A bZIP protein from halophilic archaea: structural features and dimer formation of cGvpE from Halobacterium salinarum.
The cGvpE protein of Halobacterium salinarum PHH4 has been identified as transcriptional activator for the promoter of the c-gvpA gene encoding the major gas vesicle structural protein cGvpA. Molecular modelling of the carboxy-terminal region of cGvpE suggests that this protein resembles a basic leucine-zipper protein, and mutations in the putative DNA binding domain DNAB completely abolish the activator function in Haloferax volcanii transformants. Mutations in the key residues of the putative leucine-zipper region AH6 of cGvpE confirmed that the three residues V159, L166 and L173 were essential for the activator function of cGvpE at the c-gvpA promoter, whereas the cysteine residue C180 could be altered to a leucine or an aspartate residue without the loss of this function. Mutations in basic residues of helix AH4 demonstrated the importance of the lysine K104 for the activator function of cGvpE. A cGvpE protein containing a his-tag at the C-terminus was still able to activate the expression of c-gvpA in vivo. The cGvpE his-purified from Hf. volcanii formed a dimer in Blue-native polyacrylamide gels that could be resolved into monomers by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Dimers of cGvpE were already seen using SDS-PAGE, but not with cGvpE mutant proteins containing the alterations L166E or L173E/C180L in the leucine zipper. These results imply that the hydrophobic surface of helix AH6 is indeed required for the establishment of cGvpE dimers.
Institut für Mikrobiologie und Genetik, TU Darmstadt, D-64287 Darmstadt, Germany.
Petra
P
Plösser
Felicitas
F
Pfeifer
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D017930:Genes, Reporter; D019617:Halobacterium salinarum / Q000737:chemistry; D019622:Haloferax volcanii / Q000737:chemistry* / Q000235:genetics; D016350:Leucine Zippers / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics
D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators