Background: Listeria monocytogenes scavenges iron from heme and human hemoglobin using the Hbp1 and Hbp2 proteins. Results: Crystal structures and heme transfer studies of Hbp2 reveal an unusual binding mechanism. Conclusion: Hbp2 is a novel hemoglobin-binding hemophore that rapidly delivers hemin to Hbp1 and other Hbp2 proteins. Significance: These studies provides insight into how L. monocytogenes captures heme iron.
Phosphorylation of the kinase-inducible domain (KID) of the cyclic AMP response element binding transcription factor (CREB) regulates its function through several mechanisms. Transcriptional activation occurs following phosphorylation at serine 133, but multisite phosphorylation in a neighboring region termed the CK cassette, residues 108–117, results in inhibition of CREB-mediated transcription. A molecular-level understanding of the mechanism of these opposing reactions has been lacking, in part because of the difficulty of preparing multiply-phosphorylated CREB in vitro. By substitution of a single residue, we have generated an engineered mammalian CREB in which the CK cassette can be phosphorylated in vitro by casein kinases and have characterized its interactions with cyclic AMP response element (CRE) DNA. Phosphorylation of the CK cassette promotes an intramolecular interaction between the KID domain and the site of DNA binding, the basic region of the C-terminal basic leucine zipper (bZip) domain. Competition between the phosphorylated KID domain and DNA for bZip binding results in lowered affinity of CREB for DNA. The binding free energy calculated from the dissociation constant is directly proportional to the number of phosphate groups in the CK cassette, indicating that the DNA binding is regulated by a rheostat-like mechanism. The rheostat is modulated by variation in the concentration of cations such as Mg2+ and by alternative isoforms such as the natural CREB isoform that lacks residues 162–272. Multisite phosphorylation of CREB represents a versatile mechanism by which transcription can be tuned to meet the variable needs of the cell.
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