Antiparallel Four-Stranded Coiled Coil Specified by a 3-3-1 Hydrophobic Heptad Repeat

Coiled-coil sequences in proteins commonly share a seven-amino acid repeat with nonpolar side chains at the first (a) and fourth (d) positions. We investigate here the role of a 3-3-1 hydrophobic repeat containing nonpolar amino acids at the a, d, and g positions in determining the structures of coiled coils using mutants of the GCN4 leucine zipper dimerization domain. When three charged residues at the g positions in the parental sequence are replaced by nonpolar alanine or valine side chains, stable four-helix structures result. The X-ray crystal structures of the tetramers reveal antiparallel, four-stranded coiled coils in which the a, d, and g side chains interlock in a combination of knobs-into-knobs and knobs-into-holes packing. Interfacial interactions in a coiled coil can therefore be prescribed by hydrophobic-polar patterns beyond the canonical 3-4 heptad repeat. The results suggest that the conserved, charged residues at the g positions in the GCN4 leucine zipper can impart a negative design element to disfavor thermodynamically more stable, antiparallel tetramers.


Introduction
The coiled coil is among the most ubiquitous structural motifs in proteins consisting of interwound a helices that pack in parallel or antiparallel orientations, with a distinct preference for the parallel orientation (Gruber and Lupas, 2003;Kohn et al., 1997). Coiled-coil proteins contain a short-range seven-residue (heptad) sequence repeat, (a-b-c-d-e-f-g) n , with a predominance of the aliphatic side chains Leu, Ile, Val, and Ala at the a and d positions, and polar residues generally elsewhere (Hodges et al., 1972;Lupas et al., 1991;McLachlan and Stewart, 1975;Parry, 1982). This characteristic 3-4 hydrophobic polar residue pattern is the basis of Crick's canonical ''knobs-into-holes'' core packing between a helices and represents an essential structural feature of the coiled-coil family (Crick, 1953;Hodges et al., 1972;Lupas et al., 1991;McLachlan and Stewart, 1975;Parry, 1982). Residues at the e and g positions are frequently charged, and have been shown to influence the number and orientation of chains in a supercoil through interhelical electrostatic interactions (Kohn et al., 1995(Kohn et al., , 1998Krylov et al., 1994;O'Shea et al., 1992O'Shea et al., , 1993. Substantial progress has been achieved toward understanding the main factors that specify and stabilize parallel coiledcoil conformations (Bryson et al., 1995;Gonzalez et al., 1996;Harbury et al., 1993;Ogihara et al., 1997).
The determinants of antiparallel coiled-coil structure are less well understood (Kohn et al., 1997;Monera et al., 1996;Oakley and Hollenbeck, 2001;Oakley and Kim, 1998). DeGrado and coworkers have characterized a series of peptides designed to form four-stranded, antiparallel coiled coils (Bryson et al., 1995;North et al., 2001). One member of this class, a 1 B, contains leucine residues at the a, d, and g positions and forms a helical tetramer in solution (Ho and DeGrado, 1987). In 1993, Muller-Hill and coworkers presented genetic evidence that the C-terminal heptad repeat region of the lac repressor can self-associate into an antiparallel four-helix bundle (Alberti et al., 1993). They emphasized that uncharged amino acids at the e and g positions are required to specify this tetrameric structure (Alberti et al., 1993). Fairman and coworkers subsequently showed that a 21-residue peptide corresponding to the lac repressor heptad repeat region (called Lac21) forms a stably folded, antiparallel, a-helical tetramer in solution (Fairman et al., 1995). The crystal structure of the lac repressor confirmed that residues at the a, d, and e positions of this tetrameric coiled coil participate in interhelical hydrophobic interactions (Friedman et al., 1995;Lewis et al., 1996). The Richardsons first identified conserved features of a tightly packed, antiparallel helical bundle structure, the Alacoil (Gernert et al., 1995), in which the backbones of two neighboring helices are offset by half a turn with alanine in position g (ferritin-type Alacoils) or by a full turn with alanine at position e (rop-type Alacoils). Experimental studies of the Lac21 peptide model of a ferritin-type Alacoil show that the presence of neutral amino acids at the e and g positions indeed contributes to folding and stability (Fairman et al., 1995(Fairman et al., , 1996Solan et al., 2002). In connection with this, we note that introducing alanine into the e position of the GCN4 dimeric leucine zipper creates tetramers (Krylov et al., 1994).
Recent crystallographic studies of the tetrameric domain of the SARS coronavirus S2 protein identify an antiparallel, four-stranded coiled coil in which leucine or isoleucine residues at the d positions comprise the hydrophobic core, while bulky a and g side chains pack against the outer surface of the core (Y.D., J.L., Q.Z., and M.L., unpublished data). In light of the results discussed above, this interaction involving the positions a, d, and g of the heptad repeat raises the possibility that a distinct hydrophobic residue pattern (a 3-3-1 hydrophobic repeat) might be sufficient to specify a superhelical fold in which four a helices align in an antiparallel relative orientation. To test this hypothesis, we have engineered mutations of the g side chains in the GCN4 leucine zipper model which has served as an invaluable test bed for defining determinants of coiled-coil structure (Harbury et al., 1993(Harbury et al., , 1998. We simultaneously changed three charged residues at the g positions in the classical GCN4-pR sequence to nonpolar Ala and Val side chains. In contrast to the parent dimeric structure with the helices parallel (O'Shea et al., 1991), both mutant peptides form discrete, stable, a-helical tetramers in solution. Their X-ray crystal structures reveal antiparallel, four-stranded coiled coils with combined knobs-into-knobs and knobs-into-holes packing interactions between the hydrophobic a, d, and g side chains. Thus, the 3-3-1 hydrophobic pattern in the heptad repeat represents an extension of the Alacoil motif in nature for mediating protein-protein interactions via coiled coils. Our results suggest that the structural se-lectivity of conserved, charged residues at the e and g positions of leucine zipper dimers results not only from favorable interhelical electrostatic interactions, but also from the necessary destabilization (negative design) of alternate coiled-coil conformations.

