Attractive Interhelical Electrostatic Interactions in the Proline- and Acidic-rich Region (PAR) Leucine Zipper Subfamily Preclude Heterodimerization with Other Basic Leucine Zipper Subfamilies

• Published in: The Journal of biological chemistry Vol. 275; no. 44; pp. 34826 - 34832
• Main Authors: Moll, Jonathan R., Olive, Michelle, Vinson, Charles
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.11.2000; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Base Sequence; C/EBP^a protein; CCAAT-Enhancer-Binding Proteins - chemistry; CCAAT-Enhancer-Binding Proteins - genetics; Dimerization; DNA Primers; Hydrogen-Ion Concentration; Leucine Zippers; Molecular Sequence Data; Proline - chemistry; Sequence Homology, Amino Acid; Static Electricity; vitellogenin promoter-binding protein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M004545200

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/EBPα, 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 α-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/EBPα, 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/EBPα 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.