Glutamine-rich regions of the disordered CREB transactivation domain mediate dynamic intra- and intermolecular interactions.

The cyclic AMP response element (CRE) binding protein (CREB) is a transcription factor that contains a 280-residue N-terminal transactivation domain and a basic leucine zipper that mediates interaction with DNA. The transactivation domain comprises three subdomains, the glutamine-rich domains Q1 and Q2 and the kinase inducible activation domain (KID). NMR chemical shifts show that the isolated subdomains are intrinsically disordered but have a propensity to populate local elements of secondary structure. The Q1 and Q2 domains exhibit a propensity for formation of short ß-hairpin motifs that function as binding sites for glutamine-rich sequences. These motifs mediate intramolecular interactions between the CREB Q1 and Q2 domains as well as intermolecular interactions with the glutamine-rich Q1 domain of the TATA-box binding protein associated factor 4 (TAF4) subunit of transcription factor IID (TFIID). Using small-angle X-ray scattering, NMR, and single-molecule Förster resonance energy transfer, we show that the Q1, Q2, and KID regions remain dynamically disordered in a full-length CREB transactivation domain (CREBTAD) construct. The CREBTAD polypeptide chain is largely extended although some compaction is evident in the KID and Q2 domains. Paramagnetic relaxation enhancement reveals transient long-range contacts both within and between the Q1 and Q2 domains while the intervening KID domain is largely devoid of intramolecular interactions. Phosphorylation results in expansion of the KID domain, presumably making it more accessible for binding the CBP/p300 transcriptional coactivators. Our study reveals the complex nature of the interactions within the intrinsically disordered transactivation domain of CREB and provides molecular-level insights into dynamic and transient interactions mediated by the glutamine-rich domains.

Structural Basis for Graded Inhibition of CREB:DNA Interactions by Multisite Phosphorylation.

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 substituting 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 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 a decreased 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 of 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.

Novel mechanism of hemin capture by Hbp2, the hemoglobin-binding hemophore from Listeria monocytogenes.

Iron is an essential nutrient that is required for the growth of the bacterial pathogen Listeria monocytogenes. In cell cultures, this microbe secretes hemin/hemoglobin-binding protein 2 (Hbp2; Lmo2185) protein, which has been proposed to function as a hemophore that scavenges heme from the environment. Based on its primary sequence, Hbp2 contains three NEAr transporter (NEAT) domains of unknown function. Here we show that each of these domains mediates high affinity binding to ferric heme (hemin) and that its N- and C-terminal domains interact with hemoglobin (Hb). The results of hemin transfer experiments are consistent with Hbp2 functioning as an Hb-binding hemophore that delivers hemin to other Hbp2 proteins that are attached to the cell wall. Surprisingly, our work reveals that the central NEAT domain in Hbp2 binds hemin even though its primary sequence lacks a highly conserved YXXXY motif that is used by all other previously characterized NEAT domains to coordinate iron in the hemin molecule. To elucidate the mechanism of hemin binding by Hbp2, we determined crystal structures of its central NEAT domain (Hbp2(N2); residues 183-303) in its free and hemin-bound states. The structures reveal an unprecedented mechanism of hemin binding in which Hbp2(N2) undergoes a major conformational rearrangement that facilitates metal coordination by a non-canonical tyrosine residue. These studies highlight previously unrecognized plasticity in the hemin binding mechanism of NEAT domains and provide insight into how L. monocytogenes captures heme iron.