Formation of Leucine Zipper Coiled Coils? (Molecular Modeling/A-Helices/Protein-Protein Interaction) ALEXANDER TROPSHA*T, J

Home , Helical wheel, Leucine zipper

Proc. Nall. Acad. Sci. USA Vol. 88, pp. 9488-9492, November 1991 Biochemistry Do interhelical side chain-backbone hydrogen bonds participate in formation of leucine zipper coiled coils? (molecular modeling/a-helices/protein-protein interaction) ALEXANDER TROPSHA*t, J. PHILLIP BOWEN*, FRANK K. BROWN§, AND J. S. KIZER*t¶ *Brain and Development Research Center and 1Departments of Medicine and Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599; tDepartment of Chemistry, University of Georgia, Athens, GA 30602; and §Glaxo Research Institute, 5 Moore Drive, Research Triangle Park, NC 27709 Communicated by Robert G. Parr, July 1, 1991 (received for review August 10, 1990)

ABSTRACT The leucine zipper proteins are a group of K K transcriptional regulators that dimerize to form a DNA binding L a E E G K E v E R E L 'y N R R V L domain. It has been proposed that this dimerization results L H K LI V L A s H L N L

from the hydrophobic association ofthe a-helices oftwo leucine K L zipper monomers into a coiled coil. We propose a model for a QjL~ v K EE s ~ ~ ~ VsK coiled coil based on a periodic hydrophobic-hydrophilic amino acid motif found in the leucine zipper regions of 11 transcriptional regulatory proteins. This model predicts the symmetrical N L AI formation of secondary hydrogen bonds between the polar side V L E \.J LVVj IN H G E L EE v R K chains of one helix and the peptide carbonyls of the opposite a chain, supplementing the interactions between hydrophobic K a K E side chains. Physical modeling (CPK) and in vacuo molecular A B mechanics calculations of the stability of the GCN4 leucine zipper coiled coil configured in accordance with this model FIG. 1. Helical wheel representation oftwo GCN4 leucine zipper demonstrate a greater stability for this conformer than for a monomers paired as a coiled coil according to the model of O'Shea conformer configured according to a current hydrophobic et al. (8) (A) and according to the model proposed in this paper (B). model. Molecular dynamics simulations show similar stability Each N terminus begins with the first leucine (designated as a) ofthe of the two models in vacuo but a higher stability of the postulated zipper region. Those amino acids that might occupy hydrophobic model in water. positions in the interhelical space are boxed. hydrophilic amino acids in the hydrophobic contact zone pre- Coiled coils, found in a number of proteins, are interesting dicted by the 4,3 rule, we reanalyzed the amino acid sequences examples of stable homodimers formed by the association of of leucine zipper proteins, searching for alternative motifs. We two, three, or four a-helices (1-3). The helices comprising a found that in all cases, with but a single exception, the fourth coiled coil are usually oriented in a parallel manner with residue after each leucine of the zipper region is polar (Fig. 2). corresponding N and C termini (1-3). Analysis of the primary With this motif as a starting point, a second alignment for the structure ofcoiled coils has identified a repeating seven-amino leucine zipper region is possible, as shown for the GCN4 protein acid motif. The first and the fifth positions of this heptad are (Fig. 1B). This alignment is generated by turning both left and usually occupied by hydrophobic amino acids (the 4,3 rule), so right helical wheels (Fig. LA) one-seventh of a full turn in a that one side of each helix is predominantly hydrophobic. clockwise direction. Thealignmentofthe two helices inthis way, Because of these observations, it is thought that dimerization however, would disrupt the hydrophobic contacts postulated by