Results and Discussion
Position g Mutants of the GCN4 Leucine Zipper The extensively investigated GCN4 leucine zipper has provided a key model system for analysis of the hydrophobic heptad repeat pattern and structural selectivity in coiled coils (Harbury et al., 1993;O'Shea et al., 1991). The apolar interface of the parent doublestranded coiled coil is formed by interhelical association in a parallel orientation between residues at the a and d positions (O'Shea et al., 1991). Electrostatic interactions between oppositely charged amino acid residues at the e and g positions of different helices also can contribute to dimerization specificity (Kohn et al., 1998;O'Shea et al., 1992O'Shea et al., , 1993. The recombinant leucine zipper peptide GCN4-pR includes four g positions: Lys9, Lys16, Glu23, and Leu30 ( Figure 1). To investigate the effect of a 3-3-1 hydrophobic residue repeat on helix association and interface specificity, we simultaneously mutated Lys9, Lys16, and Glu23 of GCN4-pR to the small aliphatic amino acids alanine and valine. The resulting mutants were designated GCN4-pA and GCN4-pV, respectively. These leucine zipper peptides were expressed in Escherichia coli and purified by reverse-phase HPLC.

Tetramer Formation in Solution
The circular dichroism spectra of GCN4-pA and GCN4-pV show the characteristic signature of an a helix with minima at 222 and 208 nm ( Figure 2A). On the basis of the mean residue ellipticity at 222 nm at 4ºC and The view is from the N terminus looking down the superhelical axis. Heptad repeat positions are labeled a-g. Prime ( 0 ) refers to positions from the second helix. In the mutant peptides described here, three charged residues in the dashed box at position g were changed to alanine and valine. The sequences (with the three mutated g positions underlined) are the following: GCN4-pR, MK VKQLEDK VEELLSK NYHLENE VARLKKL VGER; GCN4-pA, MK VKQLEDA VEELLSA NYHLENA VARLKKL VGER; GCN4-pV, MK VKQLEDV VEELLSV NYHLENV VARLKKL 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. (C) Sedimentation equilibrium data for a 50 mM sample of GCN4-pA at 20ºC and 28,000 rpm in PBS (pH 7.0). The data fit closely to a tetramer bundle. Curves expected for trimeric and pentameric models are indicated for comparison. The deviation in the data from the linear fit for a tetrameric model is plotted (upper). (D) Sedimentation equilibrium data for a 500 mM sample of GCN4-pV at 20ºC and 28,000 rpm in PBS (pH 7.0). 50 mM peptide concentration in neutral pH phosphatebuffered saline (PBS), each peptide contains >90% helical structure. Under these conditions, GCN4-pA and GCN4-pV undergo cooperative and reversible thermal unfolding transitions with midpoints (T m s) of 74ºC and 95ºC, respectively, as compared to a T m of 47ºC for the parent molecule GCN4-pR at the same concentration ( Figure 2B). Sedimentation equilibrium measurements indicate that GCN4-pA and GCN4-pV sediment as tetramers and exhibit no systematic dependence of molecular weight on concentration between 50 and 500 mM ( Figure 2C). Thus, both the hydrophobic g position mutants of the dimeric leucine zipper peptide form well-ordered and extremely stable quadruple-helical structures.
