Thermal unfolding in a GCN4-like leucine zipper: 13C alpha NMR chemical shifts and local unfolding curves.

13C alpha chemical shifts and site-specific unfolding curves are reported for 12 sites on a 33-residue, GCN4-like leucine zipper peptide (GCN4-lzK), ranging over most of the chain and sampling most heptad positions. Data were derived from NMR spectra of nine synthetic, isosequential peptides bearing 99% 13C alpha at sites selected to avoid spectral overlap in each peptide. At each site, separate resonances appear for unfolded and folded forms, and most sites show resonances for two folded forms near room temperature. The observed chemical shifts suggest that 1) urea-unfolded GCN4-lzK chains are randomly coiled; 2) thermally unfolded chains include significant transient structure, except at the ends; 3) the coiled-coli structure in the folded chains is atypical near the C-terminus; 4) only those interior sites surrounded by canonical interchain salt bridges fail to show two folded forms. Local unfolding curves, obtained from integrated resonance intensities, show that 1) sites differ in structure content and in melting temperature, so the equilibrium population must comprise more than two molecular conformations; 2) there is significant end-fraying, even at the lowest temperatures, but thermal unfolding is not a progressive unwinding from the ends; 3) residues 9-16 are in the lowest melting region; 4) heptad position does not dictate stability; 5) significant unfolding occurs below room temperature, so the shallow, linear decline in backbone CD seen there has conformational significance. It seems that only a relatively complex array of conformational states could underlie these findings.     

Conformational specificity of the lac repressor coiled-coil tetramerization domain

Predictive understanding of how the folded, functional shape of a native protein is encoded in the linear sequence of its amino acid residues remains an unsolved challenge in modern structural biology. Antiparallel four-stranded coiled coils are relatively simple protein structures that embody a heptad sequence repeat and rich diversity for tertiary packing of alpha-helices. To explore specific sequence determinants of the lac repressor coiled-coil tetramerization domain, we have engineered a set of buried nonpolar side chains at the a-, d-, and e-positions into the hydrophobic interior of the dimeric GCN4 leucine zipper. Circular dichroism and equilibrium ultracentrifugation studies show that this core variant (GCN4-pAeLV) forms a stable tetrameric structure with a reversible and highly cooperative thermal unfolding transition. The X-ray crystal structure at 1.9 A reveals that GCN4-pAeLV is an antiparallel four-stranded coiled coil of the lac repressor type in which the a, d, and e side chains associate by means of combined knobs-against-knobs and knobs-into-holes packing with a characteristic interhelical offset of 0.25 heptad. Comparison of the side chain shape and packing in the antiparallel tetramers shows that the burial of alanine residues at the e positions between the neighboring helices of GCN4-pAeLV dictates both the antiparallel orientation and helix offset. This study fills in a gap in our knowledge of the determinants of structural specificity in antiparallel coiled coils and improves our understanding of how specific side chain packing forms the teritiary structure of a functional protein.     

The contribution of buried polar groups to the conformational stability of the GCN4 coiled coil

The dimeric interface of the leucine zipper coiled coil from GCN4 has been used to probe the contributions of hydrophobic and hydrogen bonding interactions to protein stability. We have determined the energetics of placing Ile or Asn residues at four buried positions in a two-stranded coiled coil. As expected, Ile is favored over Asn at these buried positions, but not as much as predicted by considering only the hydrophobic effect. It appears that interstrand hydrogen bonds form between the side-chains of the buried Asn residues and these contribute to the conformational stability of the coiled-coil peptides. However, these contributions are highly dependent on the locations of the Asn pairs. The effect of an Ile to Asn mutation is greatest at the N terminus of the peptide and decreases almost twofold as we move the substitution from the N to C-terminal heptads.     

Pitch diversity in α-helical coiled coils

Two complementary methods for measuring local pitch based on heptad position in α-helical coiled coils are described and applied to six crystal structures. The results reveal a diversity of pitch values: two-stranded coiled coils appear to have pitch values near 150 Å the values for three- and four-stranded coiled coils range closer to 200 Å. The methods also provide a rapid and sensitive gauge of local coiled-coil conformation. Polar or charged residues in the apolar interface between coiled-coil helices markedly affect local pitch values, suggesting a connection between pitch uniformity and coiled-coil stability. Moreover, the identification of a skip residue (heptad frame shift) in the hemaglutinin glycoprotein of influenza virus (HA) allows interpretation of local pitch changes. These results on relatively short coiled-coil structures have relevance for the much longer fibrous proteins (many of which have skip residues) whose detailed structures are not yet established. We also show that local pitch values from molecular dynamics predictions of the GCN4 leucine zipper are in striking agreement with the high-resolution crystal structure—a result not readily discerned by direct comparison of atomic coordinates. Taken together, these methods reveal specific aspects of coiled-coil structure which may escape detection by global analyses of pitch. © 1993 Wiley-Liss, Inc.     

