Coupling of binding and differential subdomain folding of the intrinsically disordered transcription factor CREB

Abstract

The cyclic AMP response element binding protein (CREB) contains a basic leucine zipper motif (bZIP) that forms a coiled coil structure upon dimerization and specific DNA binding. Although this state is well characterized, key features of CREB bZIP binding and folding are not well understood. We used single-molecule Förster resonance energy transfer (smFRET) to probe conformations of CREB bZIP subdomains. We found differential folding of the basic region and leucine zipper in response to different binding partners; a strong and previously unreported DNA-independent dimerization affinity; folding upon binding to nonspecific DNA; and evidence of long-range interdomain interactions in full-length CREB that modulate DNA binding. These studies provide new insights into DNA binding and dimerization and have implications for CREB function.

The transition state for coupled folding and binding of a disordered DNA binding domain resembles the unbound state

“…The nanomolar value of L is also noteworthy because previously reported values differ wildly from tens of µM (73, 74) to sub-nanomolar (75). Our value is similar to that found by Bentley et al for full-length CREB (72) who suggest CREB may be largely dimeric within the cell. For reference cellular CREB concentrations are around 100-fold higher than our reported homodimerisation K D (76, 77).…”

“…The bZIP domains are observed to fold upon binding their cognate DNA sequences (2,43,(68)(69)(70). We have examined the interaction of the isolated bZIP domain of CREB with CRE and find the binding affinity is sub-nanomolar, which is slightly lower than previous estimates for the full-length protein of around 1-2 nM (52, 71), but in line with a recent 1 nM upper limit (72). Our results rationalise the observed differences in terms of the experimental approach.…”

“…helicity is propagated outwards by dimerisation. Single-molecule FRET studies of CREB with fluorescent labels at either end of the LZ (residues 310 and 337) and BR (residues 270 and 310) are consistent with dimerisation leading to structural change in the ensemble of the BR (72). Changes to helicity observed by CD for mutant proteins can add further detail.…”

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

“…1 ) is typical of eukaryotic transcription factors in having a long IDR that plays an essential functional role ( 1 , 2 ). Apart from the bZip domain, which folds upon dimerization and binding to the CRE ( 25 , 26 ), the Q1, KID (kinase inducible activation domain), and Q2 activation domains of CREB (residues 1 to 280) are predicted to be intrinsically disordered.…”