Crystal Structures of GCN4-pA and GCN4-pV To investigate the side chain packing at the a, d, and g positions in the heptad repeat in atomic detail, we determined the X-ray crystal structure of GCN4-pA at 1.5 Å resolution by the method of multiwavelength anomalous diffraction (Hendrickson, 1991) using a SeMet derivative ( Table 1). As hypothesized, GCN4-pA adopts a lefthanded superhelix consisting of four antiparallel a-helical peptide monomers in register that cross at an angle of approximately 20º ( Figure 3). The superhelix forms a cylinder with overall diameter of w24 Å and length of w48 Å . Diagonally related helices have identical relative orientation. The individual helices in the tetramer can be superimposed on each other with an rms deviation for acarbon atoms of 0.30-0.51 Å and with the largest deviations occurring at their ends. The distance between the axes of parallel helices (on diagonal) is w14 Å , whereas that between the axes of adjacent antiparallel helices is w8 Å , an Alacoil characteristic. However, the leucine residues at the d positions interact between parallel helices and point into the core of the tetramer (see Discussion). Cross-sectional layers containing leucine al-ternate between 2-fold symmetrical pairs of parallel helices. Fourteen of the 16 leucine side chains assume c 1 and c 2 dihedral angles near 265º, 175º or 2177º, 65º, corresponding to their most favored rotamers in a helices (Lovell et al., 2000;Ponder and Richards, 1987). Residues at positions a and g in the neighboring antiparallel helices pack against the leucines at d to complete the hydrophobic core.
The crystal structure of GCN4-pV at 2.0 Å resolution (Table 1) also reveals a symmetric, left-handed, antiparallel four-a helix bundle, in this case w28 Å wide and w46 Å long ( Figure 4). An exact dyad is perpendicular to the superhelical axis. The leucine side chains at the d positions interact between parallel helices (on diagonal) and stagger axially to form the hydrophobic core of the tetramer. All the dihedral angles c 1 and c 2 of the leucine residues are approximately 265º, 175º for the N-terminal two d layers and 2177º, 65º for the C-terminal two d layers in their well-populated rotamer conformations. The equally spaced a and g residues along the neighboring antiparallel helices flank the hydrophobic interface, efficiently sequestering the leucine side chains from solvent. The differential bulk of valine relative to alanine shows that interfacial interactions involving apolar residues at the a, d, and g positions of the heptad repeat play a major role in stabilizing the antiparallel tetramer structure. The stability differences between GCN4-pA and GCN4-pV are seen clearly in the solution data ( Figure 2B).
Although the superhelix pitch, radius of curvature, and residues per supercoil turn in the parallel GCN4-pIL (with isoleucine at each d position and leucine at each a) (Harbury et al., 1993) and antiparallel GCN4-pA and GCN4-pV coiled coils differ significantly (Table 2), the fraction of solvent-accessible surface area buried in the tetramer remains about the same. For example, approximately 5470 Å 2 of accessible surface area is buried in the GCN4-pA and GCN4-pV tetramers, compared with a R sym = SjI -<I>j/SI, where I is the integrated intensity of a given reflection. b Numbers in parentheses represent the statistics for the shell comprising the outer 10% (theoretical) of the data. c R cryst = SjF obs -F calc j/SF obs , R free = R cryst calculated using 10% of the reflection data chosen randomly and omitted from the start of refinement.
w5930 Å 2 of buried surface area in the GCN4-pIL tetramer (Harbury et al., 1993). Relative to the side chains of isolated helices, the leucine side chains at the d positions of GCN4-pA and GCN4-pV are completely buried; residues at the a and g positions are substantially buried; those at the e position are partly buried; whereas the b position remains completely exposed (see below). Due to the smaller size of the alanine side chain, GCN4-pA has a compressed g-d 0 interface, and therefore the resulting structure is less symmetric than that of GCN4-pV. More surface area at the c and f positions is buried in GCN4-pA than GCN4-pV (58% versus 35% at position c and 20% versus 1% at position f ).