between the two helices ofcoiled coils resultsfrom hydrophobic O'Shea et al. (8) to an even greater extent, with hydrophilic side chain-side chain contacts (4, 5). Although there are exam- amino acids constituting nearly 50%o of all the residues in the ples of coiled coils that do not conform to this model because interhelical contact zone. the potential hydrophobic side of the helix is interrupted by To examine the possible significance ofhydrophilic residues in hydrophilic amino acids (6), a more generalizable model for the the interhelical contact zone, a physical CPK model of a coiled formation of coiled coils has not been proposed. coil was assembled incorporating two 13-amino acid peptides A coiled-coil configuration was recently proposed to ex- composed of polyalanine and our proposed hydrophobic- plain the formation ofdimers by leucine zipper proteins (7, 8), hydrophilic periodic motifwith leucine and lysine at positions 1 a class of DNA transcriptional activators with a distinctive, and 4 ofeach heptad (ALK-13; Figs. 3 and 4). From this model, repetitive leucine heptad (9). This idea came from observa- it became apparent that a dimer made up of this peptide can be tions that a model peptide derived from the structure of the stabilized by hydrophobic interactions between the methylenes GCN4 regulator forms a stable, symmetrical dimer ofparallel oflysine and leucine aswell asby intermolecularhydrogenbonds a-helices in aqueous solution (8, 10) and that its primary between the e-ammoniums ofthe lysine side chains of one helix structure contains the 4,3 leucine-valine hydrophobic repeat and the peptide carbonyls ofthe leucines ofthe opposing helix. characteristic of coiled coils (Fig. lA). Apparently, these interhelical hydrogen bonds would be second- Although this hydrophobic model is consistent with the pri- ary to the preexisting intrahelical amide-carbonyl hydrogen mary structure of the GCN4 protein, simple inspection of the bonds and, therefore, bifurcated at the leucine carbonyls. More- postulated zipper region of several leucine zipper proteins re- over, these secondary hydrogen bonds wouldbe strengthenedby veals that the fifth residue ofeach heptad is hydrophobic in only the surrounding hydrophobic milieu produced by the side-chain 5(f4o ofthe cases (Fig. 2). Puzzled by the presence ofnumerous tTo whom reprint requests should be addressed at: Brain and The publication costs of this article were defrayed in part by page charge Development Research Center, School of Medicine, University of payment. This article must therefore be hereby marked "advertisement" North Carolina, 241 Biological Sciences Research Building, Chapel in accordance with 18 U.S.C. §1734 solely to indicate this fact. Hill, NC 27599-7250. 9488 Downloaded by guest on October 1, 2021 Biochemistry: Tropsha et al. Proc. Natl. Acad. Sci. USA 88 (1991) 9489

Protein Leucine zipper Peptide Leucine zipper GCN4, C-terminus C/EBP 306 RNVETQQKVLELTSDNDRLRKRVEQLSRELDTLRG 340 ALK-13 ALAAKAAALAAKA Jun 284 RIARLEEKVKTLKAQNSELASTANMLTE.QVAQLKQ 319 ALK-19 AAAALAAKAAALAAKAAAA Fos 261 LTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEF 295 ALKV-20 AAAALAAKVAALAAKVAAAA GCN4 249 RMKQLEDKVEELLSKNYHLENEVARLKKLVGER-COOH GCN4-20 AMKQLEDKVEELLSKVYHLA YAP1 88 KMKELEKKVQSLESIQQQNEVEATFLRDQLITLVN 122 CREB 307 YVKCLENRVAVLEN.QNKTLIEELKALKDLYCHKSD 341 FIG. 4. Amino acid sequences of the GCN4 leucine zipper Cys-3 123 REQALEKSAKEMSEKVTQLEGRIQALETENKYLKG 147 peptide and the peptides modeled in this study. Leucines that define the beginning of each heptad are in boldface type. Periodic hydro- CPC1 239 RLEELEAKIEELIAERDRYKNLALAHGASTE-COOH philic residues are underlined. HBP1 204 ECEELGORAEALKSENSSLRIELDRIKKEYEELLS 238 Opaque2 251 HLKELEDQVAQLKAENSCLLRRIAALNQKYNDANV 285 and by molecular dynamics simulations in water using AMBER hXBP-1 93 RMSELEQQVVDLEEENQKLLLENQLLREKTHGLVVENQELRQRL 136 (14, 15). Simulations in water and in vacuo were considered 1234567123456712345671234567 to be equally important for modeling simplified peptides since Consensus -----L--P----L--P--- -L--P----L--P--- -L- - the degree of hydration of the interface of the leucine zipper TGA1 96 YVQQLENSKLKLIQLEQELERARKQGMCVGGGVDA 130 protein dimers is currently unknown. 