Heme binding to cold shock protein D, CspD, from Vibrio cholerae.

Cold shock protein D (CspD) is one of the homologous proteins of cold shock protein A (CspA), inhibiting DNA replication by binding to single-stranded DNA. We found that CspD from Vibrio cholerae (VcCspD) possesses one heme regulatory motif (HRM) sequence and specifically binds heme with a stoichiometry of 1:1. The binding of a synthetic single-stranded DNA oligomer (ssDNA) was followed by fluorescence quenching of Trp. The fluorescence quenching associated with the addition of ssDNA was suppressed in the presence of heme, indicating that heme binding to VcCspD inhibited the formation of the VcCspD-ssDNA complex. Such heme-induced inhibition was not observed for the VcCspD mutant with replacement of Cys22 in the HRM with alanine (C22A). Heme binding at Cys22 is, therefore, essential for the inhibition of ssDNA binding for VcCspD. The growth of Escherichia coli at 37 °C was slowed when VcCspD was overexpressed, indicating that VcCspD hampers the growth of E. coli. When the production of heme in cells was promoted by the addition of a heme precursor, δ-aminolevulinic acid, the growth of E. coli expressing VcCspD was decelerated, but the growth of E. coli expressing the C22A mutant was not decelerated. These observations allow us to conclude that heme specifically binds to the HRM region in VcCspD and inhibits the binding of target ssDNA, which suggests that heme functions as a regulatory molecule for DNA replication.