Knobs-into-Knobs and Knobs-into-Holes Packing Interactions
The tetramer interfaces of GCN4-pA and GCN4-pV show nonclassical packing of side chains in the core of interacting helices. The leucine side chains at the d positions directly face each other between parallel helices (on diagonal), giving rise to a so-called knobs-into-knobs packing in these layers (Gottschalk, 2005;Lupas and Gruber, 2005). By contrast, adjacent antiparallel helices adopt a new kind of knobs-into-holes interaction in which each leucine knob at the d position of one helix packs into a hole formed by the a and g residues of one neighboring helix and by two d residues in adjacent layers along the superhelical axis ( Figure 5). This geometry results in a similar placement of atoms around the side chains at positions a and g. Knobs formed by a residues of one helix fit into holes formed by the spaces between the d and e residues on the neighboring helix and by two a residues in adjacent layers along the superhelical axis. Similarly, knobs at g positions pack into holes formed by the c and d residues of the neighboring helix and by two g residues in adjacent layers. Thus, the a, d, and g residues of the heptad repeat segregate into four geometrically distinct helix-helix interfaces. Consequently, the GCN4-pA and GCN4-pV superhelices have alternating rectangular cross-sections.

Comparison with Other Antiparallel Tetramers
We begin by considering the classification of antiparallel helices due to the Richardsons (Gernert et al., 1995). They distinguished two fundamental types of their ''Alacoils'': the ferritin type, with the helices offset by 0.25 heptad, and the rop type (Banner et al., 1987), with the helices shifted by 0.5 heptad (see Table 2). Our GCN4-pA and GCN4-pV structures apparently conform to the rop type. On the other hand, the lac repressor tetramerization domain (Kercher et al., 1997) and the WSPLB 21-52 model (Slovic et al., 2005) are of the ferritin type.
There are substantial differences in the interior packing of the tetramer among each of these structures ( Figure 6). Each type of Alacoil can exhibit at least two modes of core packing arrangements: the well-known a,d pattern (WSPLB and Rop), and a triad a,d,e (the lac repressor tetramerization domain) or a,d,g as in GCN4-pA and GCN4-pV. The packing properties in each of these cases are clearly revealed by calculating the extent of buried surface areas of the side chains at these heptad positions ( Figure 6D). We see that each packing mode has a signature in surface areas. For the a,d packing, for example, both a and d side chains show nearly complete burial at these sites. Conversely, the a,d,e and a,d,g patterns show lower burial values at either of the a and d sites. Thus, surface burial provides a useful diagnostic for each core packing interaction.

3-3-1 Hydrophobic Residue Repeat
Our results show that heptad sequence repeats with a 3-3-1 hydrophobic residue pattern can encode a stable, antiparallel four-helix structure. In this tetramer conformation, the a, d, and g residues form a left-handed apolar strip along the surface of the right-handed a helices, while their side chains mesh between interacting helices by the winding of antiparallel chains around one another. This motif adds an additional element to the current repertoire of coiled-coil structures: the hydrophobic core is formed by interlocking of residues at positions a and d in rop (Banner et al., 1987) but at positions a, d, and g in both GCN4-pA and GCN4-pV. The 3-3-1 hydrophobic residue repeat retains two structural features of coiled   (Crick, 1953): an interhelix packing angle near 20º and knobsinto-holes packing of side chains between adjacent helices. Our results support the notion that the stoichiometry and geometry of coiled coils can be determined by distinctive hydrophobic patterns within the classical heptad repeat (Lupas and Gruber, 2005).

