Measurement of Interhelical Electrostatic Interactions in the GCN4 Leucine Zipper

Lumb, Kevin J.; Kim, Peter

doi:10.1126/science.7716550

Cited by 175 publications

(187 citation statements)

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“…Heterospecificity of helix pairing can be engineered into coiled coils by taking advantage of electrostatic repulsions in homomeric structures (termed ''negative design''), 18 electrostatic attractions in heteromeric structures, or both. [19][20][21] It is likely that charge repulsion plays a role in destabilizing the ecE and ecK homotetramers since, in both cases, the peptides are largely unfolded (see Fig.…”

Section: Resultsmentioning

confidence: 99%

Design of a heterotetrameric coiled coil

et al. 2009

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We have successfully designed a simple peptide sequence that forms highly stable coiled-coil heterotetramers. Our model system is based on the GCN4-pLI parallel coiled-coil tetramer, first described by Kim and coworkers (Harbury et al., Science 1993;262:1401-1407.We introduced glutamates at all of the e and c heptad positions of one sequence (ecE) and lysines at the same positions in a second sequence (ecK). Based on a modeling study, these sidechains are close enough in space to form structure-stabilizing salt bridges. We show that ecE and ecK are highly unstable by themselves but form very stable parallel helical tetramers when mixed, as judged by circular dichroism, analytical ultracentrifugation, and disulfide crosslinking studies. The origin of the difference in stabilities between the homomeric structures and the heteromeric structures comes from a combination of the relief of electrostatic repulsions with concomitant formation of electrostatic attractive interactions based on pH and NaCl screening experiments. We quantify the stability of the heterotetrameric coiled coil from a thermodynamic analysis and compare the finding to other similar coiled-coil systems.

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Section: Resultsmentioning

confidence: 99%

Design of a heterotetrameric coiled coil

et al. 2009

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“…According to the crystallographic structure, stabilizing interactions between oppositely charged ion pairs appear to be greater than destabilizing interactions between like charged residues, thus contributing to the overall stability of the folded dimer (O'Shea et al, 1989;Lumb et al, 1994). Recently, however, Lumb and Kim (1995) have observed that the charge-charge interaction between Lys 15 and Glu 2 0 might be actually destabilizing at the salt condition of their studies (150 mM NaCI), judging by the observed stabilization after replacing the Glu residue by Gln.The existence of stabilizing or destabilizing electrostatic effects can be evaluated by measuring the salt dependence of the stability of the coiled coil. In this paper, the dependence of the thermodynamic parameters on the concentration of different salts was investigated by high-sensitivity differential scanning calorimetry.…”

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confidence: 93%

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

1995

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Abstract:The effects of different salts (LiCI, NaCI, ChoC1, KF, KCI, 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 -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-> C1-> Br-). Under all conditions studied, the coiled coil undergoes reversible thermal unfolding that can be well represented by a reaction of the form N, * 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.Keywords: calorimetry; folding thermodynamics; leucine zipper; salt effectsThe structural stability of the leucine zipper region of GCN4 has been measured spectroscopically (O'Shea et al., 1989; Krylov et al., 1994) and by high-sensitivity differential scanning calorimetry (Thompson et al., 1993 play a crucial role in the high stability of the molecule and that electrostatic interactions may also play a role in the stability of the GCN4 leucine zipper (O'Shea et al., 1991;Thompson et al., 1993; Krylov et al., 1994). Several interhelical and intrahelical electrostatic interactions are revealed by the crystal structure of GCN4 (O'Shea et al., 1991). The distances between the pairs Lys 15 and Glu 20', Glu 22 and Lys 27', and Lys 27 and Glu 22' suggest the existence of interhelical electrostatic interactions. In addition, intrahelical interactions are also apparent between Lys 8 and Glu 11 and between Glu 22 and Arg 25. According to the crystallographic structure, stabilizing interactions between oppositely charged ion pairs appear to be greater than destabilizing interactions between like charged residues, thus contributing to the overall stability of the folded dimer (O'Shea et al., 1989;Lumb et al., 1994). Recently, however, Lumb and Kim (1995) have observed that the charge-charge interaction between Lys 15 and Glu 2 0 might be actually destabilizing at the salt condition of their studies (150 mM NaCI), judging by the observed stabilization after replacing the Glu residue by Gln.The existence of stabili...

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“…However, the contributions of ionic interactions between charged end groups remains controversial . NMR measurements of pK, shifts of glutamates involved in ion pairs in the GCN4 structure suggest that the net contributions of interchain Glu-Lys electrostatic interactions to coiled-coil stability are either insignificant or slightly destabilizing (Lumb & Kim, 1995). The relative and absolute contributions of attractive and repulsiveg-e' ionic interactions are also clearly dependent on solution conditions (Monera et al, 1994;Yu et al, 1996).…”

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confidence: 99%

Oligomerization properties of GCN4 leucine zipper e and g position mutants

et al. 1997

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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 A 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 I11 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.Keywords: dimerization specificity; leucine zippers; protein structure; recombinant fusion proteins; site-directed mutagenesis a-Helical coiled coils are involved in the assembly of a wide variety of proteins. A subclass of the coiled-coils known as leucine zippers are found as short dimerization motifs in many eukaryotic transcription factors. Leucine zipper sequences are characterized by leucine appearing in every seventh position (4 over four to five heptad repeats (abcdefg), (for a review, see Hurst, 1994). Leucine zippers fold into dimeric, parallel coiled-coils, where each heptad forms two a-helical turns. Because of their small size and simple structure, leucine zippers and other short a-helical coiled coils have been used extensively as a model system to study how amino acid sequences specify structure (e.g., see Hodges et al., 1988;

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Measurement of Interhelical Electrostatic Interactions in the GCN4 Leucine Zipper

Cited by 175 publications

References 46 publications

Design of a heterotetrameric coiled coil

Design of a heterotetrameric coiled coil

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

Oligomerization properties of GCN4 leucine zipper e and g position mutants

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