Kuravsky, M. | Search bioRxiv

bioRxiv2025

A basic leucine zipper uses a dimer pathway to locate its targets in DNA mixtures

The operation of Eukaryotic transcription factors remains enigmatic. Cyclic AMP-responsive element-binding protein (CREB) is a member of the basic zipper family, a superfamily of transcription factors which operate exclusively in eukaryotes and bind DNA targets as homodimers or heterodimers. Modulation of oligomerization provides an additional opportunity for transcriptional control by this (and similar) families over monomeric transcription factors. However, when dimerization occurs - before or after target binding - is not known. We performed a suite of in vitro stopped-flow kinetic measurements, including CREB basic zippers target search amongst excess non-target DNA. The extensive dataset enabled a kinetic and thermodynamic understanding of DNA binding that demonstrated most productive search is performed by dimeric, rather than monomeric, CREB. Equilibrium is approached very rapidly under physiologically relevant concentrations, where relative flux through the monomer pathway is only around 1 in every 10,000 complexes formed. This preference of mechanism is driven by CREB monomer having a substantially higher affinity for another CREB monomer than for its DNA target. Equilibrium experiments with eight other monomeric peptides further suggest this as a common feature amongst the bZIP proteins, with only one peptide (Jun) displaying similar affinities for both. The work has implications for understanding the nature of DNA target search, as well as designing efficient artificial transcription factors and transcriptional inhibitors.

biophysics↗

bioRxiv2024

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

The basic zippers (bZIPs) are one of two large eukaryotic families of transcription factors whose DNA binding domains are disordered in isolation but fold into stable -helices upon target DNA binding. Here we systematically disrupt pre-existing helical propensity within the DNA binding region of the homodimeric bZIP domain of cAMP-response element binding protein (CREB) using Ala-Gly scanning and examine the impact on target binding kinetics. We find that the secondary structure of the transition state strongly resembles that of the unbound state. The closest residue to the dimerisation domain that has been examined is largely folded within both unbound and transition states; dimerisation apparently propagates additional helical propensity into the basic region. The results are consistent with electrostatically-enhanced DNA binding, followed by rapid folding from the folded zipper outwards. Interestingly, despite taking the exact experimental approach suggested for testing it, we find no evidence for disorder-mediated rate enhancement predicted by fly-casting theory.

biophysics↗