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A Switch Between Two-, Three-, and Four-Stranded Coiled Coils in GCN4 Leucine Zipper Mutants

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A Switch Between Two-, Three-, and Four-Stranded Coiled Coils in GCN4 Leucine Zipper Mutants A Switch Between Two-, Three-, and Four-Stranded Coiled Coils in GCN4 Leucine Zipper Mutants Pehr B. Harbury; Tao Zhang; Peter S. Kim; Tom Alber Science, New Series, Vol. 262, No. 5138. (Nov. 26, 1993), pp. 1401-1407. Stable URL: http://links.jstor.org/sici?sici=0036-8075%2819931126%293%3A262%3A5138%3C1401%3AASBTTA%3E2.0.CO%3B2-H Science is currently published by American Association for the Advancement of Science. Your use of the JSTOR archive indicates your acceptance of JSTOR's Terms and Conditions of Use, available at http://www.jstor.org/about/terms.html. JSTOR's Terms and Conditions of Use provides, in part, that unless you have obtained prior permission, you may not download an entire issue of a journal or multiple copies of articles, and you may use content in the JSTOR archive only for your personal, non-commercial use. 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For more information regarding JSTOR, please contact [email protected]. http://www.jstor.org Sun Sep 9 23:07:30 2007 fit!@ RESEARCH ARTICLES surements, each peptide was >90 percent A Switch Between Two-, Three-, helical at 4"C, neutral pH, and a concen- tration of 150 p,M (15), and each exhibited a cooperative thermal unfolding transition and Four-Stranded Coiled Coils in (Table 1). The midpoint of the thermal transition (T,) for each variant exceeded GCN4 Leucine Zipper Mutants that of the parental GCN4-pl peptide. Equilibrium analytical ultracentrifuga- Pehr B. Harbury, Tao Zhang,* Peter S. Kim, Tom Alber* tion (16) indicated that the peptides fall into three molecular weight classes: the Coiled-coil sequences in proteins consist of heptad repeats containing two characteristic peptides p-IL, p-11, and p-LI sedimented as hydrophobic positions. The role of these buried hydrophobic residues in determining the dimeric, trimeric, and tetrameric species, structuresof coiled coils was investigatedby studying mutantsof the GCN4 leucinezipper. respectively (Table 1). The oligomerization When sets of buried residues were altered, two-, three-, and four-helix structures were states of these peptides (p-IL, p-11, and formed. The x-ray crystal structure of the tetramer revealeda parallel,four-stranded coiled p-LI) were independent of peptide concen- coil. In the tetramer conformation, the local packing geometry of the two hydrophobic tration from 20 to 200 p,M. A derivative of positions in the heptad repeat is reversed relative to that in the dimer. These studies each peptide that contained the added se- demonstrate that conserved, buried residues in the GCN4 leucine zipper direct dimer quence Cys-Gly-Gly at the NH2-terminus formation. In contrast to proposals that the pattern of hydrophobic and polar amino acids also was synthesized (12). The cysteine in a protein sequence is sufficientto determinethree-dimensional structure,the shapes of residue permits disulfide bond formation, buried side chains in coiled coils are essential determinants of the global fold. and the two glycine residues provide a flexible linker. Pairing peptide monomers with a covalent disulfide bond did not change the oligomerization state of p-IL Recent evidence has suggested that the The structural variety of the coiled-coil and p-LI. In contrast, disulfide-bonded p-I1 three-dimensional structure of a protein is family and the functional requirements of sedimented with the molecular mass of a determined largely by the pattern of hydro- leucine zipper sequences suggest that geo- hexamer, consistent with the assignment of phobic (H) and polar (P) residues in the metric properties of buried, apolar amino p-I1 as a trimer in the absence of a disulfide amino acid sequence and is independent of acids may influence the overall structure of linkage. the geometric properties of the amino acid coiled coils. To investigate this possibility The peptides p-VI, p-VL, p-LV, and side chains that make up the pattern (1-3). we altered the hydrophobic core of a leu- p-LL populated multiple oligomerization This simplifying hypothesis, however, fails cine zipper molecule in a concerted fashion states (Table 1). The sizes of the complexes to account for a group of proteins composed and characterized the structures of the de- formed by these variants were determined of interacting, amphipathic a helices, the rivatives. by gel filtration using the peptides p-IL, coiled-coil family. Coiled-coil proteins Hydrophobic core mutants form two-, p-11, p-LI, and GCN4-pl as size standards have a characteristic seven-residue repeat, three-, and four-stranded structures. We (17). The peptides p-VI and p-VL ex- (a.b.c.d.e.f.g),, with hydrophobic residues systematically mutagenized the hydrophobic hibited concentration-dependent retention at positions a and d and polar residues core of the dimeric leucine zipper peptide times between 5 and 50 p,M, and deriva- generally elsewhere. Despite this shared HP GCN4-pl (12). With rare exceptions, hy- pattern, coiled-coil sequences adopt dimer- drophobic residues occupy the a and d posi- ic (4, 5), trimeric (6-8), and anti-parallel tions of the GCN4-p1 sequence, and polar tetrameric (9) conformations. residues appear at b, c, e, f, and g, generating In addition, parallel, dimeric coiled coils the characteristic (H.P.P.H.P.P.P), pat- exhibit strong preferences for specific amino tern. Because supercoiled a helices haxe acids at the hydrophobic a and d positions approximately 3.5 residues per turn, the of the heptad repeat. A striking example is spacing of the a and d positions three and provided by the leucine zipper motif, which four residues apart places .the H residues on functions to dimerize bZIP transcription one side of the helix. In the GcN4-~1 factors. Unlike other coiled coils, leucine dimer, the hydrophobic faces of two helides zippers contain leucine at -80 percent of pack against each other in a parallel orien- all d positions (5, 10). Multiple substitution tation (12, 13). Thus, the five amino acids of these leucines with similarly-sized hydro- at position a and the four leucines at position phobic residues often interferes with dimer- d from each monomer of GCN4-p1 form the ization function (11). apolar interface of the dimer (Fig. 1). We simultaneously changed four a P. B. Harbury is in the Department of Biological residues (Va19,Asn'6,Va123,Va130)and four Fig. 1. Helical wheel projection of residues Chemistry and Molecular Pharmacology, Haward Medical School, Boston, MA 021 15 and the Howard d residues (Le~~,Leu'~,Leu'~,Leu~~)of Met2 to GIu~~of the GCN4-pi sequence. View Hughes Medical Institute, Whitehead Institute, Depart- GCN4-pl to leucine, valine, or isoleucine is from the NH,-terminus, and residues in the ment of Biology, Massachusetts Institute of Technolo- (Met2 at the first a position was not first two helical turns are boxed or circled. 9 02142. gy, Cambridge Center, Cambridge, MA T. changed, Fig. 1) (14). These peptides were Heptad positions are labeled a through g. In the Zhang and T. Alber were In the Department of Bio- mutant peptides described here, the residues chemist~.Unlversitv of Utah School of Medicine. Salt named bv a two-letter code, the first letter in the dashed box at position a were collectively Lake ~itjl, UT 841i2. P. S. Kim is in the ~oward indicating the residueat the'four a positions Hughes Medical Institute, Whitehead Institute, Depart- changed to I, V, or L, and, separately, the ment of Biology, Massachusetts Institute of Technolo- and the second letter indicating the residue residues in the dashed box at position d were gy,-. 9 Cambridge Center, Cambridge, MA 02142. at the four d positions. The sequences were changed to I, V, or L (41). For example, p-LI *Present address: Department of Molecular and Cell GCN4-~1, contains leucine at the four boxed a positions Biology, University of California, Berkeley, CA 94720 On the basis of circular dichroism mea- and isoleucine at the four boxed d positions. SCIENCE VOL. 262 26 NOVEMBER 1993 tives of p-VI, p-LV, and p-LL that con- Unlike two- and three-stranded coiled peptide monomers and the disulfide bonds tained an NH2-terminalCys-Gly-Gly disul- coils, all well-characterized examples of to exchange (Fig. 2B) (21). Only ho- fide linkage eluted as multiple species. four interacting a helices exist in an anti- modimers were observed at equilibrium. Helix orientation. The observation that parallel four-helix bundle conformation Assuming that the glycyl linkers allow the the p-IL peptide remains dimeric with an (Fig. 2A) (20). To determine the helix terminal cysteines to assort randomly, the NH2-terminal disulfide linkage (Table 1) orientation of the p-LI tetramer, variants of results indicate that the p-LI peptide assem- indicates that the helices are parallel. Con- the p-LI peptide with the sequence Cys- bles into a conformation containing four sequently, the conformation of p-IL likely Gly-Gly at the NH2-terminus (denoted parallel a helices (22). To investigate the resembles the structure of GCN4-pl. The p-LI-N) or with the sequence Gly-Gly-Cys basis for the switch between parallel tetra- helices of the trimeric peptide p-I1 also are at the COOH-terminus (denoted p-LI-C) mer, dimer, and trimer conformations, the parallel because a two-dimensional double were synthesized.
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