Dimerization of leucine zippers analyzed by random selection.

The leucine zipper is a coiled coil that mediates specific dimerization of bZIP DNA-binding domains. A hydrophobic spine involving the conserved leucines runs down the coiled-coil and is thought to stabilize the dimer. We used the method of random selection to further define the primary sequence requirements for homodimer formation and heterodimer formation with Fos. When positions on either side of the hydrophobic spine of GCN4 are diversified to include the corresponding residues of Jun, a large percentage of the resulting sequences form homodimers, and a large percentage form heterodimers with Fos. Basic residues were preferred, but not essential, at position e of zippers which heterodimerize with Fos. When random sequences containing 5 heptad repeat of leucines are subject to a selection for homodimer formation, a diverse set of sequences is isolated. Certain residues are preferred at each position in the heptad repeat, although no essential primary sequence determinants could be identified. No pair of residues not involving the conserved leucines could be identified which strongly promotes homodimerization. These results suggest that factors determining leucine zipper dimerization are complex, with numerous interactions contributing to the association.     

Jun DNA-binding is modulated by mutations between the leucines or by direct interaction of fos with the TGACTCA sequence

Mutations between the leucines of the "leucine zipper" domain of Jun D can either decrease (Asn 301 to Ala) or increase (Thr 307, Ala 308, to Glu, Val) homodimer formation and specific binding to DNA even though such changes do not modify the predicted alpha-helical structure of this region. As shown previously, addition of Fos strongly increases the affinity of Jun for DNA by forming a heterodimer. The jun down mutation (Asn 301 to Ala) also diminishes DNA binding by the Fos-Jun D heterodimer. These data strongly support the coiled coil conformation of this region where residues adjacent to the leucines are also important for dimer formation. Ultraviolet cross-linking experiments have shown that both Fos and Jun directly contact the TGACTCA palindromic sequence defined as a TPA (12-O-tetradecanoyl phorbol-13-acetate) response element or TRE. Both Jun homodimers and Jun-Fos heterodimers bind this TRE as well as the cAMP responsive element (CRE or TGACGTCA) with comparable affinities. While strong c-Jun or Jun D binding requires a perfect palindrome, Jun-Fos complexes can also efficiently recognize sequences where the right half of the palindrome is less conserved (TGACTAA or TGACGCA).     

Packing and hydrophobicity effects on protein folding and stability: effects of beta-branched amino acids, valine and isoleucine, on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers.

The aim of this study was to examine the differences between hydrophobicity and packing effects in specifying the three-dimensional structure and stability of proteins when mutating hydrophobes in the hydrophobic core. In DNA-binding proteins (leucine zippers), Leu residues are conserved at positions "d," and beta-branched amino acids, Ile and Val, often occur at positions "a" in the hydrophobic core. In order to discern what effect this selective distribution of hydrophobes has on the formation and stability of two-stranded alpha-helical coiled coils/leucine zippers, three Val or three Ile residues were simultaneously substituted for Leu at either positions "a" (9, 16, and 23) or "d" (12, 19, and 26) in both chains of a model coiled coil. The stability of the resulting coiled coils was monitored by CD in the presence of Gdn.HCl. The results of the mutations of Ile to Val at either positions "a" or "d" in the reduced or oxidized coiled coils showed a significant hydrophobic effect with the additional methylene group in Ile stabilizing the coiled coil (delta delta G values range from 0.45 to 0.88 kcal/mol/mutation). The results of mutations of Leu to Ile or Val at positions "a" in the reduced or oxidized coiled coils showed a significant packing effect in stabilizing the coiled coil (delta delta G values range from 0.59 to 1.03 kcal/mol/mutation). Our results also indicate the subtle control hydrophobic packing can have not only on protein stability but on the conformation adopted by the amphipathic alpha-helices. These structural findings correlate with the observation that in DNA-binding proteins, the conserved Leu residues at positions "d" are generally less tolerant of amino acid substitutions than the hydrophobic residues at positions "a."     

Structural features of apolipoprotein B synthetic peptides that inhibit lipoprotein(a) assembly

Lipoprotein(a) [Lp(a)] is assembled via an initial noncovalent interaction between apolipoprotein B100 (apoB) and apolipoprotein(a) [apo(a)] that facilitates the formation of a disulfide bond between the two proteins. We previously reported that a lysine-rich, alpha-helical peptide spanning human apoB amino acids 4372-4392 was an effective inhibitor of Lp(a) assembly in vitro. To identify the important structural features required for inhibitory action, new variants of the apoB4372-4392 peptide were investigated. Introduction of a central leucine to proline substitution abolished the alpha-helical structure of the peptide and disrupted apo(a) binding and inhibition of Lp(a) formation. Substitution of hydrophobic residues in the apoB4372-4392 peptide disrupted apo(a) binding and inhibition of Lp(a) assembly without disrupting the alpha-helical structure. Substitution of all four lysine residues in the peptide with arginine decreased the IC50 from 40 microM to 5 microM . Complexing of the arginine-substituted peptide to dimyristoylphosphatidylcholine improved its activity further, yielding an IC50 of 1 microM. We conclude that the alpha-helical structure of apoB4372-4392, in combination with hydrophobic residues at the lipid/water interface, is crucial for its interaction with apo(a). Furthermore, the interaction of apoB4372-4392 with apo(a) is not lysine specific, because substitutions with arginine result in a more effective inhibitor.     

