ABSTRACT
The cAMP response element (CRE) binding protein (CREB) is central in the transcription regulation by cAMP, and the CREB-regulated transcriptional coactivators (CRTCs) play critical roles in CREB-mediated transcription activation. Upon stimulation, CRTCs translocate into the nucleus and complex with CREB on CRE promoters to activate target gene transcription. Their physiological importance is underscored by their function in energy balance, long-term memory, longevity and other processes. The CREB binding domain on CRTCs has been mapped, which interacts with the CREB basic leucine zipper domain that also mediates interaction with CRE-containing DNA. We report here crystal structures of a complex containing the CRTC2 CREB binding domain, the CREB basic leucine zipper domain and a CRE-containing DNA. The structures revealed that CRTC and CREB form a 2:2 complex on CRE-containing DNA, and CRTC interacts with both CREB and DNA through highly conserved residues. Structure-guided functional studies revealed that both interactions are crucial for the complex assembly and CREB stabilization on DNA. Interestingly, we found that the CRTC-DNA interaction confers selectivity toward the intrinsic DNA shape, which may play a role in selective transcription activation of the CRE genes.
Subject(s)
Cyclic AMP Response Element-Binding Protein/chemistry , Cyclic AMP Response Element-Binding Protein/metabolism , Transcription Factors/chemistry , Transcription Factors/metabolism , Binding Sites , Cell Nucleus/metabolism , Crystallography, X-Ray , DNA/metabolism , Gene Expression Regulation , Humans , Models, Molecular , Promoter Regions, Genetic , Protein Binding , Protein Conformation , Response Elements , Transcription, Genetic , Transcriptional ActivationABSTRACT
The cyanobacterial clock proteins KaiA, KaiB and KaiC interact with each other to generate circadian oscillations. We have identified the residues of the KaiA homodimer affected through association with hexameric KaiC (KaiC6mer) using a spin-label-tagged KaiA C-terminal domain protein (KaiAc) and performing electron spin resonance (ESR) analysis. Cys substitution and/or the attachment of a spin label to residues located at the bottom area of the KaiAc concave surface, a KaiC-binding groove, hindered the association of KaiAc with KaiC6mer, suggesting that the groove likely mediates the interaction with KaiC6mer. The residues affected by KaiC6mer association were concentrated in the three areas: the concave surface, a lobe-like structure (a mobile lobe near the concave surface) and a region adjacent to both the concave surface and the mobile lobe. The distance between the two E254, D255, L258 and R252 residues located on the mobile lobe decreased after KaiC association, suggesting that the two mobile lobes approach each other during the interaction. Analyzing the molecular dynamics of KaiAc showed that these structural changes suggested by ESR analysis were possible. Furthermore, the analyses identified three asymmetries in KaiAc dynamic structures, which gave us a possible explanation of an asymmetric association of KaiAc with KaiC6mer.
Subject(s)
Bacterial Proteins/metabolism , CLOCK Proteins/metabolism , Circadian Rhythm Signaling Peptides and Proteins/metabolism , Synechococcus/metabolism , Cysteine/metabolism , Electron Spin Resonance Spectroscopy , Molecular Dynamics Simulation , Phosphorylation , Protein Interaction Domains and Motifs , Protein Interaction Mapping , Spin LabelsABSTRACT
The molecular machinery of the cyanobacterial circadian clock consists of three proteins, KaiA, KaiB, and KaiC. The three Kai proteins interact with each other and generate circadian oscillations in vitro in the presence of ATP (an in vitro KaiABC clock system). KaiB consists of four subunits organized as a dimer of dimers, and its overall shape is that of an elongated hexagonal plate with a positively charged cleft flanked by two negatively charged ridges. We found that a mutant KaiB with a C-terminal deletion (KaiB(1-94)), which lacks the negatively charged ridges, was a dimer. Despite its dimeric structure, KaiB(1-94) interacted with KaiC and generated normal circadian oscillations in the in vitro KaiABC clock system. KaiB(1-94) also generated circadian oscillations in cyanobacterial cells, but they were weak, indicating that the C-terminal region and tetrameric structure of KaiB are necessary for the generation of normal gene expression rhythms in vivo. KaiB(1-94) showed the highest affinity for KaiC among the KaiC-binding proteins we examined and inhibited KaiC from forming a complex with SasA, which is involved in the main output pathway from the KaiABC clock oscillator in transcription regulation. This defect explains the mechanism underlying the lack of normal gene expression rhythms in cells expressing KaiB(1-94).
