changed to amino acids already present in SmNifA. Our artificial process thus recreated the natural evolution followed by this protein and suggests that NifA is trapped in a restricted sequence space with very limited solutions for higher activity by point mutation.
authors.affiliation: Instituto de Biotecnologia, Universidad Nacional Autónoma de México, AP 510-3, Cuernavaca, Morelos, 62250, Mexico.
authors.forename: K
authors.initials: K
authors.lastname: Juarez
authors.forename: H
authors.initials: H
authors.lastname: Flores
authors.forename: S
authors.initials: S
authors.lastname: Dávila
authors.forename: L
authors.initials: L
authors.lastname: Olvera
authors.forename: V
authors.initials: V
authors.lastname: González
authors.forename: E
authors.initials: E
authors.lastname: Morett
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D020369:Bradyrhizobium / Q000235:genetics; D019020:Directed Molecular Evolution; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D016962:Sinorhizobium meliloti / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation
chemical_list: D001426:Bacterial Proteins; C061338:NifA protein, Bacteria; D014157:Transcription Factors