12345671234567 12345 Molecular Mechanics Calculations. For our initial studies, a Consensus ----L---P--L---P--L---P------simplified 19-residue polyalanyl monomer containing the 1-4 leucine-lysine repeat (ALK-19; Fig. 4) and the corresponding FIG. 2. Amino acid sequences of the leucine zipper regions of homodimer configured in our ideal coiled-coil conformation several DNA binding proteins (from refs. 11 and 12). The position of were used to separately examine the importance of lysine- the leucine zipper in each protein is given by the numbers bracketing backbone, lysine-lysine side chain, and possible lysine- each sequence. Invariant leucine residues (L in the consensus) are in boldface type, and invariant polar hydrogen bond donors or accep- leucine side chain interactions to the overall stability of the tors (P in the consensus) are underlined. One sequence, TGA1, has coiled coil. In subsequent steps, monomers with progres- a different consensus. sively more complex primary structure (ALKV-20 and GCN4-20; Fig. 4) and the corresponding dimers configured methylenes of these same leucines and lysines. The purpose of in alternative orientations (Fig. 1) were analyzed to estimate this report is to demonstrate that formation of a coiled coil in a the difference in their stability. For these calculations, we hydrogen-bonded alignment (Fig. 1B) is energetically permissible considered the energy difference between a fully minimized and may represent an alternative or complementary model to the dimer and that of two minimized, isolated monomers as a hydrophobic-bonded coiled coil (Fig. LA). measure of the energy of dimer stabilization. The ideal coiled-coil conformation of a-helices (3.5 residues per helical turn, or roughly 7 residues per two turns) was METHODS determined empirically by monitoring the 4 and 4i backbone Our proposed model was refined by computer-assisted mo- angles of a polyalanyl a-helix within allowed helical toler- lecular mechanics and molecular dynamics calculations in ances (-40° to -60°) (16) and simultaneously setting the vacuo with the SYBYL 5.3 (Tripos Associates, St. Louis) torsion angle [Ca-CT3-Ca'-C9'] of each seventh pair of ala- molecular modeling package using the AMBER potential (13), nines equal to zero by using the SYBYL 5.3 graphics option, set conformation (i.e., finding the helical conformation when all Ca-Cj) vectors ofall the residues within any heptad repeat would be parallel). Of the many possible backbone conformations satisfying this criterion, one (4) = -44.00o, f = -55.27°) was chosen arbitrarily as a starting point for subsequent calculations. To create a symmetrical model of helix-helix interactions for the homodimer, the distances between Ca atoms of all opposing leucines and all opposing lysines were taken as a measure of the interhelical distance, and the similarity between these distances was considered as an index of the symmetry of the orientation of the helices. Different initial interhelical distances were tested empirically to achieve an optimized geometry for each dimer. The lowest energy minimum (Table 1) was achieved when two helices were initially separated by 5.6 A in the hydrogen-bonded orientation and by 5.4 A in the hydrophobic orientation. (It is important to point out that current techniques for molecular mechanics cannot ensure that the global minimum is found when a structure is optimized.) A Tripos-implemented AMBER program (14, 15) was used for the energy minimization ofthese model peptides in vacuo. Both the all-atom (14) and the united-atom (15) algorithms for the most simplified peptide (ALK-19; Fig. 4) and only the latter one for the other peptides were used. The calculations FIG. 3. CPK model of two ALK-13 monomers paired into a coiled coil. Each solid arrow indicates the secondary hydrogen bonds formed for protein minimization consisted of 100 steps of a steepest- between the £-amino group of lysine and the backbone carbonyl of descent routine followed by a conjugate-gradient routine with leucine. (Only two offour such bonds may be seen in the figure.) Open a gradient convergence criterion of rms = 0.07 kcal- arrows indicate hydrophobic contacts between leucine side chains. moh*1A-2 (1 cal = 4.184 J). The following essential param- Black, carbon; white, hydrogen; blue, nitrogen; red, oxygen. eters of minimization were used: (i) nonbonded cutoff dis- Downloaded by guest on October 1, 2021 9490 Biochemistry: Tropsha et al. Proc. Natl. Acad. Sci. USA 88 (1991) Table 1. Molecular mechanics calculations of energy minima for model peptides AMBER Utfin kcal/molt force-field Peptide* parameter set Uconf* Uel§ Total Ustabi Mono-ALK-19 All atom -7.379 84.934 77.555 Di-ALK-19, H-bonded - -14.404 136.797 122.393 -32.717 Mono-ALK-19 United atom -36.258 42.282 6.024 Di-ALK-19, H-bonded - -81.978 58.607 -23.371 -35.419 Mono-ALKV-20 -37.065 43.092 6.027 Di-ALKV-20, hydrophobic -101.355 97.884 -3.471 -15.525 Di-ALKV-20, H-bonded - -79.912 63.640 -16.272 -28.326 Mono-GCN4-20 - -27.466 -84.168 -111.634 Di-GCN4-20, hydrophobic -62.775 -252.386 -315.161 -91.893 Di-GCN4-20, H-bonded -65.444 -262.278 -327.722 -104.454 *See Fig. 4. tIt is important to note that it is the differences between energy minima rather than the magnitude of the absolute values for the energy minima that are meaningful. *Conformational energy term consists of the sum of the following energy terms: angle bending, bond stretching, out of plane, 1-4 van der Waals, and van der Waals [see ref. 13 and SYBYL 5.3 program (Tripos Associates) for details]. §Electrostatic energy term consists of the sum of the following energy terms: 1-4 electrostatic, electrostatic, and H bonds [see ref. 13 and SYBYL 5.3 program (Tripos Associates) for details]. IStabilization energy of dimer formation was calculated as Umi,,, (total) of the dimer minus Ui,,, (total) of the monomer times 2. tance = 8.0 A, (it) dielectric function, distance dependent. minimized hydrogen-bonded ALK-19 dimer is shown in Fig. The helical backbones were initially treated as "aggregates" 5. (a routine that excludes backbone atoms from optimization) Stability of a coiled coil in a hydrogen-bonded vs. a so that for the first calculations only the side chain geometry hydrophobic configuration. From calculations of the energy was minimized. When the energy-change convergence minimum of our simple polyalanyl leucine-lysine model, we threshold (88U = 0.05 kcal/mol) was achieved, all con- proceeded to apply the same minimization protocol to a straints were removed, and the conformation of the whole second model peptide of 20 amino acids similar to ALK-19, dimer was minimized. The energy of the corresponding but with valine substituted for alanine at position 5 after each monomers was minimized by the same routine. leucine and an extra C-terminal alanine (ALKV-20; Fig. 4). Solvent Accessibility. Solvent accessibility ofthe GCN4-20 From these calculations, we conclude that the ALKV-20 dimer in different configurations was calculated by using a MACROMODEL 2.0 molecular modeling package (Department dimer is some 13 kcal/mol more stable when configured of Chemistry