Structural Selectivity
One major challenge in protein design is to engineer conformational specificity in addition to thermodynamic stability (Bryson et al., 1995). Buried polar interactions of appropriate geometry have been shown to impart specificity (structural uniqueness) in protein folding and design even if they do so at the expense of stability (Hendsch et al., 1996). For example, a buried hydrogen bond between Asn residues in the GCN4 leucine zipper is responsible for maintaining the register of the two parallel helices in the dimer structure (Harbury et al., 1993). It is tempting to apply a similar argument to explain why the 3-3-1 hydrophobic repeat is not typically found in coiled-coil proteins. Crick pointed out in 1953 that coiled coils contain fewer hydrophobic side chains than globu-lar proteins (Crick, 1953). Here we find that increasing the hydrophobic side chain content of the GCN4 leucine zipper can lead to enhanced stabilization of the resulting structure. The highly stable antiparallel, symmetric 3-3-1 tetramer might present a functionally compromised conformational trap, as we argue to be the case in the SARS S2 protein (Y.D., J.L., Q.Z., and M.L., unpublished data). Consistent with this notion, there is growing evidence that polymorphic interactions between two heptad repeat regions in viral envelope proteins provide a reservoir of free energy that can be released to drive structural transitions in a precise temporal sequence that is required for membrane fusion (Y.D., J.L., Q.Z., and M.L., unpublished data). Specificity in coiled coils (in terms of oligomeric state and strand orientation) stems in part from the precise complementarity of hydrophobic residues at the a and d positions (Bryson et al., 1995;Harbury et al., 1993), and to a larger extent from buried polar interactions (hydrogen bonds and ion pairs) that mildly destabilize the native structure yet strongly destabilize alternate conformations (Hendsch et al., 1996). While interactions between charged side chains at the e and g positions have previously been implicated in determination of helix orientation, number, and heterogeneity, their precise role has been the subject of some controversy Lumb and Kim, 1995). Recent studies have revealed that electrostatic effects are complex, including both stabilizing and destabilizing interactions (Garcia-Mayoral et al., 2003;Bosshard, 2003, 2004). Changes in the free energy of the unfolded state may account for the effects of electrostatic interactions rather than differences in the stability of the native state per se. In all cases, however, the net free energy contributions of a pair of opposite charges at the e and g positions are small (w1 kcal/mol or less), although there can be a stronger repulsive interaction between similar charged side chains at these positions (Jelesarov and Bosshard, 1996;Kohn et al., 1995;Lavigne et al., 1996;O'Shea et al., 1993). The finding that replacing g residues by nonpolar side chains in a dimeric coiled coil  (D) Buried surface areas in WSPLB 21-52 (a,d packing), the lac repressor tetramerization domain (a,d,e packing), rop (a,d packing), GCN4-pA (a,d,g packing), and GCN4-pV (a,d,g packing). Percent buried surface area is expressed as the fraction of accessible side chain surface area in the isolated helix that becomes buried in the antiparallel tetramer.
can switch both stoichiometry and helix orientation to produce a tetramer structure of enhanced stability suggests that the selection of charged or polar side chains at the g positions might provide an additional mechanism to regulate structural features of interfacial interaction specificity. For example, to restrict a given interaction exclusively to a parallel coiled coil might necessitate extensive charges at the e and g positions in order to avoid the potential trap represented by the antiparallel 3-3-1 tetramer. Indeed, a significant fraction of these positions in a protein such as myosin contains charged or polar residues (Li et al., 2003). It should be noted that while the 3-4 heptad sequence regularity has led to the successful development of prediction algorithms to identify potential coiled-coil domains from primary amino acid sequence (Lupas, 1997;Wolf et al., 1997), the 3-3-1 hydrophobic pattern is poorly accommodated in these algorithms. Accurate prediction of diverse coiled-coil interaction motifs will require an expanded understanding of the principles governing hydrophobic heptad repeat patterns.

