Surface salt bridges stabilize the GCN4 leucine zipper.

We present a study of the role of salt bridges in stabilizing a simplified tertiary structural motif, the coiled-coil. Changes in GCN4 sequence have been engineered that introduce trial patterns of single and multiple salt bridges at solvent exposed sites. At the same sites, a set of alanine mutants was generated to provide a reference for thermodynamic analysis of the salt bridges. Introduction of three alanines stabilizes the dimer by 1.1 kcal/mol relative to the wild-type. An arrangement corresponding to a complex type of salt bridge involving three groups stabilizes the dimer by 1.7 kcal/ mol, an apparent elevation of the melting temperature relative to wild type of about 22 degrees C. While identifying local from nonlocal contributions to protein stability is difficult, stabilizing interactions can be identified by use of cycles. Introduction of alanines for side chains of lower helix propensity and complex salt bridges both stabilize the coiled-coil, so that combining the two should yield melting temperatures substantially higher than the starting species, approaching those of thermophilic sequences.     

De novo simulations of the folding thermodynamics of the GCN4 leucine zipper.

Entropy Sampling Monte Carlo (ESMC) simulations were carried out to study the thermodynamics of the folding transition in the GCN4 leucine zipper (GCN4-lz) in the context of a reduced model. Using the calculated partition functions for the monomer and dimer, and taking into account the equilibrium between the monomer and dimer, the average helix content of the GCN4-lz was computed over a range of temperatures and chain concentrations. The predicted helix contents for the native and denatured states of GCN4-lz agree with the experimental values. Similar to experimental results, our helix content versus temperature curves show a small linear decline in helix content with an increase in temperature in the native region. This is followed by a sharp transition to the denatured state. van't Hoff analysis of the helix content versus temperature curves indicates that the folding transition can be described using a two-state model. This indicates that knowledge-based potentials can be used to describe the properties of the folded and unfolded states of proteins.     

Electrostatic contributions to the stability of the GCN4 leucine zipper structure

Ion pairs are ubiquitous in X-ray structures of coiled coils, and mutagenesis of charged residues can result in large stability losses. By contrast, pK_a values determined by NMR in solution often predict only small contributions to stability from charge interactions. To help reconcile these results we used triple-resonance NMR to determine pK_a values for all groups that ionize between pH 1 and 13 in the 33 residue leucine zipper fragment, GCN4p. In addition to the native state we also determined comprehensive pK_a values for two models of the GCN4p denatured state: the protein in 6 M urea, and unfolded peptide fragments of the protein in water. Only residues that form ion pairs in multiple X-ray structures of GCN4p gave large pK_a differences between the native and denatured states. Moreover, electrostatic contributions to stability were not equivalent for oppositely charged partners in ion pairs, suggesting that the interactions between a charge and its environment are as important as those within the ion pair. The pH dependence of protein stability calculated from NMR-derived pK_a values agreed with the stability profile measured from equilibrium urea-unfolding experiments as a function of pH. The stability profile was also reproduced with structure-based continuum electrostatic calculations, although contributions to stability were overestimated at the extremes of pH. We consider potential sources of errors in the calculations, and how pK_a predictions could be improved. Our results show that although hydrophobic packing and hydrogen bonding have dominant roles, electrostatic interactions also make significant contributions to the stability of the coiled coil.     

Oligomerization properties of GCN4 leucine zipper e and g position mutants.

Putative intersubunit electrostatic interactions between charged amino acids on the surfaces of the dimer interfaces of leucine zippers (g-e'' ion pairs) have been implicated as determinants of dimerization specificity. To evaluate the importance of these ionic interactions in determining the specificity of dimer formation, we constructed a pool of > 65,000 GCN4 leucine zipper mutants in which all the e and g positions are occupied by different combinations of alanine, glutamic acid, lysine, or threonine. The oligomerization properties of these mutants were evaluated based on the phenotypes of cells expressing lambda repressor-leucine zipper fusion proteins. About 90% of the mutants do not form stable homooligomers. Surprisingly, approximately 8% of the mutant sequences have phenotypes consistent with the formation of higher-order (> dimer) oligomers, which can be classified into three types based on sequence features. The oligomerization states of mutants from two of these types were determined by characterizing purified fusion proteins. The Type I mutant behaved as a tetramer under all tested conditions, whereas the Type III mutant formed a variety of higher-order oligomers, depending on the solution conditions. Stable homodimers comprise less than 3% of the pool; several g-e'' positions in these mutants could form attractive ion pairs. Putative repulsive ion pairs are not found among the homodimeric mutants. However, patterns of charged residues at the e and g positions do not seem to be sufficient to predict either homodimer or heterodimer formation among the mutants.     

Dissociation and unfolding of GCN4 leucine zipper in the presence of sodium dodecyl sulfate.

The dissociation and unfolding behavior of the GCN4 leucine zipper has been studied using SDS titration. Circular dichroism (CD) spectra showed that the alpha-helix content of the leucine zipper (20 microM) decreased during the sodium dodecyl sulfate (SDS) titration. However, the alpha-helix content of the leucine zipper still remained significant in the presence of 1 mM SDS, with little change detected when the SDS concentration further increased to 2 mM. The dimer dissociation of the leucine zipper is also a co-operative process during SDS titration; with no dimer remaining when SDS concentration reached 1 mM, as shown by electrophoresis and the the theta(222)/theta(208) ratio. Our results indicate that SDS efficiently induces leucine zipper dimer dissociation with the monomers still partially folded. The experimental results provide important evidence for the previous model that partial helix formation precedes dimerization in coiled coil folding.     

An intrahelical salt bridge within the trigger site stabilizes the GCN4 leucine zipper

We previously reported that a helical trigger segment within the GCN4 leucine zipper monomer is indispensable for the formation of its parallel two-stranded coiled coil. Here, we demonstrate that the intrinsic secondary structure of the trigger site is largely stabilized by an intrahelical salt bridge. Removal of this surface salt bridge by a single amino acid mutation induced only minor changes in the backbone structure of the GCN4 leucine zipper dimer as verified by nuclear magnetic resonance. The mutation, however, substantially destabilized the dimeric structure. These findings support the proposed hierarchic folding mechanism of the GCN4 coiled coil in which local helix formation within the trigger segment precedes dimerization.     

A calorimetric characterization of the salt dependence of the stability of the GCN4 leucine zipper.

The effects of different salts (LiCl, NaCl, ChoCl, KF, KCl, and KBr) on the structural stability of a 33-residue peptide corresponding to the leucine zipper region of GCN4 have been studied by high-sensitivity differential scanning calorimetry. These experiments have allowed an estimation of the salt dependence of the thermodynamic parameters that define the stability of the coiled coil. Independent of the nature of the salt, a destabilization of the coiled coil is always observed upon increasing salt concentration up to a maximum of approximately 0.5 M, depending on the specific cation or anion. At higher salt concentrations, this effect is reversed and a stabilization of the leucine zipper is observed. The effect of salt concentration is primarily entropic, judging from the lack of a significant salt dependence of the transition enthalpy. The salt dependence of the stability of the peptide is complex, suggesting the presence of specific salt effects at high salt concentrations in addition to the nonspecific electrostatic effects that are prevalent at lower salt concentrations. The data is consistent with the existence of specific interactions between anions and peptide with an affinity that follows a reverse size order (F- > Cl- > Br-). Under all conditions studied, the coiled coil undergoes reversible thermal unfolding that can be well represented by a reaction of the form N2<==>2U, indicating that the unfolding is a two-state process in which the helices are only stable when they are in the coiled coil conformation.     

Electrostatic interactions in the GCN4 leucine zipper: substantial contributions arise from intramolecular interactions enhanced on binding.

The GCN4 leucine zipper is a peptide homodimer that has been the subject of a number of experimental and theoretical investigations into the determinants of affinity and specificity. Here, we utilize this model system to investigate electrostatic effects in protein binding using continuum calculations. A particularly novel feature of the computations made here is that they provide an interaction-by-interaction breakdown of the electrostatic contributions to the free energy of docking that includes changes in the interaction of each functional group with solvent and changes in interactions between all pairs of functional groups on binding. The results show that (1) electrostatic effects disfavor binding by roughly 15 kcal/mol due to desolvation effects that are incompletely compensated in the bound state, (2) while no groups strongly stabilize binding, the groups that are most destabilizing are charged and polar side chains at the interface that have been implicated in determining binding specificity, and (3) attractive intramolecular interactions (e.g., backbone hydrogen bonds) that are enhanced on binding due to reduced solvent screening in the bound state contribute significantly to affinity and are likely to be a general effect in other complexes. A comparison is made between the results obtained in an electrostatic analysis carried out calculationally and simulated results corresponding to idealized data from a scanning mutagenesis experiment. It is shown that scanning experiments provide incomplete information on interactions and, if overinterpreted, tend to overestimate the energetic effect of individual side chains that make attractive interactions. Finally, a comparison is made between the results available from a continuum electrostatic model and from a simpler surface-area dependent solvation model. In this case, although the simpler model neglects certain interactions, on average it performs rather well.     

Factors influencing accuracy of computer-built models: a study based on leucine zipper GCN4 structure.

A three-dimensional model of the leucine zipper GCN4 built from its amino acid sequence had been reported previously by us. When the two alternative x-ray structures of the GCN4 dimer became available, the root mean square (r.m.s.) shifts between our model and the structures were determined as approximately 2.7 A on all atoms. These values are similar to the r.m.s. shift of 2.8 A between the two GCN4 structures in the different crystal forms (C2 and P2(1)2(1)2(1)). CONGEN conformational searches were run to better understand the conditions that may determine the preference of different conformers in different environments and to test the sensitivity of our current modeling techniques. With a judicious choice of CONGEN search parameters, the backbone r.m.s. deviation improved to 0.8 A and 2.5 A on all atoms. The side-chain conformations of Val and Leu at the helical interface were well reproduced (1.2 A r.m.s.), and the large side-chain misplacements occurred with only a small number of charged amino acids and a tyrosine. Inclusion of the crystal environment (C2 symmetry), as a passive background, into the side-chain conformational search further improved the accuracy of the model to an r.m.s. deviation of 2.1 A. Conformational searches carried out in the two different crystal environments and employing the AMBER protein/DNA forcefield, as implemented in CONGEN, gave the r.m.s. values of 2.2 A (for the C2 symmetry) and 2.5 A (for the P2(1)2(1)2(1) symmetry). In the C2 symmetry crystal, as much as 40% of the surface of each dimer was involved in crystal contacts with other dimers, and the charged residues on the surface often interacted with immobilized water molecules. Thus, occasional large r.m.s. deviations between the model and the x-ray side chains were due to specific conditions that did not occur in solution.     

Correlation between knowledge-based and detailed atomic potentials: application to the unfolding of the GCN4 leucine zipper.

The relationship between the unfolding pseudo free energies of reduced and detailed atomic models of the GCN4 leucine zipper is examined. Starting from the native crystal structure, a large number of conformations ranging from folded to unfolded were generated by all-atom molecular dynamics unfolding simulations in an aqueous environment at elevated temperatures. For the detailed atomic model, the pseudo free energies are obtained by combining the CHARMM all-atom potential with a solvation component from the generalized Born, surface accessibility, GB/SA, model. Reduced model energies were evaluated using a knowledge-based potential. Both energies are highly correlated. In addition, both show a good correlation with the root mean square deviation, RMSD, of the backbone from native. These results suggest that knowledge-based potentials are capable of describing at least some of the properties of the folded as well as the unfolded states of proteins, even though they are derived from a database of native protein structures. Since only conformations generated from an unfolding simulation are used, we cannot assess whether these potentials can discriminate the native conformation from the manifold of alternative, low-energy misfolded states. Nevertheless, these results also have significant implications for the development of a methodology for multiscale modeling of proteins that combines reduced and detailed atomic models.     

Ultracentrifuge and circular dichroism studies of folding equilibria in a retro GCN4-like leucine zipper

Equilibrium ultracentrifuge and circular dichroism (CD) studies of a retropeptide of a GCN4-like leucine zipper in neutral saline buffer are reported as functions of temperature. Ultracentrifuge results indicate the presence of three oligomeric species: monomer, dimer, and tetramer, in quantifiable amounts, and the data provide values for the standard DeltaG, DeltaH, and DeltaS for interconversion. CD at 222 nm displays the strong concentration dependence characteristic of dissociative unfolding, but also shows a helicity far below that of the parent propeptide. Remarkably enough, the CD at 222 nm shows an extremum in the region between 0 and 20 degrees C. At higher T, the usual cooperative unfolding is observed. Comparable data are presented for a mutant retropeptide, in which a single asparagine residue is restored to the characteristic heptad position it occupies in the propeptide. The mutant shows marked differences from its unmutated relative in both thermodynamic properties and CD, although the oligomeric ensemble also comprises monomers, dimers, and tetramers. The mutant is closer in helicity to the parent propeptide but is less stable. These findings do not support either of the extant views on retropeptides. The behavior seen is consistent neither with the view that retropeptides should have the same structure as propeptides nor with the view that they should have the same structure but opposite chirality. The simultaneous availability of oligomeric population data and CD allows the latter to be dissected into individual contributions from monomers, dimers, and tetramers. This dissection yields explanations for the observed extrema in curves of CD (222 nm) versus T and reveals that the dimer population in both retropeptides undergoes "cold denaturation."     

Trimerization specificity in HIV-1 gp41: analysis with a GCN4 leucine zipper model

The envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) consists of a complex of two noncovalently associated subunits, gp120 and gp41. Formation of gp120/gp41 oligomers is thought to be dependent on a 4-3 hydrophobic (heptad) repeat located in the amino-terminal region of the gp41 molecule. We have investigated the role of this heptad repeat in determining the oligomeric structure of gp41 by introducing its buried core residues into the first (a) and fourth (d) positions of the GCN4 leucine-zipper dimerization domain. The mutant peptides fold into trimeric, helical structures, as shown by circular dichroism and equilibrium sedimentation centrifugation. The 2.4 A resolution crystal structure of one such trimer reveals a parallel three-stranded, alpha-helical coiled coil. Thus, the buried core residues from the gp41 heptad repeat direct trimer formation. We suggest that the conserved amino-terminal heptad repeat within the gp41 ectodomain possesses trimerization specificity.     

Helix capping.

Helix-capping motifs are specific patterns of hydrogen bonding and hydrophobic interactions found at or near the ends of helices in both proteins and peptides. In an alpha-helix, the first four >N-H groups and last four >C=O groups necessarily lack intrahelical hydrogen bonds. Instead, such groups are often capped by alternative hydrogen bond partners. This review enlarges our earlier hypothesis (Presta LG, Rose GD. 1988. Helix signals in proteins. Science 240:1632-1641) to include hydrophobic capping. A hydrophobic interaction that straddles the helix terminus is always associated with hydrogen-bonded capping. From a global survey among proteins of known structure, seven distinct capping motifs are identified-three at the helix N-terminus and four at the C-terminus. The consensus sequence patterns of these seven motifs, together with results from simple molecular modeling, are used to formulate useful rules of thumb for helix termination. Finally, we examine the role of helix capping as a bridge linking the conformation of secondary structure to supersecondary structure.     

Numerical study of the entropy loss of dimerization and the folding thermodynamics of the GCN4 leucine zipper

A lattice-based model of a protein and the Monte Carlo simulation method are used to calculate the entropy loss of dimerization of the GCN4 leucine zipper. In the representation used, a protein is a sequence of interaction centers arranged on a cubic lattice, with effective interaction potentials that are both of physical and statistical nature. The Monte Carlo simulation method is then used to sample the partition functions of both the monomer and dimer forms as a function of temperature. A method is described to estimate the entropy loss upon dimerization, a quantity that enters the free energy difference between monomer and dimer, and the corresponding dimerization reaction constant. As expected, but contrary to previous numerical studies, we find that the entropy loss of dimerization is a strong function of energy (or temperature), except in the limit of large energies in which the motion of the two dimer chains becomes largely uncorrelated. At the monomer-dimer transition temperature we find that the entropy loss of dimerization is approximately five times smaller than the value that would result from ideal gas statistics, a result that is qualitatively consistent with a recent experimental determination of the entropy loss of dimerization of a synthetic peptide that also forms a two-stranded alpha-helical coiled coil.     

MALDI-TOF mass spectrometry characterization of recombinant hydrophobic mutants containing the GCN4 basic region/leucine zipper motif

We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to characterize hydrophobic, alanine-rich mutants of the basic region/leucine zipper (bZIP) protein GCN4. These bacterially expressed proteins were generated to probe how small, alpha-helical proteins bind specific DNA sites. Enzymatic digestion mapping combined with MALDI-TOF MS characterization of protein fragments allowed us to resolve mass discrepancies between the expected and observed molecular mass measurements. Changes in mass were attributed to posttranslational modifications (PTMs) by proteolytic cleavage of the initiating methionine residue, carbamylation at the amino terminus, oxidation of histidine side chains, and oxidative addition of beta-mercaptoethanol (BME) at the cysteine side chain. Proteins can undergo a wide variety of co-translational modifications and PTMs during growth, isolation, and purification. Such changes in mass can only be detected by a high-resolution technique such as MALDI, which in conjunction with enzymatic digestion mapping, becomes a powerful methodology for characterization of protein structure.     

Thermodynamics and kinetics of a folded-folded' transition at valine-9 of a GCN4-like leucine zipper

Spin inversion transfer (SIT) NMR experiments are reported probing the thermodynamics and kinetics of interconversion of two folded forms of a GCN4-like leucine zipper near room temperature. The peptide is 13Calpha-labeled at position V9(a) and results are compared with prior findings for position L13(e). The SIT data are interpreted via a Bayesian analysis, yielding local values of T1a, T1b, kab, kba, and Keq as functions of temperature for the transition FaV9 right arrow over left arrow FbV9 between locally folded dimeric forms. Equilibrium constants, determined from relative spin counts at spin equilibrium, agree well with the ratios kab/kba from the dynamic SIT experiments. Thermodynamic and kinetic parameters are similar for V9(a) and L13(e), but not the same, confirming that the molecular conformational population is not two-state. The energetic parameters determined for both sites are examined, yielding conclusions that apply to both and are robust to uncertainties in the preexponential factor (kT/h) of the Eyring equation. These conclusions are 1) the activation free energy is substantial, requiring a sparsely populated transition state; 2) the transition state's enthalpy far exceeds that of either Fa or Fb; 3) the transition state's entropy far exceeds that of Fa, but is comparable to that of Fb; 4) "Arrhenius kinetics" characterize the temperature dependence of both kab and kba, indicating that the temperatures of slow interconversion are not below that of the glass transition. Any postulated free energy surface for these coiled coils must satisfy these constraints.     

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.