Columbia University, New York), a probe radius of 1.4 A, and an algorithm similar to that developed by Lee and Richards (17). Molecular Dynamics Simulations. In vacuo. Initially, dynamic simulations of the energetically minimized GCN4-20 dimers in both alternative configurations were carried out in vacuo. These calculations consisted of 2 psec of heating (0-300 K) followed by 150 psec of simulations at 300 K. In water. Further dynamic simulations of the minimized GCN4-20 dimers in water were carried out using AMBER (15). For these calculations, the GCN4-20 dimer in the hydrogen- bonded configuration was solvated with 1534 water mole- cules producing an 8-A shell of water. For the GCN4-20 dimer in the hydrophobic configuration, 1589 water mole- cules were used for solvation, also in an 8-A shell. Both configurations were then simulated under periodic boundary conditions with a constant dielectric, a 6.5-A cutoff, a 1-fsec time step, and a nonbonded list table update every 50 steps. The simulations consisted of an initial minimization of the water in the potential field of the peptides, a heating of the entire system to 300 K over 2 psec, and, finally, a 240-psec simulation for the hydrogen-bonded configuration and a 200-psec simulation for the hydrophobic configuration. RESULTS Moleular Mechanics. Stability of the hydrogen-bonded ALK-19 homodimer vs. two isolated monomers. Our calcula- FIG. 5. Computer-drawn illustration of fully minimized, hydro- tions suggest that the lowest energy conformer of the dimer gen-bonded coiled coil (ALK-19). N termini of both helices are that is based on our physical CPK model (Fig. 3) is =30 toward the top of the figure. Dotted lines represent intra- and kcal/mol more stable than the lowest energy conformation of interhelical hydrogen bonds. Secondary hydrogen bonds between the two independent monomers, mainly due to favorable the lysine side chains and the peptide backbone and interactions electrostatic interactions (Table 1). The structure of the fully between the hydrophobic side chains ofthe four leucines are evident. Downloaded by guest on October 1, 2021 Biochemistry: Tropsha et al. Proc. Natl. Acad. Sci. USA 88 (1991) 9491 according to our proposed hydrogen-bonded model than when configured according to the alternative model (Table 1). As a further test of our model, we applied the same minimization protocol to a 20-amino acid portion ofthe actual GCN4 zipper in each of the alternative orientations (Fig. 1A vs. Fig. 1B). To highlight our comparison of the energy 8- minimum of a hydrogen-bonded GCN4 dimer with that of a hydrophobic dimer, valine was substituted for asparagine in the actual GCN4 sequence (cf. GCN4 and GCN4-20; Fig. 4). (This substitution would actually bias the results of our 6 calculations in favor of the hydrophobic model since the hydrophobic model predicts an enhanced stability of the coiled coil if this position were to be occupied by a hydrophobic residue.) Our calculations indicate that the hydrogen- bonded configuration of the GCN4 dimer is thermodynami- cally favorable and considerably more stable (:13 kcal/mol) than the hydrophobic configuration. Furthermore, from these calculations we conclude that this difference in stabi-

