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 B-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-VBP, 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 VBP 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/EBPalpha 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.
Moll
J R
JR
Laboratory of Metabolism, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Olive
M
M
Vinson
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers; D019281:Dimerization; D006863:Hydrogen-Ion Concentration; D016350:Leucine Zippers; D008969:Molecular Sequence Data; D011392:Proline / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity
D022762:CCAAT-Enhancer-Binding Proteins; D017931:DNA Primers; D011392:Proline