A seven-helix coiled coilJie Liu*, Qi Zheng*, Yiqun Deng*, Chao-Sheng Cheng*, Neville R. Kallenbach†, and Min Lu*‡

*Department of Biochemistry, Weill Medical College of Cornell University, New York, NY 10021; and †Department of Chemistry,New York University, New York, NY 10003

Edited by Janet M. Thornton, European Bioinformatics Institute, Cambridge, United Kingdom, and approved August 28, 2006(received for review June 12, 2006)

Coiled-coil proteins contain a characteristic seven-residue se-quence repeat whose positions are designated a to g. The inter-acting surface between �-helices in a classical coiled coil is formedby interspersing nonpolar side chains at the a and d positions withhydrophilic residues at the flanking e and g positions. To explorehow the chemical nature of these core amino acids dictates theoverall coiled-coil architecture, we replaced all eight e and gresidues in the GCN4 leucine zipper with nonpolar alanine sidechains. Surprisingly, the alanine-containing mutant forms a stable�-helical heptamer in aqueous solution. The 1.25-Å resolutioncrystal structure of the heptamer reveals a parallel seven-strandedcoiled coil enclosing a large tubular channel with an unusualheptad register shift between adjacent staggered helices. Theoverall geometry comprises two interleaved hydrophobic helicalscrews of interacting cross-sectional a and d layers that have notbeen seen before. Moreover, asparagines at the a positions play anessential role in heptamer formation by participating in a set ofburied interhelix hydrogen bonds. These results demonstrate thatheptad repeats containing four hydrophobic positions can directassembly of complex, higher-order coiled-coil structures with richdiversity for close packing of �-helices.

protein design � protein structure � helix–helix interfaces � buried polarinteractions � cavity

Helix–helix interactions are ubiquitous in the native structureof proteins and in associations among proteins, including

supramolecular assemblies and transmembrane receptors thatmediate cellular signaling and transport. Coiled coils afford aunique model system for elucidating principles of molecularrecognition between helices (1–4). The conformation of coiledcoils is prescribed by a characteristic 7-aa repeat, the 3–4 heptadrepeat denoted as a-b-c-d-e-f-g (5). Positions a and d are typicallyoccupied by hydrophobic amino acids such as Leu, Ile, Val, andAla, whereas residues at positions e and g are frequently polaror charged (5–8). Crick’s (9) classical analysis proposed that thenonpolar a and d side chains associate by means of complemen-tary ‘‘knobs-into-holes’’ packing to form supercoiled �-helicalribbons. Beyond the role of hydrophobic side chains at the corea and d positions, coiled coils can use intra- and interhelicalelectrostatic interactions to tune their stability, especially thosebetween the flanking e and g positions of neighboring chains(10–18). Despite their seeming regularity in sequence andpacking patterns, coiled coils exhibit remarkable diversity in thenumber and arrangement of the associating helices: two- tofive-stranded structures have been identified with parallel orantiparallel helix orientation (2). The structural parameters ofdimers have been described by Crick (9), and general rulesgoverning the close packing of �-helices in hexamers anddodecamers have been deduced (19, 20).

Analysis of coiled coils provides insight into the generalproblem of protein folding by simplifying the geometrical andtopological context. Although the hydrophobic cores of globularproteins can be repacked quite freely, formation of an apolarcore in coiled coils is spatially constrained, allowing dissection oftheir structure and folding in unprecedented detail (21, 22). Thecanonical knobs-into-holes packing of coiled coils confers ex-quisite sensitivity to the stereochemical properties of the core a

and d residues. Interior packing of the side chains at the a andd positions, in fact, has been shown to dominate the globalarchitecture of coiled coils (23). Polar side chains at the a andd positions also can destabilize coiled-coil structure yet imposea high degree of conformational selectivity (4, 24). Moreover,ionic interactions between the e and g residues have been shownto influence the specificity of coiled-coil assembly (10–18). Forexample, repulsive or attractive interactions between the e andg side chains can control the extent of homo- versus heterodimer-ization in model coiled coils (10). This current level of under-standing of the determinants of coiled-coil structure has allowedformulation of robust rules for coiled-coil design, embodied inalgorithms that are available for this purpose. Thus, it seemsremarkable that fundamental elements of coiled-coil formationstill remain incompletely understood. Here, we report a strikingexample resulting from an unanticipated role of nonpolar sidechains at the normally charged e and g positions.

Recent experiments show that the presence of apolar aminoacids at either the e or g position of the heptad repeat can specifyformation of antiparallel four-helix structures (see refs. 17 and18). In these tetramers, the buried a, d, e and a, d, g side chainsinterlock in nonclassical combinations of knobs-against-knobsand knobs-into-holes packing to form the hydrophobic core. Thelac repressor type of core packing, for example, is of the a, d, eclass, with the helices offset by 0.25 heptad (25, 26); on the otherhand, in the severe acute respiratory syndrome coronavirus S2protein, the packing involves the a, d, g side chains with the helixregister shifted by 0.5 heptad (27). Evidently, patterning ofhydrophobic and polar residues beyond the canonical 3–4 coiled-coil sequence can lead to distinctive three-dimensional struc-tures with characteristic supercoil shapes and exceptional sta-bility. We have previously investigated the basis of a, d, g corepacking by characterizing variants of the GCN4 leucine zipperthat adopt antiparallel tetrameric structures in response tohydrophobic substitutions at the g positions (17). Here, we reportthe engineering of a GCN4 leucine-zipper variant containingexclusively alanine residues at the e and g positions (Fig. 1).Surprisingly this mutant peptide folds into a hitherto unreportedseven-stranded coiled coil consisting of two interleaved parallelhelical screws in which the helices are staggered in phase with theheptad repeat. This heptamer highlights the need to extendcurrent rules for coiled-coil folding and assembly to includenonpolar side chains at the e and g positions. In addition, itprovides a soluble model for analyzing intrahelical interactions

Author contributions: J.L. and M.L. designed research; J.L., Q.Z., Y.D., and C.-S.C. performedresearch; J.L., Q.Z., Y.D., C.-S.C., N.R.K., and M.L. analyzed data; and N.R.K. and M.L. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

Abbreviation: ABS, acetate-buffered saline.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org (PDB ID code 2HY6).

‡To whom correspondence should be addressed at: Department of Biochemistry, WeillMedical College of Cornell University, 1300 York Avenue, New York, NY 10021. E-mail:mlu@med.cornell.edu.

© 2006 by The National Academy of Sciences of the USA

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in more complex protein interfaces and makes available a uniquenanoscale model cavity to explore by using different solutes.

Results and DiscussionDesign of GCN4-pAA. We chose the dimeric GCN4 leucine zipperas our engineering scaffold because it represents the prototypicaland best-studied model with which to decipher the structuraldeterminants of coiled-coil assembly and geometry (23). Thehydrophobic interface between the two �-helical chains of theleucine zipper is formed by interlocking of residues at positionsa, d, e, and g (28). Interhelical ionic interactions betweencharged side chains at the e and g positions also can contributeto dimerization specificity (28). Our previous studies havedemonstrated that replacing g residues with nonpolar aminoacids in the recombinant leucine-zipper peptide GCN4-pR canswitch both stoichiometry and helix orientation to produce astable antiparallel tetramer structure (17). This finding suggeststhat the selection of hydrophobic residues at both the e and gpositions of the 3–4 heptad repeat might offer another mecha-nism of mediating association of �-helices. To investigate thispossibility, we engineered a GCN4-pR variant called GCN4-pAA with alanine substitutions at four e and four g positions(Fig. 1). Alanine was selected for its minimal apolar side chainand high helix-formation propensity (29). Moreover, becausealanine has relatively low hydrophobicity, we surmised that bulkyapolar side chains at the a and d positions of the parentalsequence might still act as the dominant factor in the foldingreaction of GCN4-pAA.

