Consensus and variant cAMP-regulated enhancers have distinct CREB-binding properties

Recent determination of the cAMP response element-binding protein (CREB) ba sic leucine zipper (bZIP) consensus CRE crystal structure revealed key dime rization and DNA binding features that are conserved among members of the C REB/CREM/ATF-1 family of transcription factors. Dimerization appeared to be mediated by a Tyr(307)-Glu(312) interhelical hydrogen bond and a Glu(319)- Arg(314) electrostatic interaction. An unexpected hexahydrated Mg2+ ion wa s centered above the CRE in the dimer cavity. In the present study, we rela ted these features to CREB dimerization and DNA binding. A Y307F substituti on reduced dimer stability and DNA binding affinity, whereas a Y307R mutati on produced a stabilizing effect. Mutation of Glu(319) to Ala or Lys attenu ated dimerization and DNA binding. Mg2+ ions enhanced the binding affinity of wild-type CREB to the palindromic CRE by similar to 20-fold but did not do so for divergent CREs, Similarly, mutation of Lys(304), which mediates t he CREB interaction with the hydrated Mg2+, blocked CREB binding to the pal indromic but not the variant CRE sequences. The distinct binding characteri stics of the K304A mutants to the consensus and variant CRE sequences indic ate that CREB binding to these elements is differentially regulated by Mg2 ions, We suggest that CREB binds the consensus and variant CRE sequences t hrough fundamentally distinct mechanisms.