lization energy between the two models is due to more .5- , 150,000 favorable electrostatic interactions and hydrogen bond for- 0 50,000 100,000 150,000 mation (Table 1), as anticipated from the CPK models. Solvent Accessibility. Calculations of the solvent accessi- a, 13.00 B bility of the buried lysines of the GCN4-20 dimer indicate co that two of the three hydrogens of each E-ammonium group of every lysine side chain are completely buried and involved 0~~~~~~~~~~~~~~~,, in the formation of hydrogen bonds. One lysine hydrogen bond is formed with the backbone carbonyl ofa leucine ofthe opposing helix, and a second is formed with the 8-carboxyl of 10.25- the side chain of the glutamate that is two positions toward the N terminus-i.e., Lys-8-Glu-6 and Lys-15-Glu-13, re- spectively. The third hydrogen is partially exposed to solvent and is free to form another hydrogen bond with either solvent or a polar group ofthe same protein. This finding is in keeping with the observation that every potential hydrogen bond of a 4.750 ,S, buried polar group is actually present in the structure of a protein as determined by x-ray crystallography (18). The hydrogen-bonded configuration ofGCN4-20 (Fig. 1B) exposes to solvent those hydrophobic residues that occupy positions e and E' in the hydrophobic model (Fig. 1A). Furthermore, the solvent accessible area ofthe methylenes of the side chains of all lysine, leucine, and valine residues is greater (422.5 A2 vs. 289.9 A2) in the hydrogen-bonded model. Total surface accessible to solvent, however, is nearly identical for each model (2432.1 A2 vs. 2436.1 A2). Molecular Dynamis. In vacuo. Both the hydrogen-bonded and 2.00 hydrophobic configurations ofthe GCN4-20 dimers were stable 50,000 100,000 150,000 200,000 250,000 over the course ofmolecular dynamics simulations in vacuo. For Time, fsec the hydrogen-bonded dimer, the E-ammonium groups ofthree of four lysines remained within 3.0 A of the carbonyls of the FIG. 6. Monitoring of the four possible interhelical hydrogen corresponding leucines in the opposing helix, indicating the bonds during dynamic simulations of a hydrogen-bonded GCN4-20 stability of the proposed hydrogen bonds over the course of the dimer in vacuo (A) and in water (B). Ordinal distance is in A and simulation (Fig. 6A). After -60 psec, the fourth interhelical abscissal time is in fsec. For clarity, the amino acids of the first monomer are numbered from 1 to 20 and the amino acids of the hydrogen bond was broken due to the formation ofan intrahelical second monomer are numbered from 21 to 40. The four possible hydrogen bond between the e-ammonium of lysine and oxygen hydrogen bonds are Lys-8-Leu-25 (-), Lys-15-Leu-32 .. I of the asparagine side chain. Lys-28-Leu-5 (----), and Lys-35-Leu-12 (- --). In water. During molecular dynamics simulations in water, the hydrophobic configuration of the GCN4-20 dimer ap- peared stable. On the other hand, of the four interhelical, the hydrophobic 4,3 motif proposed earlier (8). The first secondary hydrogen bonds between the e-ammonium groups residue ofthe alternative motif is leucine. The fourth residue of lysines and the carbonyls of leucines in the hydrogen- of this motif is a hydrophilic amino acid (Fig. 2). If such a bonded configuration, two were weakened and eventually motifis important in the formation ofthe leucine zipper coiled broken by exposure to water (Fig. 6B). Of the other two coil, then a mechanism for coiled-coil formation must be hydrogen bonds, one stabilized at -3 A and the other proposed that can define a role for hydrophilic amino acids in stabilized at 4 A. stabilizing such a dimer. Based on our models, it may be possible for two leucine zipper a-helices to form a coiled coil that is stabilized by hydrogen bonding between the side DISCUSSION chains of polar residues and the peptide backbone of the Analysis of the primary structure of 11 leucine zipper tran- opposing helix. scriptional regulators suggests the presence of a previously Molecular mechanics and molecular dynamics calculations in unrecognized periodic motif that is more generalizable than vacuoindicate that a hydrogen-bonded coiled coil would stabilize Downloaded by guest on October 1, 2021 9492 Biochemistry: Tropsha et al. Proc. Natl. Acad Sci. USA 88 (1991) dimer formation at least as efficiently as the hydrophobic