An Intrahelical Salt Bridge within the Trigger Site Stabilizes the GCN4 Leucine Zipper*

Richard A. KammererDagger §, Victor A. Jaravine, Sabine FrankDagger , Therese SchulthessDagger , Ruth LandwehrDagger , Ariel LustigDagger , Carlos García-Echeverría||, Andrei T. Alexandrescu, Jürgen EngelDagger , and Michel O. Steinmetz**DaggerDagger

From the Departments of Dagger  Biophysical Chemistry and  Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland and the || Oncology Research and ** Functional Genomics Area, Novartis Pharma AG, CH-4002 Basel, Switzerland

Received for publication, November 20, 2000, and in revised form, December 26, 2000




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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.




    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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The role of intrinsic secondary structure in promoting structure formation is one of the central questions in protein folding and a major subject of debate. Because of its simplicity and regularity, the coiled-coil structural motif has frequently been used as a model system for experimental studies of protein folding. One of the simplest and most widely characterized coiled coils is the parallel two-stranded 33-residue leucine zipper from the yeast transcriptional activator GCN4 (1). We recently postulated that a segment of the GCN4 leucine zipper, termed the "trigger sequence," drives coiled-coil formation by stabilizing an alpha -helical segment within the polypeptide monomer (2). This mechanism has been confirmed by two independent research groups (3, 4). Trigger sequences are conserved in many coiled coils and are required for dimerization of the GCN4 leucine zipper (2), the actin-bundling protein cortexillin I from Dictyostelium discoideum (5, 6), for specific trimer formation of the macrophage scavenger receptor (7), and the stability of keratin intermediate filaments (8). We demonstrated that trigger sequences represent autonomous helical folding units, which differ from arbitrarily chosen heptad repeats by their ability to mediate coiled-coil formation (2, 5). Because favorable side chain interactions are critical for monomer helix stability of the isolated GCN4 trigger sequence (2), the intrahelical surface salt bridge between Glu22 and Arg25 seen in crystal structures of GCN4 (1, 9, 10) potentially could affect the stability of the leucine zipper dimer. To test this hypothesis, we designed a GCN4 leucine zipper mutant, designated GCN4p-R25A, in which this salt bridge is missing. As controls, we generated two additional variants in which the other observed salt bridge between Lys8 and Glu11 outside of the trigger sequence (GCN4p-E11A) or both ionic interactions (GCN4p-E11A/R25A) were removed. For the purposes of this study, only Glu11 or/and Arg25 was selected for substitution because these residues at heptad c positions are not thought to be essential for maintaining the coiled-coil structure (11, 12). In contrast, Lys8 and Glu22 at heptad g positions were fixed because these residues contribute to the hydrophobic core of the leucine zipper (1). To exclude the possibility of a helix destabilizing effect due to a lower helix propensity of the mutant residue, alanine was used for substitution. Alanine was reported to have the highest helix propensity in several peptides (13, 14), at the f position in coiled coils (11) and at exposed helical sites in proteins (15, 16). The mutant proteins investigated in this study are shown in Fig. 1A.


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Plasmid Constructions, Protein Expression, and Peptide Synthesis-- Synthetic genes encoding the wild-type and mutant proteins were prepared with optimal codon usage for Escherichia coli (17) and ligated into the BamHI/EcoRI site of pPEP-T (2).

E. coli JM109(DE3) host strain (Promega) was used for expression. Production and purification of 6xHis-tagged fusion proteins by affinity chromatography on Ni2+-Sepharose (Novagen) and separation of recombinant GCN4 proteins from the carrier protein were performed as described in the manufacturer's instructions. Recombinant proteins were dialyzed against 10 mM sodium phosphate buffer (pH 7.0) containing 150 mM sodium chloride.

Peptides were assembled on an automated continuous flow synthesizer employing standard methodologies (18). The purity of the peptides was verified by reversed-phase analytical high pressure liquid chromatography, and the identity of the final products was verified by amino acid and mass spectral analyses. Protein and peptide concentrations were determined by tyrosine absorbance at 280 nm in 6 M GuHCl (19).

Biophysical Characterization-- Nuclear magnetic resonance (NMR) experiments were acquired on a 600-MHz Bruker DRX spectrometer. NMR assignments for GCN4p-wt and GCN4p-R25A were based on three-dimensional NOESY1-HSQC (mixing time, tau  = 100 ms), TOCSY-HSQC (tau  = 60 ms), HMQC-NOESY-HMQC (tau  = 150 ms ), and HNHA experiments (20, 21). The experiments were recorded on 2.4 mM samples of GCN4p-R25A, and 1.4 mM samples of GCN4p-wt (monomer concentrations) in 90% H2O, 10% D2O containing 150 mM sodium chloride, pH 6.2 ± 0.2, at a temperature of 35 °C. 3JHNHalpha coupling constants were calculated from the ratio of diagonal to cross-peak intensities in three-dimensional HNHA spectra using the approach described by Vuister and Bax (20). Hydrogen-deuterium exchange in the GCN4p-R25A mutant was monitored using a 1.4 mM solution (monomer concentration), freshly dissolved in D2O containing 150 mM NaCl, at a pD of 6.1 and a temperature of 5 °C.