Buried polar residues in coiled-coil interfaces

Coiled coils, estimated to constitute 3-5% of the encoded residues in most genomes, are characterized by a heptad repeat, (abcdefg)(n), where the buried a and d positions form the interface between multiple alpha-helices. Although generally hydrophobic, a substantial fraction ( approximately 20%) of these a- and d-position residues are polar or charged. We constructed variants of the well-characterized coiled coil GCN4-p1 with a single polar residue (Asn, Gln, Ser, or Thr) at either an a or a d position. The stability and oligomeric specificity of each variant were measured, and crystal structures of coiled-coil trimers with threonine or serine at either an a or a d position were determined. The structures show how single polar residues in the interface affect not only local packing, but also overall coiled-coil geometry as seen by changes in the Crick supercoil parameters and core cavity volumes.     

Genetic analysis of the leucine heptad repeats of Lac repressor: evidence for a 4-helical bundle.

Gel-filtration experiments indicate that a peptide (P2) composed of the basic region of GCN4 fused to the leucine heptad repeats of Lac repressor forms tetrameric aggregates. Gel-shift experiments were performed to determine the orientation of the helices in the tetrameric P2 aggregate. Sandwich-complex formation of peptide P2 with two DNA fragments containing two symmetrical CRE binding sites (5'-ATGACGTCAT-3') at a distance of 21 bp suggests antiparallel aggregation of the Lac leucine heptad repeats. Thus, we conclude that the leucine heptad repeats of Lac repressor have the ability to form homomeric 4-helical bundles with an antiparallel arrangement of the helices. This topology enables the two DNA fragments in the sandwich complexes to be held together by two tetramers of peptide P2. Replacement of the uncharged amino acids of the helical g and e positions of peptide P2 by the corresponding charged residues of GCN4 (peptide P4) results in a dimeric and parallel aggregation of the leucine heptad repeats, and consequently abolishes the potential to form sandwich structures. Similarly, a hybrid Lac repressor in which the GCN4 leucine zipper replaces the natural Lac leucine heptad repeats forms dimers only. It regains the ability to form tetramers when the charged amino acids in helical positions g and e are replaced by uncharged alanines.     

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.     

Stabilization of coiled-coil peptide domains by introduction of trifluoroleucine

Substitution of leucine residues by 5,5,5-trifluoroleucine at the d-positions of the leucine zipper peptide GCN4-p1d increases the thermal stability of the coiled-coil structure. The midpoint thermal unfolding temperature of the fluorinated peptide is elevated by 13 degrees C at 30 microM peptide concentration. The modified peptide is more resistant to chaotropic denaturants, and the free energy of folding of the fluorinated peptide is 0.5-1.2 kcal/mol larger than that of the hydrogenated form. A similarly fluorinated form of the DNA-binding peptide GCN4-bZip binds to target DNA sequences with affinity and specificity identical to those of the hydrogenated form, while demonstrating enhanced thermal stability. Molecular dynamics simulation on the fluorinated GCN4-p1d peptide using the Surface Generalized Born implicit solvation model revealed that the coiled-coil binding energy is 55% more favorable upon fluorination. These results suggest that fluorination of hydrophobic substructures in peptides and proteins may provide new means of increasing protein stability, enhancing protein assembly, and strengthening receptor-ligand interactions.     

Crystal structure of a super leucine zipper,an extended two‐stranded super long coiled coil

Coiled coil is a ubiquitous structural motif in proteins, with two to seven alpha helices coiled together like the strands of a rope, and coiled coil folding and assembly is not completely understood. A GCN4 leucine zipper mutant with four mutations of K3A, D7A, Y17W, and H18N has been designed, and the crystal structure has been determined at 1.6 Å resolution. The peptide monomer shows a helix trunk with short curved N‐ and C‐termini. In the crystal, two monomers cross in 35° and form an X‐shaped dimer, and each X‐shaped dimer is welded into the next one through sticky hydrophobic ends, thus forming an extended two‐stranded, parallel, super long coiled coil rather than a discrete, two‐helix coiled coil of the wild‐type GCN4 leucine zipper. Leucine residues appear at every seventh position in the super long coiled coil, suggesting that it is an extended super leucine zipper. Compared to the wild‐type leucine zipper, the N‐terminus of the mutant has a dramatic conformational change and the C‐terminus has one more residue Glu 32 determined. The mutant X‐shaped dimer has a large crossing angle of 35° instead of 18° in the wild‐type dimer. The results show a novel assembly mode and oligomeric state of coiled coil, and demonstrate that mutations may affect folding and assembly of the overall coiled coil. Analysis of the formation mechanism of the super long coiled coil may help understand and design self‐assembling protein fibers.     

