Attractive interhelical electrostatic interactions in the proline- and acidic-rich region (PAR) leucine zipper subfamily preclude heterodimerization with other basic leucine zipper subfamilies

Basic region-leucine zipper (B-ZIP) proteins homo- or heterodimerize to bind sequence-specific double-stranded DNA. We present circular dichroism (CD) thermal denaturation data on vitellogenin promoter-binding protein (VBP), a member of the PAR subfamily of E-ZIP proteins that also includes thyroid embryonic factor, hepatocyte leukemia factor, and albumin site D-binding protein. VBP does not heterodimerize with B-ZIP domains from C/EBP alpha, JUND, or FOS, We describe a dominant negative protein, A-VEP, that contains the VBP leucine zipper and an acidic amphipathic protein sequence that replaces the basic region critical for DNA binding. The acidic extension forms a coiled coil structure with the VEP basic region in the VBP A-VBP heterodimer, This new alpha -helical structure extends the leucine zipper N-terminally, stabilizing the complex by 2.0 kcal/mol, A-VBP abolishes DNA binding of VBP in an equimolar competition assay, but does not affect DNA binding even at 100-fold excess of CREB, C/EBP alpha, or FOS/JUND, Likewise, proteins containing the acidic extension appended to seven other leucine zippers do not inhibit VBP DNA binding. We show that conserved g <-> e' or i, i' + 5 salt bridges are sufficient to confer specificity to VBP by mutating the C/EBP alpha leucine zipper to contain the g <-> e' salt bridges that characterize VBP, A-VBP heterodimerizes with this mutant C/EBP, preventing it from binding to DNA These conserved g <-> e' electrostatic interactions define the specificity of the PAR subfamily of B-ZIP proteins and preclude interaction with other B-ZIP subfamilies.