Analytical ultracentrifugation and CD spectroscopy were performed as described previously (2, 5).


    RESULTS AND DISCUSSION
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The secondary structures, thermal stabilities, and subunit stoichiometries of the recombinant GCN4 polypeptide chain fragments (Fig. 1A) were analyzed by CD spectroscopy (Fig. 1, B and C) and analytical ultracentrifugation (Table I). Consistent with the previously reported observations (2, 22), the wild-type GCN4 leucine zipper (GCN4p-wt) formed a dimer with a CD spectrum typical of alpha -helix structure and with a sigmoidal thermal unfolding profile. GCN4p-E11A also folded into a dimer with an alpha -helix content and a thermal stability comparable with those of the wild-type protein. In contrast, substitution of Arg25 in the coiled-coil trigger site resulted in a loss of helix content at 5 °C and an 18 °C decrease in Tm relative to the wild-type protein (Table I). With the exception of a slight difference in Tm, the results for the GCN4p-E11A/R25A double mutant were similar to those for the GCN4p-R25A single mutant.



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Fig. 1.   The surface salt bridge between Glu22 and Arg25 stabilizes the GCN4 leucine zipper dimer. A, sequences of the recombinant GCN4 proteins used in this study. The numbers refer to the amino acid positions within GCN4p-1 (1). Heptad repeats are represented as blocks of seven amino acid residues, and heptad positions are indicated by lowercase letters. The 3,4-hydrophobic repeat with mostly hydrophobic residues at heptad positions a and d is indicated in bold. Residues that form intrahelical salt bridges in crystal structures of GCN4 (1, 9, 10) are indicated by arrows. The trigger site that drives coiled-coil formation (2) is shaded in gray. B and C, CD spectra recorded at 5 °C (B) and thermal unfolding profiles (C) of GCN4p-wt (open circles), GCN4p-E11A (closed circles), GCN4p-R25A (open triangles), and GCN4p-E11A/R25A (closed triangles). Thermal stability of the peptides was monitored by the change of the CD signal at 222 nm. Polypeptide chain concentrations were 75 µM in 10 mM sodium phosphate buffer (pH 7.0) containing 150 mM sodium chloride.


                              
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Table I
Mean molar residue ellipticities, melting temperatures, and molecular masses of the wild-type and mutant GCN4 leucine zipper fragments and peptides
All fragments were analyzed in 10 mM sodium phosphate buffer (pH 7.0) containing 150 mM sodium chloride, except GCN4p16-31 and GCN4p16-31(R25A), which were analyzed in 1 mM sodium phosphate buffer (pH 7.4). The corresponding amino acid sequences of the GCN4 fragments and the GCN4-derived peptides are shown in Figs. 1 and 4, respectively. nd, not determined.

These findings demonstrate that the GCN4 leucine zipper coiled coil is sensitive to the mutation in the trigger site. Both GCN4-pR25A and GCN4p-E11A/R25A show sigmoidal thermal unfolding profiles characteristic of a cooperative two-state coiled-coil to random-coil transition. To further investigate the structural consequences of the R25A mutation in the coiled-coil trigger site, 15N-labeled GCN4p-wt and GCN4-pR25A proteins were examined by NMR. Chemical shift indices for Halpha resonances and 3JHNHalpha coupling constants measured from a three-dimensional HNHA experiment are summarized for the wild-type and R25A mutant proteins in Fig. 2. Residues Lys3 through Lys28 of the GCN4p-R25A mutant show negative Halpha chemical shift indices (Fig. 2A) and 3JHNHalpha coupling constants below 6 Hz (Fig. 2B), consistent with alpha -helical structure (21, 23). The four C-terminal residues give Halpha and 3JHNHalpha values suggestive of conformational averaging. The same conformational averaging is evident for the four C-terminal residues in the wild-type protein (Fig. 2, C and D). The differences in Halpha chemical shifts and 3JHNHalpha coupling constants between GCN4p-wt and GCN4p-R25A are summarized in Fig. 2, C and D. In GCN4p-R25A, residues Val23 through Lys27 flanking the R25A mutation, show small down-field Halpha chemical shifts and increased 3JHNHalpha coupling constants relative to the wild-type protein. The changes in these parameters suggest that the dynamic fraying of the C terminus in the wild-type might extend toward the site of the R25A substitution in the mutant. Nevertheless, the differences between the wild-type and mutant are small, and the 3JHNHalpha and Halpha chemical shifts for residues Val23 through Lys27 in GCN4p-R25A remain within the limits for alpha -helical structure (23). On dissolving the GCN4p-R25A mutant in D2O at pD 6.1, only two amide protons (Asn16, and Glu32) are protected from hydrogen-deuterium exchange after 12 min at 5 °C, and all amide protons are exchanged after 40 min. In contrast, the 35-residue construct LZ, which has an identical sequence to residues Met2 through Glu32 of GCN4p-wt, shows much stronger hydrogen exchange protection (24). At pD 6.2, the amide protons of residues Met2 through Glu32 persist for 12 h and the seven slowest exchanging amide protons from residues Asn21 through Lys27 persist for at least 2 days (24).



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Fig. 2.   Summary of Halpha chemical shift indices and 3JHNHalpha coupling constants. A, differences in Halpha chemical shifts between GCN4p-R25A and random-coil values (23). B, 3JHNHalpha coupling constants for GCN4p-R25A. C, differences in Halpha chemical shifts between the mutant and the wild-type protein. D, differences in 3JHNHalpha coupling constants between GCN4p-R25A and the GCN4p-wt. The position of the R25A substitution is indicated.

Taken together, the NMR results suggest that the R25A substitution induces only small differences in the alpha -helix backbone structure of GCN4p-R25A compared with GCN4p-wt. Based on the present data we cannot exclude changes in side chain packing as a result of the mutation. The extremely fast hydrogen exchange observed for GCN4p-R25A suggests that whereas the alpha -helix backbone structure is conserved in the R25A mutant, its stability is much lower than in the wild-type protein. This conclusion is also supported by proteolytic susceptibility experiments of the wild-type and mutant peptides. Whereas the GCN4p-wt was resistant to trypsin cleavage at molar ratio of 10,000 to 1, GCN4p-R25A was completely digested after 2 h under the same conditions (data not shown). These findings indicate that the entire GCN4 leucine zipper dimer is destabilized by the R25A mutation in a cooperative manner.

To assess whether monomer alpha -helix stability within the GCN4 trigger sequence is favored by the observed intrahelical salt bridge between Glu22 and Arg25 in the leucine zipper dimer (1, 9, 10), we produced the two synthetic 16-residue peptides shown in Fig. 3. The GCN4p16-31 peptide corresponds to the wild-type trigger sequence (2), whereas GCN4p16-31(R25A) corresponds to the R25A mutation in which the putative salt bridge between Glu22 and Arg25 is missing. As previously reported (2), GCN4p16-31 is monomeric with significant helicity (Fig. 3B; Table I). In contrast, the peptide with the R25A substitution showed a 40% loss of helical structure according to [Theta ]222 (Fig. 3B; Table I). The shift of the two minima at 205 and 222 nm in the CD spectrum also indicate a substantial shift in the equilibrium from helix to random coil.



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Fig. 3.   The salt bridge between Glu22 and Arg25 stabilizes the autonomous helical folding unit that drives assembly of the GCN4 leucine zipper dimer. A, sequences of the synthetic GCN4 peptides. Residues that form the salt bridge are indicated by an arrow. B, CD spectra of GCN4p16-31 (closed circles) and GCN4p16-31(R25A) (open circles) recorded at 5 °C at peptide concentrations of 50 µM in 1 mM sodium phosphate buffer (pH 7.4). C, effect of ionic strength on the helicity of the wild-type and mutant peptide at 5 °C and at a peptide concentration of 50 µM in 1 mM sodium phosphate buffer (pH 7.4).

Helix formation of the GCN4p16-31 and GCN4p16-31(R25A) peptides was further monitored as a function of salt concentration at physiological pH. As shown in Fig. 3C, an increase in sodium chloride concentration strongly disfavors alpha -helical structure formation in the wild-type peptide. This suggests that net electrostatic interactions are favorable and helix-stabilizing. A decrease in -[Theta ]222 with increasing salt concentration is also observed for GCN4p16-31(R25A). The difference in helicity of the two CD signals, however, indicates that a major component of the interaction between Glu22 and Arg25 cannot be screened by external salts. These findings suggest that the interaction observed at high salt is not a simple electrostatic interaction and that hydrogen bonding between the side chains of Glu22 and Arg25 is a prominent determinant of the stability of alpha -helix monomer structure in the trigger site. This conclusion is consistent with a recent study by Smith and Scholtz (25) who report that in designed helical peptides the energetics of the interaction between the fully charged ion pairs can be diminished by salt, but the interaction is not completely screened at high ionic strength.

