Identification of a co-repressor binding site in catabolite control protein CcpA.
The catabolite control protein CcpA is the central regulator of carbon catabolite repression in Bacilli and other Gram-positive bacteria. A comparison of 12 CcpA-like sequences with regulators from the LacI/GalR family defines a CcpA subfamily based on extensive similarities found among CcpAs and not in 32 other members of the family. These amino acids are clustered in three blocks in the CcpA sequence. Their interpretation, assuming a PurR-like fold, reveals that almost all of them are surface exposed and form a continuous patch on the N-terminal subdomain of the protein core extending into the DNA reading head. We introduced nine single amino acid exchanges in the subfamily specific residues of CcpA from Bacillus megaterium. Six mutants, namely CcpA47RS, 79AE, 89YE, 295YR, 299YE and 303RD, are inactive or severely impaired in catabolite repression, underlining their relevance for CcpA function. They are negatively transdominant over wild-type CcpA demonstrating their ability to correctly fold for dimerization. Five of them are unable or impaired in binding HPr-Ser-46-P in vitro, establishing a correlation between catabolite repression efficiency and HPr-Ser-46-P binding. These results support the hypothesis that the conserved region in CcpA is the HPr-Ser-46-P binding site.
Kraus
A
A
Lehrstuhl für Mikrobiologie, Institut für Mikrobiologie, Biochemie und Genetik der Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany.
Küster
E
E
Wagner
A
A
Hoffmann
K
K
Hillen
W
W
D000595:Amino Acid Sequence; D001410:Bacillus megaterium / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins; D001665:Binding Sites; D002244:Carbon / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid