A Seven-Helix Coiled Coil

Jie 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- and d residues. Interior packing of the side chains at the a and quence repeat whose positions are designated a to g. The inter- d positions, in fact, has been shown to dominate the global acting surface between ␣-helices in a classical coiled coil is formed architecture of coiled coils (23). Polar side chains at the a and by interspersing nonpolar side chains at the a and d positions with d positions also can destabilize coiled-coil structure yet impose hydrophilic residues at the ﬂanking e and g positions. To explore a high degree of conformational selectivity (4, 24). Moreover, how the chemical nature of these core amino acids dictates the ionic interactions between the e and g residues have been shown overall coiled-coil architecture, we replaced all eight e and g to influence the specificity of coiled-coil assembly (10–18). For residues in the GCN4 leucine zipper with nonpolar alanine side example, repulsive or attractive interactions between the e and chains. Surprisingly, the alanine-containing mutant forms a stable g side chains can control the extent of homo- versus heterodimer- ␣-helical heptamer in aqueous solution. The 1.25-Å resolution ization in model coiled coils (10). This current level of under- crystal structure of the heptamer reveals a parallel seven-stranded standing of the determinants of coiled-coil structure has allowed coiled coil enclosing a large tubular channel with an unusual formulation of robust rules for coiled-coil design, embodied in heptad register shift between adjacent staggered helices. The algorithms that are available for this purpose. Thus, it seems overall geometry comprises two interleaved hydrophobic helical remarkable that fundamental elements of coiled-coil formation screws of interacting cross-sectional a and d layers that have not still remain incompletely understood. Here, we report a striking been seen before. Moreover, asparagines at the a positions play an example resulting from an unanticipated role of nonpolar side essential role in heptamer formation by participating in a set of chains at the normally charged e and g positions. buried interhelix hydrogen bonds. These results demonstrate that Recent experiments show that the presence of apolar amino BIOPHYSICS heptad repeats containing four hydrophobic positions can direct acids at either the e or g position of the heptad repeat can specify assembly of complex, higher-order coiled-coil structures with rich formation of antiparallel four-helix structures (see refs. 17 and diversity for close packing of ␣-helices. 18). In these tetramers, the buried a, d, e and a, d, g side chains interlock in nonclassical combinations of knobs-against-knobs protein design ͉ protein structure ͉ helix–helix interfaces ͉ buried polar and knobs-into-holes packing to form the hydrophobic core. The interactions ͉ cavity lac repressor type of core packing, for example, is of the a, d, e class, with the helices offset by 0.25 heptad (25, 26); on the other elix–helix interactions are ubiquitous in the native structure hand, in the severe acute respiratory syndrome coronavirus S2 Hof proteins and in associations among proteins, including protein, the packing involves the a, d, g side chains with the helix supramolecular assemblies and transmembrane receptors that register shifted by 0.5 heptad (27). Evidently, patterning of mediate cellular signaling and transport. Coiled coils afford a hydrophobic and polar residues beyond the canonical 3–4 coiled- unique model system for elucidating principles of molecular coil sequence can lead to distinctive three-dimensional struc- recognition between helices (1–4). The conformation of coiled tures with characteristic supercoil shapes and exceptional sta- coils is prescribed by a characteristic 7-aa repeat, the 3–4 heptad bility. We have previously investigated the basis of a, d, g core a b c d e f g a d repeat denoted as ------(5). Positions and are typically packing by characterizing variants of the GCN4 leucine zipper occupied by hydrophobic amino acids such as Leu, Ile, Val, and that adopt antiparallel tetrameric structures in response to Ala, whereas residues at positions e and g are frequently polar hydrophobic substitutions at the g positions (17). Here, we report or charged (5–8). Crick’s (9) classical analysis proposed that the the engineering of a GCN4 leucine-zipper variant containing nonpolar a and d side chains associate by means of complemen- exclusively alanine residues at the e and g positions (Fig. 1). tary ‘‘knobs-into-holes’’ packing to form supercoiled ␣-helical Surprisingly this mutant peptide folds into a hitherto unreported ribbons. Beyond the role of hydrophobic side chains at the core a and d positions, coiled coils can use intra- and interhelical seven-stranded coiled coil consisting of two interleaved parallel electrostatic interactions to tune their stability, especially those helical screws in which the helices are staggered in phase with the between the flanking e and g positions of neighboring chains heptad repeat. This heptamer highlights the need to extend (10–18). Despite their seeming regularity in sequence and current rules for coiled-coil folding and assembly to include packing patterns, coiled coils exhibit remarkable diversity in the nonpolar side chains at the e and g positions. In addition, it number and arrangement of the associating helices: two- to provides a soluble model for analyzing intrahelical interactions five-stranded structures have been identified with parallel or antiparallel helix orientation (2). The structural parameters of Author contributions: J.L. and M.L. designed research; J.L., Q.Z., Y.D., and C.-S.C. performed dimers have been described by Crick (9), and general rules research; J.L., Q.Z., Y.D., C.-S.C., N.R.K., and M.L. analyzed data; and N.R.K. and M.L. wrote governing the close packing of ␣-helices in hexamers and the paper. dodecamers have been deduced (19, 20). The authors declare no conﬂict of interest. Analysis of coiled coils provides insight into the general This article is a PNAS direct submission. problem of protein folding by simplifying the geometrical and Abbreviation: ABS, acetate-buffered saline. topological context. Although the hydrophobic cores of globular Data deposition: The atomic coordinates and structure factors have been deposited in the proteins can be repacked quite freely, formation of an apolar Protein Data Bank, www.pdb.org (PDB ID code 2HY6). core in coiled coils is spatially constrained, allowing dissection of ‡To whom correspondence should be addressed at: Department of Biochemistry, Weill their structure and folding in unprecedented detail (21, 22). The Medical College of Cornell University, 1300 York Avenue, New York, NY 10021. E-mail: canonical knobs-into-holes packing of coiled coils confers ex- [email protected]. quisite sensitivity to the stereochemical properties of the core a © 2006 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604871103 PNAS ͉ October 17, 2006 ͉ vol. 103 ͉ no. 42 ͉ 15457–15462 Downloaded by guest on September 25, 2021 Fig. 1. Helical wheel projection of residues Met-1 to Arg-34 of the GCN4-pAA sequence. The view is from the N terminus. Heptad repeat positions are labeled 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 e and g positions set in italics) are as follows: GCN4-pR, MK VKQLEDK VEELLSK NYHLENE VARLKKL VGER; GCN4-pAA, MK VKQLADA VEELASA NYHLANA VARLAKA VGER. The GCN4-pR sequence contains an additional Met-Lys-Val and no Arg-Met at its N terminus but is otherwise identical to GCN4-p1.

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

Results and Discussion Design of GCN4-pAA. We chose the dimeric GCN4 leucine zipper as our engineering scaffold because it represents the prototypical and best-studied model with which to decipher the structural determinants of coiled-coil assembly and geometry (23). The hydrophobic interface between the two ␣-helical chains of the leucine zipper is formed by interlocking of residues at positions a, d, e, and g (28). Interhelical ionic interactions between charged side chains at the e and g positions also can contribute to dimerization specificity (28). Our previous studies have demonstrated that replacing g residues with nonpolar amino acids in the recombinant leucine-zipper peptide GCN4-pR can switch both stoichiometry and helix orientation to produce a stable antiparallel tetramer structure (17). This finding suggests that the selection of hydrophobic residues at both the e and g Fig. 2. The GCN4-pAA peptide forms a seven-stranded helical bundle. (A)CD positions of the 3–4 heptad repeat might offer another mecha- spectrum at 20°C in ABS (pH 5.2) and 50 ␮M peptide concentration. (B) nism of mediating association of ␣-helices. To investigate this Thermal melt monitored by CD at 222 nm. (C) Representative sedimentation equilibrium data of a 100 ␮M sample at 20°C and 18,000 rpm in ABS (pH 5.2). possibility, we engineered a GCN4-pR variant called GCN4- The data ﬁt closely to a heptameric complex. (Upper) The deviation in the data pAA with alanine substitutions at four e and four g positions from the linear ﬁt for a heptameric model is plotted. (Fig. 1). Alanine was selected for its minimal apolar side chain and high helix-formation propensity (29). Moreover, because alanine has relatively low hydrophobicity, we surmised that bulky temperature of Ϸ95°C (Fig. 2B). Sedimentation equilibrium ex- apolar side chains at the a and d positions of the parental periments indicate that GCN4-pAA forms a clean heptamer and sequence might still act as the dominant factor in the folding exhibits no systematic dependence of molecular mass on peptide reaction of GCN4-pAA. concentration between 30 and 300 ␮M (Fig. 2C). Thus, alanine residues at the e and g positions of the dimeric leucine zipper serve Solution Properties of GCN4-pAA. The GCN4-pAA peptide is only to direct the formation of a stable, well ordered seven-helix bundle marginally soluble in aqueous solutions in the pH range of 6.5–8.5 in solution. (Ͻ10 ␮M) but attains solubility of Ͼ1 mM in acetate-buffered saline (ABS) (50 mM sodium acetate, pH 5.2͞150 mM NaCl), Structure of the Heptamer. To investigate the basis for the switch which might be attributed to the high hydrophobic side-chain between dimer and heptamer conformations, the x-ray crystal content and high isoelectric point (pI ϭ 8.21) of the engineered structure of the GCN4-pAA peptide was determined at 1.25-Å peptide. CD measurements at 50 ␮M peptide concentration in ABS resolution (Table 1). The GCN4-pAA heptamer consists of show that GCN4-pAA is Ϸ90% helical at 20°C (Fig. 2A) and seven parallel ␣-helical peptide monomers wrapped in a gradual displays a cooperative thermal unfolding transition with a melting left-handed superhelix with several unique features (Fig. 3). The

15458 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604871103 Liu et al. Downloaded by guest on September 25, 2021 Table 1. Crystallographic data and refinement statistics five hexanediol molecules (a precipitant that is present in the crystallization buffer). Given the fact that large internal cavities Data collection in natural proteins are unstable in the aqueous milieu, the Resolution, Å 47.5–1.25 heptameric channel formed from the parallel GCN4-pAA ␣- No. of unique reflections 51,370 helices may offer a valuable model for investigating how cavities Redundancy 2.0 alter protein folding and stability. Completeness, % 92.2 (91.4) Rmerge,* % 4.6 Helix Interactions. Although the superhelical radius differs sig- I͞␴(I) 12.2 (2.3) nificantly in the parallel GCN4-pLI tetramer (with leucine at Space group P1 each a position and isoleucine at each d) (23) and GCN4-pAA Unit-cell parameters a ϭ 31.5 Å, b ϭ 35.3 Å, c ϭ heptamer, these coiled-coil structures can still maintain similar 49.0 Å pitch and residues per supercoil turn (Table 2). Moreover, the ␣ ϭ 86.5°, ␤ ϭ 104.3°, ␥ ϭ surface area of each helix is less buried in the heptamer (1,453 99.9° Å2 per helix) than in the tetramer (1,640 Å2 per helix) because No. of molecules, AU 7 of the smaller size of the e and g alanine side chains and the lower Solvent content, % 36.7 burial values of the a and d side chains in GCN4-pAA. Relative Refinement to the side chains of isolated helices, residues at the a, d, e, and Resolution, Å 47.5–1.25 g positions of the heptamer are substantially buried (Ͼ75%); No. of reflections 51,370 residues at the b and c positions are partly buried (Ϸ25%), No. of protein atoms 1,612 whereas the f position remains completely exposed. The distance No. of water molecules 179 between the axes of neighboring helices (except for the A and G No. of hexane-1,6-diols 8 chains) is 7.6 Å, whereas the distance between the axis of helix ͞ † ͞ Rcryst Rfree, % 18.1 20.9 A and the axis of helix D is 17.1 Å, and the distance between the rmsd bond lengths, Å 0.009 axis of helix A and the axis of helix E is 17.7 Å (Fig. 3F). The A rmsd bond angles, ° 1.1 and G helices are 0.9 Å farther apart; this gap is due to a heptad Average B factors, Å2 23.3 phase shift. rmsd B values, Å2 1.7 BIOPHYSICS A Buried Hydrogen-Bonding Network. Values in parentheses refer to the highest-resolution shell, 1.27–1.25 Å. AU, To achieve efficient side- asymmetric units. chain packing, Asn-17 residues at an a position form a network *Rmerge ϭ͚͉I Ϫ͗I͉͚͘͞ I, where I is the integrated intensity of a given reflection. of hydrogen bonds within the hydrophobic channel of the † Rcryst ϭ͚͉Fo Ϫ Fc͉͚͞ Fobs, Rfree ϭ Rcryst calculated by using 5% of the reflection GCN4-pAA heptamer (Fig. 3D). Substitution of Asn-17 with data chosen randomly and omitted from the start of refinement. valine, serine, or threonine causes the resulting molecule to unfold in solution, whereas introducing glutamine into this position creates a variant with hydrodynamic properties identical superhelix includes Ϸ30 residues (3–32) from each chain (the to those of the Asn-17-containing peptide. In addition, our initial two N- and C-terminal-most residues are not well defined in crystallographic analysis indicates that the Gln-17 side chain the electron density maps). This seven-helix bundle has a makes good interhelical packing interactions and is well accom- straight supercoil axis and forms a cylinder that is Ϸ48Åin modated without significantly altering the seven-helix structure. length and Ϸ31 Å in diameter. The individual helices in the Thus, interhelix hydrogen bonds between the Asn side chains are heptamer can be superimposed on each other with an rmsd for essential for the folding and assembly of GCN4-pAA and likely ␣ C atoms of 0.14–0.40 Å. act to nucleate and maintain the heptad phase register of the Serendipitously, the seven parallel helices in the GCN4-pAA seven parallel helices in the heptamer. Our results reinforce the structure are offset from each other by one amino acid residue, notion that buried polar interactions of appropriate geometry causing a heptad shift in register between the A and G helices can impart structural specificity in coiled coils even if they do so (Fig. 3 A and B). Consequently, residues at positions a and d at the expense of thermodynamic stability (23). make unique side-to-side contacts with side chains of neighboring helices throughout the core of the heptamer, leading to the Core Packing. The heptamer interface shows nonclassical meshing formation of two distinctive screws of interacting cross-sectional of interfacial side chains in knobs-into-holes packing (Fig. 4 E layers (Fig. 3A). One of these helical screws is formed by the 21 and J). Looking down the superhelical axis from the N terminus, valine and 7 asparagine side chains at the a positions (Fig. 3D; knobs formed by a residues of one helix fit into holes formed by the Val-3 side chains at the N terminus lack core packing), and the a and g side chains and two adjacent d side chains of the the second is formed by the 28 leucine side chains at the d counterclockwise-related monomer. Similarly, knobs at d posi- positions (Fig. 3E). All of the a and d side chains except one tions pack into holes formed by the d and e side chains and two (Leu-27 of the C helix) adopt the most preferred rotamer adjacent a side chains of the clockwise-related monomer. By conformations in ␣-helices (30). Alanine residues at positions e contrast, the alanine side chains at e positions pack into trian- and g along the neighboring helices flank the a and d side chains gular spaces between the c, d, and g residues of the neighboring to efficiently sequester the hydrophobic interface from the helix; those at g positions pack into triangular spaces between the solvent (Fig. 3B). a, b, and e residues. Thus, the a, b, c, d, e, and g residues of the heptad repeat segregate into two geometrically distinct helix– Hydrophobic Channel. The GCN4-pAA heptamer contains a 7-Å- helix interfaces so as to create two continuous hydrophobic wide central channel that is formed by spaces in the middle of seams between the seven ␣-helical chains. As far as we know, each a and d layer and lined by hydrophobic side chains; the these interleaved parallel helical screws have not been seen channel spans the superhelix and is open at both ends (Fig. 3 B before in natural proteins. and C). Within this channel, the experimentally phased map The seven parallel ␣-helical peptide monomers in the hep- shows a strong string of electron density on the coiled-coil tamer structure have crossing angles of Ϸ10° for both neighbor- heptamer axis between residues 6 and 22, separate from the ing helices and relative to the superhelical axis of the heptamer. protein portion of the map. For the purpose of crystallographic Thus, the heptamer conforms to a coiled-coil model proposed by model building and refinement, we have modeled this string as Crick (9) to explain ␣-helix packing in ␣-keratin: an interhelix

Liu et al. PNAS ͉ October 17, 2006 ͉ vol. 103 ͉ no. 42 ͉ 15459 Downloaded by guest on September 25, 2021 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, and green 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 spheres identify residues at the a positions, green spheres identify residues at the d positions, yellow spheres identify residues at the e positions, and pink spheres identify residues at the g positions. (C) Molecular surface representation of the heptamer. The solvent-accessible surface is colored according to the local electrostatic potential, ranging from ϩ32 V in dark blue (most positive) to Ϫ30 V in deep red (most negative). (D) Cross-section of the superhelix in the Asn-17(a) layer. The 1.25-Å 2Fo Ϫ Fc electron density map at 1.5␴ contour is shown with the reﬁned molecular model. Hydrogen bonds are denoted by pink dotted lines. (E) Omit map showing the Leu-20(d) layer in a 2Fo Ϫ Fc difference Fourier synthesis (contoured at 1.5␴). (F) Helical wheel projection of the heptamer showing interhelical packing interactions. The view is from the N terminus. Heptad positions are labeled a through g.

packing angle near 20°C with knobs-into-holes packing of side Protein Structure. Accurate prediction of side-chain packing and chains between adjacent helices. In addition, the C␣–C␣ and its influence on tertiary conformation is an important objective C␣–C␤ vectors at the a and d layers of the heptamer are 51.4° and in modern protein structure and design efforts. De novo protein 218.6°, respectively (Fig. 4 E and J). The local packing geometry design and engineering of coiled-coil interfaces have been used in the hydrophobic interface of the heptamer thus follows a to test and enhance our growing ability to predict folded general trend observed in the structures of parallel coiled coils structures (23, 32). Previous studies have analyzed the potential containing two to five helices (31). This trend shows how residues of van der Waals interactions beyond the a and d side chains to at the b, c, e, and g positions of the heptad repeat become generate higher-order coiled-coil structures (20, 33–36). The increasingly buried as the number of strands grows (Fig. 4). The dimeric GCN4 leucine zipper represents the simplest protein– results suggest that the amino acid sequence at these core protein interface with which to study the detailed relationship positions will play a crucial role in determining the higher-order between local side-chain interactions and global three- structures of coiled coils. dimensional architecture. In the heptamer conformation, the

Table 2. Structural parameters of GCN4 leucine-zipper variants Parameter GCN4-pIL GCN4-pA GCN4-pV GCN4-pAA

Superhelix

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

␣-Helix radius, r1,Å 2.25 2.29 2.29 2.29 Residues per ␣-helical turn, n 3.58 3.61 3.63 3.61 Rise per residue, h,Å 1.53 1.51 1.51 1.50

Angular frequency, ␻1, ° per residue Ϫ100.8 Ϫ99.7 Ϫ99.3 Ϫ99.7

15460 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604871103 Liu et al. Downloaded by guest on September 25, 2021 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 N terminus 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 hole on 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-pII trimer (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) Packing at 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.

core a and d residues from the parent dimer sequence form two sodium acetate, pH 5.2͞150 mM NaCl) and 50 ␮M peptide. continuous helical screws because of local packing of these Thermal stability was assessed by monitoring [␪]222 as a function nonpolar side chains. Conserved asparagines at a positions direct of temperature under the same conditions. A [␪]222 value of parallel helix orientation by forming a network of buried hydro- Ϫ35,000° cm2 dmolϪ1 was taken to correspond to 100% helix gen bonds. The lateral displacement of adjacent helices in a (41). Sedimentation equilibrium measurements were carried out BIOPHYSICS sevenfold screw symmetry observed in the heptamer structure is on an XL-A analytical ultracentrifuge (Beckman Coulter, Ful- unique among known coiled-coil structures. lerton, CA) equipped with an An-60 Ti rotor at 20°C. Peptide Although the mechanism by which the geometric details of samples were dialyzed overnight against ABS (pH 5.2), loaded packing among side chains at the a, d, e, and g positions confer at initial concentrations of 30, 100, and 300 ␮M, and analyzed at specificity toward the native state cannot be predicted at present, rotor speeds of 18,000 and 21,000 rpm. Data sets were fitted to the availability of the seven-helix coiled coil provides a soluble a single-species model. Random residuals were observed in all model for detailed exploration of individual side-chain–side- cases. chain interactions within a large-scale structure. The properties of the nanoscaled central cavity in this structure merit further Crystallization and Structure Determination. GCN4-pAA was crys- investigation as well: How do various solutes influence the cavity tallized at room temperature by using the hanging drop vapor- and the stability of the heptamer? We anticipate that additional diffusion method by equilibrating against reservoir buffer (0.1 M fundamental rules prescribing helix-to-helix packing in coiled sodium citrate, pH 5.4͞0.1 M KH2PO4͞1.4 M hexanediol), a coils remain to be discovered and will provide insight into the solution containing 1 ␮lof6mg⅐mlϪ1 peptide in water, and 1 ␮l subtle relationship between amino acid sequence and the higher of reservoir buffer. Crystals belonged to space group P1(a ϭ levels of structure adopted by this diverse class of proteins. 31.5 Å, b ϭ 35.3 Å, c ϭ 49.0 Å, ␣ ϭ 86.5°, ␤ ϭ 104.3°, and ␥ ϭ 99.9°) and contained seven monomers in the asymmetric unit. Materials and Methods The crystals were harvested in 0.1 M sodium citrate, pH 5.4͞0.1 Protein Expression and Purification. The recombinant peptide MKH2PO4͞1.7 M hexanediol͞15% glycerol and frozen in liquid GCN4-pAA and its variants were expressed in Escherichia coli nitrogen. Diffraction data were collected on beamline X4C at BL21(DE3)͞pLysS by using a modified pET3a vector (Novagen, the National Synchrotron Light Source (Brookhaven, NY). San Diego, CA). Substitutions were introduced into the Reflection intensities were integrated and scaled with DENZO pGCN4-pA plasmid (17) by using the method of Kunkel et al. and SCALEPACK (42). Initial phases were determined by (37) and were verified by DNA sequencing. Cells were lysed by molecular replacement with Phaser (43), using the structure of glacial acetic acid and centrifuged to separate the soluble the GCN4-pA monomer (17) as a search model. Seven fraction from inclusion bodies. The soluble fraction containing GCN4-pA molecules were oriented and placed in the asymmet- peptide was subsequently dialyzed into 5% acetic acid overnight ric unit with a Z score of 7.8 and final refined log-likelihood gain at 4°C. The GCN4-pAA(V) construct was appended to the of 431.7, corresponding to the seven GCN4-pAA molecules. This TrpLEЈ leader sequence (38) and cloned into the pET24a vector model and the data set for GCN4-pAA were directly fed to (Novagen). GCN4-pAA(V) was purified from inclusion bodies Arp͞Warp (44), which provided a largely complete asymmetric and cleaved from the TrpLEЈ leader sequence with cyanogen unit of the seven chains and allowed Ϸ93% of the final model to bromide as described in ref. 39. Final purification of all peptides be interpreted. The resulting experimental electron density map was performed by reverse-phase HPLC on a C18 preparative was of excellent quality and showed the location of most of the column using a water-acetonitrile gradient in the presence of side chains. Crystallographic refinement of the GCN4-pAA 0.1% trifluoroacetic acid. Peptide identities were confirmed by structure was carried out by using Refmac (45). Density inter- electrospray mass spectrometry (Voyager Elite; PerSeptive Bio- pretation and manual model building were performed with O systems, Framingham, MA). Peptide concentrations were de- (46). The final model (Rcryst ϭ 18.1% and Rfree ϭ 20.9% for the termined by using the method of Edelhoch (40). resolution range 47.5–1.25 Å) consists of residues 3–33 (monomer A), 3–33 (monomer B), 3–33 (monomer C), 3–34 (monomer Biophysical Analysis. CD spectra were measured on a 62A͞DS CD D), 3–33 (monomer E), 1–32 (monomer F), and 3–32 (monomer spectrometer (Aviv, Lakewood, NJ) at 4°C in ABS (50 mM G) in the asymmetric unit, eight hexane-1,6-diols, and 179 water

Liu et al. PNAS ͉ October 17, 2006 ͉ vol. 103 ͉ no. 42 ͉ 15461 Downloaded by guest on September 25, 2021 molecules. Bond lengths and bond angles of the model have GCN4-pAA were used in the calculations. To calculate an omit rmsds from ideality of 0.009 Å and 1.1°, respectively. All protein map, target residues were removed, and the remaining atoms residues are in the most favored regions of the Ramachandran were shaken randomly by 0.3 Å to minimize model bias and then plot. refined. Figures were generated by using SETOR (50), Insight II (Accelrys, San Diego, CA), and GRASP (51). Structure Analysis. Coiled-coil parameters were calculated by using TWISTER (47). The rmsds were calculated with LSQKAB We thank John Schwanof at the National Synchrotron Light Source in the CCP4i program suite (48). Buried surface areas were (beamline X4C) for support, Sergei Strelkov for help with the superhelical parameter calculations, and David Eliezer for comments on the calculated from the differences of the accessible side-chain manuscript. This work was supported by National Institutes of Health surface areas of the seven-helix complex and of the individual Grant AI42382, the Irma T. Hirschl Trust, and a Fellowship from the helical monomers by using CNS 1.0 (49). Residues 3–32 of National Science Council of Taiwan (to C.-S.C.).

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