Buried asparagines determine the dimerization specificities of leucine zipper mutants

Abstract

Regulation of gene expression by many transcription factors is controlled by specific combinations of homo-and heterodimers through a short ␣-helical coiled-coil known as a leucine zipper. The dimer interface of a leucine zipper involves side chains of the residues at the a, d, e, and g positions of the (abcdefg) n heptad repeat. To understand the basis for the specificity of dimer formation, we characterized GCN4 leucine zipper mutants with all 16 possible permutations and combinations of isoleucines and asparagines at four a positions in the dimer interface, using a genetic test for the specificity of dimer formation by repressor-leucine zipper fusions. Heterodimers were detected by loss of repressor activity in the presence of a fusion to a dominant-negative mutant form of the DNA-binding domain of repressor. Reconstruction experiments using leucine zippers from GCN4, Jun, Fos, and C͞EBP showed that this assay distinguishes pairs that form heterodimers from those that do not. We found that the mutants have novel dimerization specificities determined by the positioning of buried asparagine residues at the a positions. The pattern of buried polar residues could also explain the dimerization specificities of some naturally occurring leucine zippers. The altered specificity mutants described here should be useful for the construction of artificial regulatory circuitry.The stoichiometry and specificity with which proteins interact is a key control point in many biological processes. For example, common dimerization domains allow transcription factors in the bZIP or bHLH-LZ families to form a variety of homo-and heterodimers with different properties. By expressing different sets of subunits under different conditions, cells can generate complex regulatory circuits from a relatively small number of genes. The correct functioning of this complex regulatory machinery depends on each of the component proteins assembling only with specific partners.Leucine zippers are an excellent model system to study how the stability and specificity of protein-protein interactions are determined. High-resolution x-ray crystallographic and NMR structures are available for several leucine zippers (1-7). As ␣-helical coiled coils, leucine zippers have simple secondary and tertiary structures. Moreover, the large number of naturally occurring leucine zipper proteins includes a wide variety of distinct and overlapping dimerization specificities. At the amino acid sequence level, leucine zippers are characterized by leucine appearing in every seventh position (d) over 4 to 5 heptad repeats (abcdefg) n . The hydrophobic core of the dimer interface is formed by residues at the a and d positions (Fig. 1); the solvent-accessible e and g positions are frequently occupied by charged amino acids (8, 9). In the crystal structures of leucine zippers, including GCN4 homodimers and Jun-Fos heterodimers, intersubunit salt bridges are seen between oppositely charged amino acids at the g (ith heptad) and eЈ (i ϩ 1th heptad of the other monomer)...

The K domain mediates heterodimerization of theArabidopsisfloral organ identity proteins, APETALA3 and PISTILLATA

“…In leucine zipper dimers, the i þ 5 ionic interaction also promotes the formation of specific dimer pairs and prevents the formation of non-specific dimer pairs (O'Shea et al, 1992;Vinson et al, 1993;Zeng et al, 1997). We propose that the interaction between Glu-97 in PI and Arg-102 in AP3 facilitates specific heterodimerization between AP3 and PI and prevents formation of homodimers and other AP3-and PI-containing heterodimers.…”

“…The leucine zipper proteins GCN4 and Jun contain asparagine at an a position in the central region of the leucine zipper motif (Figure 4b) (Zeng et al, 1997). Similarly, AP3 and PI encode asparagine at position 98 in the central region of K1.…”

“…In GCN4, an a-position asparagine is proposed to impart specificity for formation of a two-stranded, parallel coiled coil (Gonzalez et al, 1996;Harbury et al, 1993;Junius et al, 1995;Zeng et al, 1997). The a-position asparagine helps position the helices in a parallel orientation and disfavors anti-parallel arrangements that necessitate asparagine side chains packing against non-polar chains.…”

N@a and N@d: Oligomer and Partner Specification by Asparagine in Coiled-Coil Interfaces

“…13 From considerable work, the generally accepted view is that, at a sites in coiled-coil dimers, Asn residues make side chain-side chain hydrogen bonds to partly offset the cost of burying a polar group, but that this is not possible in other oligomers and topologies. [35][36][37][38] Studies have also been performed to quantify the contribution of these Asn-Asn pairs to dimer stability at least. [39][40][41] To summarize a large body of literature, N@a is destabilising, but it specifies parallel dimer over alternative states such as antiparallel dimer and parallel trimer.…”

Independent and Interchangeable Multimerization Domains of the AbrB, Abh, and SpoVT Global Regulatory Proteins

“…The in vivo assay developed by James Hu and colleagues for detection of protein domains having multimerization activity was used in this study. Methods for constructing and assaying the fusions were according to previously published protocols and information supplied by the Hu Laboratory as part of their generous package of vectors, strains, and phages required for the technique (15,32,33). The principle of the assay is that the DNA-binding N-terminal domain (DBD) of the cI repressor is unable to bind its cognate operator sequences unless fused to a peptide segment that can self-associate to form dimers or higher-order multimeric complexes.…”