model helical hydrogen bonds. [Parenthetically, interhelical hydro- proposed by O'Shea et al. (8). Molecular dynamics simulations gen bonding is also possible with a polar amino acid in the in water, however, suggest a decreased stability ofthe hydrogen- fifth position ofthe leucine heptad. In the exceptional case of bonded dimerdue tothe solvation oftwoofthefoure-ammonium TGA1 (Fig. 2), the fourth residue after each leucine is groups ofthelysines. The hydrophobic model, ontheotherhand, hydrophobic and the fifth one is hydrophilic, the inverse of appears more stable during dynamics simulations in water. the same motif.] It is not possible at this time to be certain Solventaccessibility calculations alsoindicate agreaterexposure which of these two mechanisms is the more likely, but it is of hydrophobic residues to solvent in the hydrogen-bonded certain that no matter what orientation is assumed by leucine model. These observations demonstrate that a low-dielectric zipper coiled coils as they dimerize, the high frequency of environment is a necessary prerequisite for stabilin of the hydrophilic amino acids within the dimer-forming regions model hydrogen-bonded dimer. Although such conditions are argues for a fundamental role for polar residues in the unlikely to obtain when modeling the behavior of complex formation of the leucine zipper coiled-coil interface. biological proteins with simple oligopeptides in water, the microenvironment ofcoiled-coil proteins within the cell is currently We wish tothank Drs. Michael Cory, Jan Hermans, and Lee Pedersen undefined, but it is certainly not adequately modeled by pure for cntically reviewing the manuscnipt, and Drs. Aaron Miller and Tom water. Thus, the proximity of membranes, phospholipid deter- DardenfortheirhelpfUl counselregardinghardwarecommunications and the graphics software. We would also like to thank Tripos Associates for gents, amphiphilic gels, and salts will decrease the dielectric a software/hardware grant. This work was partially supported by Grants constant of the protein microenvironment. Furthermore, other HD 03110 and MH 33127 from the National Institutes of Health. folded domains of the same protein may radically alter the exposure ofthe coiled-coil interface to water. For example, the 1. Crick, F. H. C. (1953) Acta Crystallogr. 6, 689-697. C-terminal a-helix of bovine pancreatic trypsin inhibitor is 2. Schultz, G. E. & Schirmer, R. H. (1979) Principles ofProtein capped by a serine backbone hydrogen bond (N cap) within the Structure (Springer, New York). native folded protein (19). This hydrogen bond is stable during 3. Cohen, C. & Parry, D. A. D. (1990) Proteins Struct. Funct. free molecular dynamics stimulations of the whole protein in Genet. 7, 1-15. water; it is lost, however, when the isolated helix, shorn of 4. Lau, S. Y. M., Taneja, A. K. & Hodges, R. S. (1984) J. Biol. surrounding structure, is Chem. 259, 13253-13261. dynamically simulated in water (A.T., 5. Cohen, C. & Parry, D. A. D. (1986) Trends Biochem. Sci. 11, unpublished observations). 245-248. Ifhydrophilic amino acids are involved in the formation of 6. McLachlan, A. D. & Karn, J. (1983) J. Mol. Biol. 164,605-626. coiled coils, their presence in the interhelical contact zone 7. O'Shea, E. K., Rutkowski, R., Stafford, W. F. & Kim, P. S. would augment any hydrophobic interactions and increase (1989) Science 245, 646-648. the stability of coiled-coil proteins in two different ways 8. O'Shea, E. K., Rutkowski, R. & Kim, P. S. (1989) Science 243, depending on the environment: The first would be to enhance 538-542. the stability of a coiled coil in a low-dielectric microenviron- 9. Landschulz, W. H., Johnson, P. F. & McKnight, S. L. (1988) ment such as the interior of a cell or a complex protein by Science 240, 1759-1764. 