Solution Properties of GCN4-pAA. The GCN4-pAA peptide is onlymarginally soluble in aqueous solutions in the pH range of 6.5–8.5(�10 �M) but attains solubility of �1 mM in acetate-bufferedsaline (ABS) (50 mM sodium acetate, pH 5.2�150 mM NaCl),which might be attributed to the high hydrophobic side-chaincontent and high isoelectric point (pI � 8.21) of the engineeredpeptide. CD measurements at 50 �M peptide concentration in ABSshow that GCN4-pAA is �90% helical at 20°C (Fig. 2A) anddisplays a cooperative thermal unfolding transition with a melting

temperature of �95°C (Fig. 2B). Sedimentation equilibrium ex-periments indicate that GCN4-pAA forms a clean heptamer andexhibits no systematic dependence of molecular mass on peptideconcentration between 30 and 300 �M (Fig. 2C). Thus, alanineresidues at the e and g positions of the dimeric leucine zipper serveto direct the formation of a stable, well ordered seven-helix bundlein solution.

Structure of the Heptamer. To investigate the basis for the switchbetween dimer and heptamer conformations, the x-ray crystalstructure of the GCN4-pAA peptide was determined at 1.25-Åresolution (Table 1). The GCN4-pAA heptamer consists ofseven parallel �-helical peptide monomers wrapped in a gradualleft-handed superhelix with several unique features (Fig. 3). The

Fig. 1. Helical wheel projection of residues Met-1 to Arg-34 of the GCN4-pAAsequence. The view is from the N terminus. Heptad repeat positions arelabeled a through g. GCN4-pAA differs from the recombinant dimeric leucine-zipper peptide GCN4-pR by alanine substitutions at four e and four g positions(bold). The sequences of GCN4-pR and GCN4-pAA (with the eight mutated eand g positions set in italics) are as follows: GCN4-pR, MK VKQLEDK VEELLSKNYHLENE VARLKKL VGER; GCN4-pAA, MK VKQLADA VEELASA NYHLANAVARLAKA VGER. The GCN4-pR sequence contains an additional Met-Lys-Valand no Arg-Met at its N terminus but is otherwise identical to GCN4-p1.

Fig. 2. The GCN4-pAA peptide forms a seven-stranded helical bundle. (A) CDspectrum at 20°C in ABS (pH 5.2) and 50 �M peptide concentration. (B)Thermal melt monitored by CD at 222 nm. (C) Representative sedimentationequilibrium data of a 100 �M sample at 20°C and 18,000 rpm in ABS (pH 5.2).The data fit closely to a heptameric complex. (Upper) The deviation in the datafrom the linear fit for a heptameric model is plotted.

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superhelix includes �30 residues (3–32) from each chain (thetwo N- and C-terminal-most residues are not well defined inthe electron density maps). This seven-helix bundle has astraight supercoil axis and forms a cylinder that is �48 Å inlength and �31 Å in diameter. The individual helices in theheptamer can be superimposed on each other with an rmsd forC� atoms of 0.14–0.40 Å.

Serendipitously, the seven parallel helices in the GCN4-pAAstructure are offset from each other by one amino acid residue,causing a heptad shift in register between the A and G helices(Fig. 3 A and B). Consequently, residues at positions a and dmake unique side-to-side contacts with side chains of neighbor-ing helices throughout the core of the heptamer, leading to theformation of two distinctive screws of interacting cross-sectionallayers (Fig. 3A). One of these helical screws is formed by the 21valine and 7 asparagine side chains at the a positions (Fig. 3D;the Val-3 side chains at the N terminus lack core packing), andthe second is formed by the 28 leucine side chains at the dpositions (Fig. 3E). All of the a and d side chains except one(Leu-27 of the C helix) adopt the most preferred rotamerconformations in �-helices (30). Alanine residues at positions eand g along the neighboring helices flank the a and d side chainsto efficiently sequester the hydrophobic interface from thesolvent (Fig. 3B).

Hydrophobic Channel. The GCN4-pAA heptamer contains a 7-Å-wide central channel that is formed by spaces in the middle ofeach a and d layer and lined by hydrophobic side chains; thechannel spans the superhelix and is open at both ends (Fig. 3 Band C). Within this channel, the experimentally phased mapshows a strong string of electron density on the coiled-coilheptamer axis between residues 6 and 22, separate from theprotein portion of the map. For the purpose of crystallographicmodel building and refinement, we have modeled this string as

five hexanediol molecules (a precipitant that is present in thecrystallization buffer). Given the fact that large internal cavitiesin natural proteins are unstable in the aqueous milieu, theheptameric channel formed from the parallel GCN4-pAA �-helices may offer a valuable model for investigating how cavitiesalter protein folding and stability.

Helix Interactions. Although the superhelical radius differs sig-nificantly in the parallel GCN4-pLI tetramer (with leucine ateach a position and isoleucine at each d) (23) and GCN4-pAAheptamer, these coiled-coil structures can still maintain similarpitch and residues per supercoil turn (Table 2). Moreover, thesurface area of each helix is less buried in the heptamer (1,453Å2 per helix) than in the tetramer (1,640 Å2 per helix) becauseof the smaller size of the e and g alanine side chains and the lowerburial values of the a and d side chains in GCN4-pAA. Relativeto the side chains of isolated helices, residues at the a, d, e, andg positions of the heptamer are substantially buried (�75%);residues at the b and c positions are partly buried (�25%),whereas the f position remains completely exposed. The distancebetween the axes of neighboring helices (except for the A and Gchains) is 7.6 Å, whereas the distance between the axis of helixA and the axis of helix D is 17.1 Å, and the distance between theaxis of helix A and the axis of helix E is 17.7 Å (Fig. 3F). The Aand G helices are 0.9 Å farther apart; this gap is due to a heptadphase shift.

A Buried Hydrogen-Bonding Network. To achieve efficient side-chain packing, Asn-17 residues at an a position form a networkof hydrogen bonds within the hydrophobic channel of theGCN4-pAA heptamer (Fig. 3D). Substitution of Asn-17 withvaline, serine, or threonine causes the resulting molecule tounfold in solution, whereas introducing glutamine into thisposition creates a variant with hydrodynamic properties identicalto those of the Asn-17-containing peptide. In addition, our initialcrystallographic analysis indicates that the Gln-17 side chainmakes good interhelical packing interactions and is well accom-modated without significantly altering the seven-helix structure.Thus, interhelix hydrogen bonds between the Asn side chains areessential for the folding and assembly of GCN4-pAA and likelyact to nucleate and maintain the heptad phase register of theseven parallel helices in the heptamer. Our results reinforce thenotion that buried polar interactions of appropriate geometrycan impart structural specificity in coiled coils even if they do soat the expense of thermodynamic stability (23).

Core Packing. The heptamer interface shows nonclassical meshingof interfacial side chains in knobs-into-holes packing (Fig. 4 Eand J). Looking down the superhelical axis from the N terminus,knobs formed by a residues of one helix fit into holes formed bythe a and g side chains and two adjacent d side chains of thecounterclockwise-related monomer. Similarly, knobs at d posi-tions pack into holes formed by the d and e side chains and twoadjacent a side chains of the clockwise-related monomer. Bycontrast, the alanine side chains at e positions pack into trian-gular spaces between the c, d, and g residues of the neighboringhelix; those at g positions pack into triangular spaces between thea, b, and e residues. Thus, the a, b, c, d, e, and g residues of theheptad repeat segregate into two geometrically distinct helix–helix interfaces so as to create two continuous hydrophobicseams between the seven �-helical chains. As far as we know,these interleaved parallel helical screws have not been seenbefore in natural proteins.

The seven parallel �-helical peptide monomers in the hep-tamer structure have crossing angles of �10° for both neighbor-ing helices and relative to the superhelical axis of the heptamer.Thus, the heptamer conforms to a coiled-coil model proposed byCrick (9) to explain �-helix packing in �-keratin: an interhelix

Table 1. Crystallographic data and refinement statistics

Data collectionResolution, Å 47.5–1.25No. of unique reflections 51,370Redundancy 2.0Completeness, % 92.2 (91.4)Rmerge,* % 4.6I��(I) 12.2 (2.3)Space group P1Unit-cell parameters a � 31.5 Å, b � 35.3 Å, c �

49.0 Å� � 86.5°, � � 104.3°, � �

99.9°No. of molecules, AU 7Solvent content, % 36.7

RefinementResolution, Å 47.5–1.25No. of reflections 51,370No. of protein atoms 1,612No. of water molecules 179No. of hexane-1,6-diols 8Rcryst�Rfree,† % 18.1�20.9rmsd bond lengths, Å 0.009rmsd bond angles, ° 1.1Average B factors, Å2 23.3rmsd B values, Å2 1.7

Values in parentheses refer to the highest-resolution shell, 1.27–1.25 Å. AU,asymmetric units.*Rmerge � � �I � �I���� I, where I is the integrated intensity of a given reflection.†Rcryst � � �Fo � Fc��� Fobs, Rfree � Rcryst calculated by using 5% of the reflectiondata chosen randomly and omitted from the start of refinement.

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packing angle near 20°C with knobs-into-holes packing of sidechains between adjacent helices. In addition, the C�–C� andC�–C� vectors at the a and d layers of the heptamer are 51.4° and218.6°, respectively (Fig. 4 E and J). The local packing geometryin the hydrophobic interface of the heptamer thus follows ageneral trend observed in the structures of parallel coiled coilscontaining two to five helices (31). This trend shows how residuesat the b, c, e, and g positions of the heptad repeat becomeincreasingly buried as the number of strands grows (Fig. 4). Theresults suggest that the amino acid sequence at these corepositions will play a crucial role in determining the higher-orderstructures of coiled coils.

Protein Structure. Accurate prediction of side-chain packing andits influence on tertiary conformation is an important objectivein modern protein structure and design efforts. De novo proteindesign and engineering of coiled-coil interfaces have been usedto test and enhance our growing ability to predict foldedstructures (23, 32). Previous studies have analyzed the potentialof van der Waals interactions beyond the a and d side chains togenerate higher-order coiled-coil structures (20, 33–36). Thedimeric GCN4 leucine zipper represents the simplest protein–protein interface with which to study the detailed relationshipbetween local side-chain interactions and global three-dimensional architecture. In the heptamer conformation, the

Fig. 3. Crystal structure of GCN4-pAA. (A) Lateral view of the heptamer (residues 5–31). Red van der Waals surfaces identify residues at the a positions, andgreen surfaces identify residues at the d positions. (B) Axial view of the heptamer. The view is from the N terminus looking down the superhelical axis. Red spheresidentify residues at the a positions, green spheres identify residues at the d positions, yellow spheres identify residues at the e positions, and pink spheres identifyresidues at the g positions. (C) Molecular surface representation of the heptamer. The solvent-accessible surface is colored according to the local electrostaticpotential, ranging from 32 V in dark blue (most positive) to �30 V in deep red (most negative). (D) Cross-section of the superhelix in the Asn-17(a) layer. The1.25-Å 2Fo � Fc electron density map at 1.5� contour is shown with the refined molecular model. Hydrogen bonds are denoted by pink dotted lines. (E) Omitmap showing the Leu-20(d) layer in a 2Fo � Fc difference Fourier synthesis (contoured at 1.5�). (F) Helical wheel projection of the heptamer showing interhelicalpacking interactions. The view is from the N terminus. Heptad positions are labeled a through g.

Table 2. Structural parameters of GCN4 leucine-zipper variants

Parameter GCN4-pIL GCN4-pA GCN4-pV GCN4-pAA

SuperhelixSupercoil radius, r0, Å 7.1 6.9 7.4 11.4Residues per supercoil turn, n 133.1 110.9 107.3 141.3Supercoil pitch, P, Å 198 161 156 202Buried surface area, Å2 per helix 1,640 1,368 1,368 1,453

�-Helix�-Helix radius, r1, Å 2.25 2.29 2.29 2.29Residues per �-helical turn, n 3.58 3.61 3.63 3.61Rise per residue, h, Å 1.53 1.51 1.51 1.50Angular frequency, �1, ° per residue �100.8 �99.7 �99.3 �99.7

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core a and d residues from the parent dimer sequence form twocontinuous helical screws because of local packing of thesenonpolar side chains. Conserved asparagines at a positions directparallel helix orientation by forming a network of buried hydro-gen bonds. The lateral displacement of adjacent helices in asevenfold screw symmetry observed in the heptamer structure isunique among known coiled-coil structures.

Although the mechanism by which the geometric details ofpacking among side chains at the a, d, e, and g positions conferspecificity toward the native state cannot be predicted at present,the availability of the seven-helix coiled coil provides a solublemodel for detailed exploration of individual side-chain–side-chain interactions within a large-scale structure. The propertiesof the nanoscaled central cavity in this structure merit furtherinvestigation as well: How do various solutes influence the cavityand the stability of the heptamer? We anticipate that additionalfundamental rules prescribing helix-to-helix packing in coiledcoils remain to be discovered and will provide insight into thesubtle relationship between amino acid sequence and the higherlevels of structure adopted by this diverse class of proteins.

Materials and MethodsProtein Expression and Purification. The recombinant peptideGCN4-pAA and its variants were expressed in Escherichia coliBL21(DE3)�pLysS by using a modified pET3a vector (Novagen,San Diego, CA). Substitutions were introduced into thepGCN4-pA plasmid (17) by using the method of Kunkel et al.(37) and were verified by DNA sequencing. Cells were lysed byglacial acetic acid and centrifuged to separate the solublefraction from inclusion bodies. The soluble fraction containingpeptide was subsequently dialyzed into 5% acetic acid overnightat 4°C. The GCN4-pAA(V) construct was appended to theTrpLE leader sequence (38) and cloned into the pET24a vector(Novagen). GCN4-pAA(V) was purified from inclusion bodiesand cleaved from the TrpLE leader sequence with cyanogenbromide as described in ref. 39. Final purification of all peptideswas performed by reverse-phase HPLC on a C18 preparativecolumn using a water-acetonitrile gradient in the presence of0.1% trif luoroacetic acid. Peptide identities were confirmed byelectrospray mass spectrometry (Voyager Elite; PerSeptive Bio-systems, Framingham, MA). Peptide concentrations were de-termined by using the method of Edelhoch (40).

Biophysical Analysis. CD spectra were measured on a 62A�DS CDspectrometer (Aviv, Lakewood, NJ) at 4°C in ABS (50 mM

sodium acetate, pH 5.2�150 mM NaCl) and 50 �M peptide.Thermal stability was assessed by monitoring [�]222 as a functionof temperature under the same conditions. A [�]222 value of�35,000° cm2 dmol�1 was taken to correspond to 100% helix(41). Sedimentation equilibrium measurements were carried outon an XL-A analytical ultracentrifuge (Beckman Coulter, Ful-lerton, CA) equipped with an An-60 Ti rotor at 20°C. Peptidesamples were dialyzed overnight against ABS (pH 5.2), loadedat initial concentrations of 30, 100, and 300 �M, and analyzed atrotor speeds of 18,000 and 21,000 rpm. Data sets were fitted toa single-species model. Random residuals were observed in allcases.

