A BETA-SHEET PEPTIDE INHIBITOR OF E47 DIMERIZATION AND DNA-BINDING

Background: Many transcription factors are active only in their dimeri c form, including the basic-helix-loop-helix (bHLH) family of transcri ption factors. The disruption of the dimer therefore presents a means of inhibiting the biological functions of such transcription factors. E47 is a homodimeric bHLH transcription factor with a four-helix bundl e dimerization interface. Here, we investigate the concept of dimeriza tion inhibition using peptides derived from the dimerization domain of E47. Results: We have synthesized several peptides corresponding to t he E47 dimerization interface that inhibit E47 DNA-binding activity wi th IC50 values in the range of 3.6-120 mM. Interestingly, helix II, a peptide corresponding to the carboxy-terminal helix of the E47 dimeriz ation interface, adopted a beta-sheet structure in solution, as shown using circular dichroism (CD), and inhibited the binding of E47 to DNA at equimolar concentrations. Size-exclusion chromatography, analytica l ultracentrifugation and cross-linking experiments verified that this peptide prevented E47 dimerization. Furthermore, CD experiments provi ded evidence that helix II could induce a beta-sheet secondary structu re upon the highly alpha-helical E47 bHLH domain. Conclusions: This st udy is the first demonstration of dissociative inhibition in the bHLH class of transcription factors and also provides an example of beta-sh eet induction in an alpha-helical protein. Future experiments will pro be the structural determinants of the beta-sheet secondary structure i n helix II and investigate the generality of the dissociative strategy in other transcription factor families.