10. Oas, T. G., McIntosh, L. P., O'Shea, E. K., Dahlquist, F. W. forming an interhelical hydrogen bond. The second might be & Kim, P. S. (1990) Biochemistry 29, 2891-2894. to enhance the stability of the coiled coil in a high-dielectric 11. Vinson, C. R., Sigler, P. B. & McKnight, S. L. (1989) Science microenvironment by the "snorkeling" (20) of the charged 246, 911-916. end ofthe side chain out ofthe hydrophobic interface, leaving 12. Liou, H.-C., Boothby, M. R., Finn, P. W., Davidson, R., the hydrophobic portions of the side chain within. Nabavi, N., Zeleznik-Le, N. J., Ting, J. P.-Y. & Glimcher, The proposal that secondary, intermolecular hydrogen L. H. (1990) Science 247, 1581-1584. bonds may contribute to the structure of a coiled coil is 13. Clark, M., Cramer, R. D. & Opdenbosch, N. V. (1989) J. buttressed by several experimental observations: (i) (a) The Comput. Chem. 10, 982-1012. well-known role of secondary hydrogen bonding in DNA 14. Weiner, S. J., Kollman, P. A., Nguyen, E. T. & Case, D. A. (1986) J. Comput. Chem. 7, 230-252. triple-helix formation (21-24), (b) the recent discovery that 15. Weiner, S. J., Kollman, P. A., Case, D. A., Singh, U. C., secondary hydrogen bonds stabilize bifurcated "propeller" Ghio, C., Alagona, G., Profeta, S. & Weiner, P. (1984) J. Am. conformations in double-stranded DNA (25), and (c) the Chem. Soc. 106, 765-784. frequent occurrence of secondary hydrogen bonds in the 16. Ramachandran, G. N. & Sasisekharan, V. (1968) Adv. Protein well-refined crystal structures of proteins and small mole- Chem. 23, 283-437. cules (26); (it) the postulated role of primary hydrogen 17. Lee, B. & Richards, F. M. (1971) J. Mol. Biol. 55, 379-400. bonding between polar side chains and the peptide backbone 18. Baker, E. N. & Hubbard, R. E. (1984) Prog. Biophys. Mol. in the formation of N- and C-terminal a-helical caps (27, 28); Biol. 44, 97-179. (iii) the calculations of hydrophilic-hydrophobic amino acid 19. Richardson, J. S. (1985) Methods Enzymol. 115, 359-380. 20. Segrest, J. P., De Loof, H., Dohlman, J. G., Brouillette, C. G. frequencies in the a-helical dimer-forming regions of a-fi- & Anantharamaiah, G. M. (1990) Proteins Struct. Funct. brous proteins (3); (iv) the observation that lysines (and Genet. 8, 103-117. asparagines) are often found within the hydrophobic interface 21. Hoogsteen, K. (1959) Acta Crystallogr. 12, 822-823. of the internal coiled coils of myosin (6). 22. Christophe, D., Cabrer, B., Bacolla, A., Targovnik, H., Pohl, As detailed above, analysis of the dimer-forming regions of V. & Vassart, G. (1985) Nucleic Acids Res. 13, 5127-5144. leucine zipper proteins suggests the presence of two possible 23. Lyamichev, V. I., Mirkin, S. M. & Frank-Kamenetskii, M. D. amino acid motifs, seven residues long, each beginning with (1986) J. Biomol. Struct. Dynam. 3, 667-669. leucine. The first is a hydrophobic motif wherein the fifth 24. Hanvey, J. C., Shimizu, M. & Wells, R. D. (1988) Proc. Natl. amino acid ofeach leucine heptad would be hydrophobic (the Acad. Sci. USA 85, 6292-6296. 25. Coll, M., Frederick, C. A., Wang, A. H.-J. & Rich, A. (1987) 4,3 repeat), implying that the helical dimer is stabilized only Proc. Natl. Acad. Sci. USA 84, 8385-8389. by hydrophobic interactions. Such a pattern, however, is 26. Burley, S. K. & Petsko, G. A. (1988) Adv. Protein Chem. 39, found in only 50%o of possible heptads. The second, our 125-189. alternative motif, predicts that the fourth amino acid of each 27. Richardson, J. S. & Richardson, D. C. (1988) Science 240, leucine heptad is polar, a pattern that is found in nearly 90%o 1648-1652. of possible heptads and that suggests the formation of inter- 28. Presta, L. G. & Rose, G. D. (1988) Science 240, 1632-1641. Downloaded by guest on October 1, 2021