Peptide Expression and Purification
The peptides GCN4-pA and GCN4-pV were expressed in E. coli BL21(DE3)/pLysS using a modified pET3a vector. Cells were lysed by glacial acetic acid and centrifuged to separate the soluble fraction from inclusion bodies. The soluble fraction was subsequently dialyzed into 5% acetic acid overnight at 4ºC. Peptides from the soluble fraction were purified to homogeneity by reverse-phase HPLC on a C18 preparative column using a water-acetonitrile gradient in the presence of 0.1% trifluoroacetic acid and lyophilized. The identity of each peptide was confirmed by mass spectrometry (PerSeptive Biosystems Voyager Elite, Cambridge, MA); all molecular masses were found to be within 1 dalton of the expected mass. Selenomethionine (SeMet)-substituted GCN4-pA was produced for multiwavelength anomalous diffraction analysis in amino acidsupplemented minimal media and purified as described above (Doublie, 1997).

Biophysical Analysis
Circular dichroism (CD) spectra were measured on an Aviv 62A/DS CD spectrometer (Lakewood, NJ) at 4ºC in PBS (50 mM sodium phosphate/150 mM NaCl [pH 7.0]) and 50 mM peptide. Thermal stability was assessed by monitoring [q] 222 as a function of temperature under the same conditions. A [q] 222 value of -33,000 deg cm 2 dmol 21 was taken to correspond to 100% helix (Chen et al., 1974). Values of T m were estimated by evaluating the maximum of the first derivative of [q] 222 versus temperature data (Cantor and Schimmel, 1980). Peptide concentrations were determined by absorbance at 280 nm in 6 M guanidine hydrochloride, using an extinction coefficient of 1280 cm 21 M 21 for tyrosine (Edelhoch, 1967). Sedimentation equilibrium experiments were performed on a Beckman XL-A analytical ultracentrifuge (Fullerton, CA) at 20ºC as described (Shu et al., 1999). Peptide solutions were dialyzed overnight against PBS (pH 7.0), loaded at initial concentrations of 50, 150, and 500 mM, and analyzed at rotor speeds of 25,000 and 28,000 rpm. Data sets were fitted to a single-species model. Random residuals were observed in all cases.

Data Collection and Structure Determination
All data sets were collected on beamline X4A at the National Synchrotron Light Source. Data for all crystals were indexed and reduced with DENZO/SCALEPACK (Otwinowski and Minor, 1997). The CCP4 set of programs was used for truncating and scaling the data sets (CCP4, 1994). One selenium site (corresponding to the GCN4-pA M14 position) in the asymmetric unit was located with Solve (Terwilliger and Berendzen, 1999), and approximately 80% of the polypeptide chain was traced automatically into the electron density maps. Iterative rounds of model building with O (Jones et al., 1991), refinement with Refmac (Murshudov et al., 1997), and addition of ordered solvent clarified the trace except for a few residues at the helix termini. The structures of GCN4-pA and GCN4-pV were solved by molecular replacement with Phaser (Storoni et al., 2004) using the GCN4-pA/SeMet structure as a search model. All main chain torsional angles fall within the helical regions of the Ramachandran plot. Figures were generated using SETOR (Evans, 1993) and Insight II (Accelrys, San Diego, CA).

Structure Analysis
Superhelix phase (f 0 ) and translation of the supercoiled helix along the superhelical axis (Z trans ) were obtained using an analytical equation for an antiparallel coiled coil (North et al., 2001;Slovic et al., 2005), whereas other coiled-coil parameters were calculated with TWISTER (Strelkov and Burkhard, 2002). Residues 26-51 of WSPLB 21-52 (PDB code: 1YOD), residues 341-355 of lac repressor (PDB code: 1LBI), residues 7-23 of rop (PDB code: 1ROP), residues 3-31 of GCN4-pA and GCN4-pV, and residues 2-30 of GCN4-pIL (PDB code: 1GCL) were used in the calculations. Buried surface areas were calculated from the difference of the accessible side chain surface areas of the tetramer structure and of the individual helical monomers using CNS 1.0 (Brunger et al., 1998). Residues 340-357 of helices A and B of lac repressor (PDB code: 1LBI), residues 31-51 of the A and B chains of WSPLB 21-52 (PDB code: 1YOD), and residues A6-A28 and A33-A54 of rop (PDB code: 1ROP) were used in the calculations. The three most N-terminal residues and the three most C-terminal residues of GCN4-pA and GCN4-pV were omitted from the calculation to minimize end effects.