We have previously reported that a helical trigger segment within the GCN4 leucine zipper monomer is indispensable for coiled-coil formation (2). Our findings demonstrated that this trigger sequence represents an autonomous helical folding unit. Our derived model proposed that productive coiled-coil formation of the GCN4 leucine zipper requires a contact of two trigger sequences at some stage in the folding pathway. Helical trigger sites are ideal for the initiation of coiled-coil formation because they ideally position the hydrophobic core residues and possible interhelical salt bridges for the recognition process. Moreover, such a mechanism would align the two chains in parallel register. Further folding of the coiled coil then proceeds in a zipper-like fashion from the C terminus and finally results in a stable leucine zipper dimer. This conclusion was in contrast to the GCN4 leucine zipper folding mechanism proposed by Sosnick and coworkers (26, 27) who reported a heterogeneous transition state that contains only little if any secondary structure. However, using diffusion collision theory, the folding data of Sosnick et al. (26) have been reinterpreted by Myers and Oas (3). These authors concluded that partial helix formation within the C-terminal part of the GCN4 leucine zipper monomer precedes dimerization, which is in line with our predicted folding model (2). Moreover, Zitzewitz et al. (4) subsequently carried out a detailed mutational and kinetic analysis of the GCN4 coiled coil. They found that the two C-terminal heptads are indeed the likely source of the nucleating helices and that helix-helix recognition between preformed elements of secondary structure plays an important role in the folding reaction.

Together, these considerations are consistent with the hierarchic view of protein folding. Hierarchic protein folding is defined as a process in which folding begins with structures that are local in sequence and marginal in stability (28). These local structures interact to finally fold into the native conformation. Accordingly, the GCN4 leucine zipper monomer can be considered as an intrinsically disordered protein with a marginally stable helical trigger site within its C terminus. Specific recognition of two trigger sites induces folding of the unstructured N terminus to finally yield the fully alpha -helical coiled-coil dimer. Consistent with the model of hierarchic protein folding, the synthetic peptide GCN4p16-31 encompassing the coiled-coil trigger site is ~50% helical in the absence of tertiary and quaternary interactions (Fig. 3). The intrahelical surface salt bridge between Glu22 and Arg25 plays a key role in stabilizing the helical structure of the coiled-coil trigger peptide. As a consequence, introduction of the monomer helix destabilizing mutation R25A, which does not affect the contact sites between helices, resulted in destabilization of the GCN4 leucine zipper dimer without causing significant changes in backbone structure (Fig. 2).

These findings are consistent with recent evidence. By introducing interactive combinations of intrahelical salt bridges into the GCN4 leucine zipper, Spek et al. (29) observed an increase in thermal stability of 22 °C of the mutant protein relative to the wild-type. As these mutations affect residues of the trigger sequence, a stabilizing effect on the monomer helix can be expected. Zhu et al. (30) reported that the effect on GCN4 dimer stability of an IIe to Asn mutation at the a position is greater at the N terminus of the peptide and decreases almost 2-fold as the substitution was moved toward the C-terminal heptads. This result supports the concept that monomer helix stability within the trigger site is more critical than the precise nature of the core residues. Finally, a hierarchical folding mechanism for coiled coils has recently also been suggested by Yu et al. (31).

A plausible explanation for the destabilizing effect of the R25A mutation is an increase in the conformational entropy of the monomer chains, which results in a reduction of the favorable entropic contribution associated with GCN4 coiled-coil formation (32). In contrast, only minor differences in enthalpies are anticipated for the two proteins due to disruption of the salt bridge, because the contact sites between helices are identical for the wild-type and mutant. Therefore, a key function of the salt bridge between Glu22 and Arg25 is to limit the possible number of monomer chain conformations and to provide an ideal scaffold for the interaction of the critical core residues. This mechanism ideally allows the two-stranded coiled coil to fold in parallel register. Consistent with the concept of hierarchic protein folding, helical structure formation of the trigger site is determined largely by local sequence information.


    ACKNOWLEDGEMENTS

We are indebted to Dr. J. van Oostrum for support.


    FOOTNOTES

* This work was supported by grants from the Swiss National Science Foundation (to J. E. and A. T. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: School of Biological Sciences, University of Manchester, Manchester M13 9PT, UK. To whom correspondence may be addressed: richard.kammerer@man.ac.uk.

Dagger Dagger Present address: Paul Scherrer Inst., Life Sciences, CH-5232 Villigen PSI, Switzerland. To whom correspondence may also be addressed: michel.steinmetz{at}psi.ch.

Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M010492200


    ABBREVIATIONS

The abbreviations used are: NOESY, nuclear Overhauser enhancement spectroscopy; HMQC, heteronuclear multi quantum coherence; HSQC, heteronuclear single quantum coherence; TOCSY, total correlation spectroscopy; wt, wild type; HNHA, (amide proton)-(nitrogen)-(alpha -proton) correlation.


    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES


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