The K domain mediates heterodimerization of the Arabidopsis floral organ identity proteins,APETALA3 and PISTILLATA

MADS genes in plants encode key developmental regulators of vegetative and reproductive development. The majority of well-characterized plant MADS proteins contain two conserved domains, the DNA-binding MADS domain and the K domain. The K domain is predicted to form three amphipathic alpha-helices referred to as K1, K2, and K3. In this report, we define amino acids and subdomains important for heterodimerization between the two Arabidopsis floral organ identity MADS proteins APETALA3 (AP3) and PISTILLATA (PI). Analysis of mutants defective in dimerization demonstrates that K1, K2 and the region between K1 and K2 are critical for the strength of AP3/PI dimerization. The majority of the critical amino acids are hydrophobic indicating that the K domain mediates AP3/PI interaction primarily through hydrophobic interactions. Specially, K1 of AP3 and PI resembles a leucine zipper motif. Most mutants defective in AP3/PI heterodimerization in yeast exhibit partial floral organ identity function in transgenic Arabidopsis. Our results also indicate that the motif containing Asn-98 and specific charged residues in K1 (Glu-97 in PI and Arg-102 in AP3) are important for both the strength and specificity of AP3/PI heterodimer formation.     

Structural similarity between legumin and vicilin storage proteins from legumes.

The primary structures for several members of both the vicilin and legumin families of storage proteins were examined using a computer routine based on amino acid physical characteristics. The comparison algorithm revealed that sequences from the two families could be aligned and share a number of predicted secondary structural features. The COOH-terminal half of the subunits in both families displayed a highly conserved core region that was largely hydrophobic and in which a high proportion of the residues were predicted to be in beta-sheet conformations. The central region of the molecules which contained mixed areas of predicted helical and sheet conformations showed more variability in residue selection than the COOH-terminal regions. The NH2-terminal segments of subunits from the two different families could not be aligned though they characteristically had a high proportion of residues predicted to be in helical conformations. The feature which most clearly distinguished subunits between the two families was an inserted span in the legumin group with a high proportion of acidic amino acids located between the central and COOH-terminal domains. Residues in this insertion were predicted to exist mainly in helical conformation. Since considerable size variation occurs in this area amongst the legumin subunits, alterations in this region may have a minimal detrimental effect on the structure of the proteins.     

Structure–Critical Distribution of Aromatic Residues in the Fibronectin Type III Protein Family

Over a thousand individual Fibronectin type III (FnIII) domain sequences, extracted from more than 60 different FnIII-dependent protein super-structures, were downloaded from curated database resources. Three regions of extreme sequence conservation within the well-characterized FnIII β-sandwich structure were respectively defined by near absolute conservation of a tryptophan (Trp) in β-strand-B, tyrosines (Tyr) in both β-strand-C and β-strand-F, and a leucine (Leu) residue in the unstructured region immediately preceding β-strand-F. Employing these four conserved landmarks, the entire FnIII sequence dataset was vertically registered to align the three conserved regions, and the cumulative distribution of all other amino acid functionality was determined and plotted relative to these landmark residues. Conserved aromatic sites were each found to be flanked by aliphatic residues that assure localization of these sites to the inaccessible hydrophobic interface between major sheet structures. Mapping the location of conserved aromatic sites in numerous PDB structures demonstrated the consistent pair-wise co-localization of the indole side-chain of the conserved strand-B Trp site to within 0.35 nm of the phenolic side-chain of the strand-C Tyr site located 8–14 amino acids distal. Likewise, the side-chain of the strand-F Tyr site co-localized to within 0.45 nm of the aliphatic side-chain of the conserved Leu that uniformly precedes it by six residues. While classic hydropathy-based theories would deem the “burying” of Tyr and Trp side-chains and/or their association with hydrophobic FnIII core residues thermodynamically unnecessary, alternative contributions of conserved Trp and Tyr residues, and particularly the role of the absolutely conserved tyrosine phenolic –OH in native FnIII structure–function are considered. A more global role for conserved FnIII aromaticity is also discussed in light of the aromatic conservation observed in other well-established protein families.     

Nanotubules formed by highly hydrophobic amphiphilic alpha-helical peptides and natural phospholipids

We previously reported that the 18-mer amphiphilic alpha-helical peptide, Hel 13-5, consisting of 13 hydrophobic residues and five hydrophilic amino acid residues, can induce neutral liposomes (egg yolk phosphatidylcholine) to adopt long nanotubular structures and that the interaction of specific peptides with specific phospholipid mixtures induces the formation of membrane structures resembling cellular organelles such as the Golgi apparatus. In the present study we focused our attention on the effects of peptide sequence and chain length on the nanotubule formation occurring in mixture systems of Hel 13-5 and various neutral and acidic lipid species by means of turbidity measurements, dynamic light scattering measurements, and electron microscopy. We designed and synthesized two sets of Hel 13-5 related peptides: 1) Five peptides to examine the role of hydrophobic or hydrophilic residues in amphiphilic alpha-helical structures, and 2) Six peptides to examine the role of peptide length, having even number residues from 12 to 24. Conformational, solution, and morphological studies showed that the amphiphilic alpha-helical structure and the peptide chain length (especially 18 amino acid residues) are critical determinants of very long tubular structures. A mixture of alpha-helix and beta-structures determines the tubular shapes and assemblies. However, we found that the charged Lys residues comprising the hydrophilic regions of amphiphilic structures can be replaced by Arg or Glu residues without a loss of tubular structures. This suggests that the mechanism of microtubule formation does not involve the charge interaction. The immersion of the hydrophobic part of the amphiphilic peptides into liposomes initially forms elliptic-like structures due to the fusion of small liposomes, which is followed by a transformation into tubular structures of various sizes and shapes.