Crystallization and Structure Determination. GCN4-pAA was crys-tallized at room temperature by using the hanging drop vapor-diffusion method by equilibrating against reservoir buffer (0.1 Msodium citrate, pH 5.4�0.1 M KH2PO4�1.4 M hexanediol), asolution containing 1 �l of 6 mg�ml�1 peptide in water, and 1 �lof reservoir buffer. Crystals belonged to space group P1 (a �31.5 Å, b � 35.3 Å, c � 49.0 Å, � � 86.5°, � � 104.3°, and � �99.9°) and contained seven monomers in the asymmetric unit.The crystals were harvested in 0.1 M sodium citrate, pH 5.4�0.1M KH2PO4�1.7 M hexanediol�15% glycerol and frozen in liquidnitrogen. Diffraction data were collected on beamline X4C atthe National Synchrotron Light Source (Brookhaven, NY).Reflection intensities were integrated and scaled with DENZOand SCALEPACK (42). Initial phases were determined bymolecular replacement with Phaser (43), using the structure ofthe GCN4-pA monomer (17) as a search model. SevenGCN4-pA molecules were oriented and placed in the asymmet-ric unit with a Z score of 7.8 and final refined log-likelihood gainof 431.7, corresponding to the seven GCN4-pAA molecules. Thismodel and the data set for GCN4-pAA were directly fed toArp�Warp (44), which provided a largely complete asymmetricunit of the seven chains and allowed �93% of the final model tobe interpreted. The resulting experimental electron density mapwas of excellent quality and showed the location of most of theside chains. Crystallographic refinement of the GCN4-pAAstructure was carried out by using Refmac (45). Density inter-pretation and manual model building were performed with O(46). The final model (Rcryst � 18.1% and Rfree � 20.9% for theresolution range 47.5–1.25 Å) consists of residues 3–33 (mono-mer A), 3–33 (monomer B), 3–33 (monomer C), 3–34 (monomerD), 3–33 (monomer E), 1–32 (monomer F), and 3–32 (monomerG) in the asymmetric unit, eight hexane-1,6-diols, and 179 water

Fig. 4. Comparison of helix–helix interfaces in parallel coiled coils. (A) Parallel packing at position a (180°) in the GCN4-p1 dimer (23). The view is from the Nterminus looking down the superhelical axis. The C�–C� bond of each knob (blue) is oriented parallel to the C�–C� vector (red) at the base of the recipient holeon the neighboring helix. The C�–C� bonds of the e and g residues are shown in pink and cyan, respectively. (B) Acute packing at position a (120°) in the GCN4-pIItrimer (52). (C) Perpendicular packing at position a (90°) in the GCN4-pLI tetramer (23). (D) Packing at position a (72°) in the Trp-14 pentamer (31). (E) Packingat position a (51.4°) in the GCN4-pAA heptamer. (F) Perpendicular packing at position d (90°) in the dimer. (G) Acute packing at position d (150°) in the trimer.(H) Parallel packing at position d (180°) in the tetramer. (I) Packing at position d (198°) in the pentamer. (J) Packing at position d (218.6°) in the heptamer.

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molecules. Bond lengths and bond angles of the model havermsds from ideality of 0.009 Å and 1.1°, respectively. All proteinresidues are in the most favored regions of the Ramachandranplot.

Structure Analysis. Coiled-coil parameters were calculated byusing TWISTER (47). The rmsds were calculated with LSQKABin the CCP4i program suite (48). Buried surface areas werecalculated from the differences of the accessible side-chainsurface areas of the seven-helix complex and of the individualhelical monomers by using CNS 1.0 (49). Residues 3–32 of

GCN4-pAA were used in the calculations. To calculate an omitmap, target residues were removed, and the remaining atomswere shaken randomly by 0.3 Å to minimize model bias and thenrefined. Figures were generated by using SETOR (50), Insight II(Accelrys, San Diego, CA), and GRASP (51).

We thank John Schwanof at the National Synchrotron Light Source(beamline X4C) for support, Sergei Strelkov for help with the super-helical parameter calculations, and David Eliezer for comments on themanuscript. This work was supported by National Institutes of HealthGrant AI42382, the Irma T. Hirschl Trust, and a Fellowship from theNational Science Council of Taiwan (to C.-S.C.).

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