Site-specific experiments on folding/unfolding of Jun coiled coils: thermodynamic and kinetic parameters from spin inversion transfer nuclear magnetic resonance at leucine-18

The 32-residue leucine zipper subsequence, called here Jun-lz, associates in benign media to form a parallel two-stranded coiled coil. Studies are reported of its thermal unfolding/folding transition by circular dichroism (CD) on samples of natural isotopic abundance and by both equilibrium and spin inversion transfer (SIT) nuclear magnetic resonance (NMR) on samples labeled at the leucine-18 alpha-carbon with 99% 13C. The data cover a wide range of temperature and concentration, and show that Jun-lz unfolds below room temperature, being far less stable than some other leucine zippers such as GCN4. 13C-NMR shows two well-separated resonances. We ascribe the upfield one to 13C spins on unfolded single chains and the downfield one to 13C spins on coiled-coil dimers. Their relative intensities provide a measure of the unfolding equilibrium constant. In SIT NMR, the recovery of the equilibrium magnetization after one resonance is inverted is modulated in part by the unfolding and folding rate constants, which are accessible from the data. Global Bayesian analysis of the equilibrium and SIT NMR data provide values for the standard enthalpy, entropy, and heat capacity of unfolding, and show the latter to be unusually large. The CD results are compatible with the NMR findings. Global Bayesian analysis of the SIT NMR data yields the corresponding activation parameters for unfolding and folding. The results show that both reaction directions are activated processes. Activation for unfolding is entropy driven, enthalpy opposed. Activation for folding is strongly enthalpy opposed and somewhat entropy opposed, falsifying the idea that the barrier for folding is solely due to a purely entropic search for properly registered partners. The activation heat capacity is much larger for folding, so almost the entire overall change is due to the folding direction. This latter finding, if it applies to GCN4 leucine zippers, clears up an extant apparent disagreement between folding rate constants for GCN4 as determined by chevron analysis and NMR in differing temperature regimes.     

Antiparallel four-stranded coiled coil specified by a 3-3-1 hydrophobic heptad repeat

Coiled-coil sequences in proteins commonly share a seven-amino acid repeat with nonpolar side chains at the first (a) and fourth (d) positions. We investigate here the role of a 3-3-1 hydrophobic repeat containing nonpolar amino acids at the a, d, and g positions in determining the structures of coiled coils using mutants of the GCN4 leucine zipper dimerization domain. When three charged residues at the g positions in the parental sequence are replaced by nonpolar alanine or valine side chains, stable four-helix structures result. The X-ray crystal structures of the tetramers reveal antiparallel, four-stranded coiled coils in which the a, d, and g side chains interlock in a combination of knobs-into-knobs and knobs-into-holes packing. Interfacial interactions in a coiled coil can therefore be prescribed by hydrophobic-polar patterns beyond the canonical 3-4 heptad repeat. The results suggest that the conserved, charged residues at the g positions in the GCN4 leucine zipper can impart a negative design element to disfavor thermodynamically more stable, antiparallel tetramers.     

Stabilities of leucine zipper dimers estimated by an empirical free energy method.

The leucine zipper motif is a characteristic amino acid sequence found in dimeric DNA-binding proteins. Computer-generated models for leucine zippers were constructed as alpha-helical coiled dimers with leucine repeated every seventh residue. An empirical Gibbs free energy, delta G, function which incorporates hydrophobic force, electrostatic interactions, and conformational entropy loss as the major intermolecular interactions was used to estimate the delta G of dimer formation in fos, jun, and GCN4 zipper sequences. The calculations showed that complexes known to form stable homo- or heterodimers have favorable (negative) delta G, while other less stable complexes have unfavorable (positive) delta G. Leucines in position d of the coiled coil contribute large hydrophobic stabilization energies while residues in the a position contribute less to dimer stability. Hydrophobic contributions show little sequence specificity, however, and do not contribute significantly to homo/heterodimer preference. Charged residues in the e and g positions, on the other hand, determine homo/heterodimer specificity. In GCN4 homodimers, residues GLU el, Glu b2, Lys g2, and Lys e4 greatly contribute to dimer stability. The preferential stability of fos-jun heterodimer over the jun-jun and fos-fos homodimers is primarily due to the side chains Asp b1, Glu g1, Asp b2, Glu e2, Glu g2, Glu g3, and Lys a5 of the fos helix, and Arg c1, Lys g1, Lys b2, Lys e2, Arg e4, and Glu g4 of the jun helix.