Investigating potential allostery in the transcription factor CREB

Intrinsically disordered proteins (IDPs) are incredibly prevalent in eukaryotic transcription factors as disorder in structure allows for great flexibility and multi-faceted pathways of signal transduction and protein regulation. The basic leucine zipper (bZIP) is one of the three eukaryotic disordered DNA-binding domains. One particular basic zipper, cAMP-response element binding protein (CREB), is predicted to be one of the most disordered proteins in the human genome. CREB is a major signal transducer of key signalling pathways including cAMP, Ca2+ etc., and regulate the transcription of over 4,000 genes. Ubiquitously expressed in all eukaryotic cells, CREB has been found to be essential in cell survival and memory formation. The misregulation of CREB in vivo has been attributed to various forms of cancer and hence must be tightly regulated by many sources.
Despite being one of the most well-studied transcription factors in terms of signal transduction, the mechanism of CREB DNA binding and search is yet to be elucidated. It is known that CREB must form a homodimer, bound to its specific DNA sequence - the cAMP response element (CRE) and activated by serine-133 phosphorylation, in order to promote downstream gene transcription by recruiting co-factors and members of the basal transcription complex. However, the exact order of events that occur leading up to cofactor recruitment remains unknown.
The Shammas lab, through biophysical and structural studies of the DNA-binding domain (bZIP), found that the basic zipper strictly follows a dimer pathway in binding to CRE DNA. This study aims to contextualise this finding by performing biophysical characterisation on a HaloTagged full length CREB construct. First, an expression and purification protocol was developed and optimised to purify HaloCREB free from nucleic acid and protein contamination. Fluorescent anisotropy, stopped-flow kinetics and other biophysical techniques were then used to estimate various key parameters in determining the DNA binding and search pathway in CREB.
This study found HaloCREB to exhibit similar binding kinetics as the bZIP construct. A fast association rate of 1010 M-1 s-1 combined with a dissociation constant of 0.1 s-1 yields a remarkable Kd,CRE,kinetics of subnanomolar scale. However, a 10-fold mismatch between the Kd from equilibrium and kinetics study suggests that, if the results are reproducible, a two-state reaction is insufficient to describe how HaloCREB binds to DNA. This infers the involvement of the monomer binding pathway.
In comparison with existing literature and the bZIP data collected by the Shammas laboratory, my finding suggests that HaloCREB does not interfere with CREB DNA-binding. This observation is consistent with other in vivo studies. Moreover, a 3-fold difference in homodimerisation Kd and a 5-fold difference in association and dissociation binding constants separates HaloCREB from the bZIP construct. While small, these changes reveal possible, yet to be understood contribution of the rest of the CREB protein in modulating the DNA-binding kinetics of CREB.
Lastly, using an experimental set-up that is a better approximation to the cellular environment, I observed a slow-down in association rates when CRE DNA is in presence of 200-fold excess non-target DNA. This novel result highlights the need to consider the effect of non-target DNA in the biophysical characterisation of transcription factors. The estimation of biophysical parameters presented in this study will enable a model to be developed for a holistic understanding of the binding and searching mechanism of CREB.

Keywords: IDP
Kinetics
Biophysics
CREB
Intrinsically disordered proteins
Transcription factor
Advisor: Shammas, S