PROTEINS: Structure, Function, and Genetics 14:425-429 (1992)

REVIEW ARTICLE ~

What Is the Pitch of the a-Helical Coiled Coil? George N. Phillips, Jr. Department of Biochemistry and Cell Biology and the W.M. Keck Center for Computational Biology, Rice University, Houston, Texas 77251 ABSTRACT The a-helical, coiled-coil protein motif is increasingly recognized in a variety of functional classes of proteins. The pitch of a coiled coil, or rate of winding of the a-helices around each other, is a key determinant of both intra- and intermolecularinteractions. Experimental measurements of the pitch of parallel two-strandedcoiled coils of muscle proteins, and examination of the recently determined structure of another two-stranded coiled coil, the GCN4 transcription factor protein, suggest that the pitch has an average value of about 140 hi. This value is consistent with the observed number of residues per turn in a-helices of globular proteins, the determinant of the interhelical packing within the coiled-coil motif. An understanding of the structural determinants of this value for the pitch and possible variations will be important in defining the interactions of coiled-coil proteins with other macromolecules. o 1992 Wiey-Liss, Inc. Key words: coiled-coil protein motif, proteins, molecular interaction, interhelical packing Coiled-coil proteins represent common motif found in the apparently finite repertoire of protein folds. According to one estimate, more than 200 coiled-coil proteins have been identified from sequences in the GENBANK.ll In a coiled coil, hydrophobic amino acid side chains from two (or more) a-helices interlock to stabilize the association of the helices. Because of the regular geometry of the ahelix, a pattern of hydrophobic residues in a “heptad” pattern, with the first and fourth residues being hydrophobic, is necessary for this tertiary structural motif. The other positions in each heptad repeat are usually hydrophilic, thus following the usual hydrophobes-inside, hydrophiles-outside paradigm for water soluble proteins. This coiled-coil motif has been found experimentally in proteins from a wide variety of organisms and has a wide variety of functions.3v4This class of proteins was originally recognized as the “k-m-e-f” (keratin, myosin, epidermin, fibrinogen) group of proteins by Astbury,’ has been found in viral and 0 1992 WILEY-LISS,

INC.

bacterial surface antigens, cytoskeletal proteins, catabolite activator protein,” and has been recently rediscovered as a “leucine zipper” motif in transcription factors of the bZIP class. The key aspect of the tertiary structure of a coiled coil is its pitch, or rate of winding of the a-helices around each other (see Fig. 1).The pitch determines the azimuthal disposition of the amino acid side chains in the overall structure, and hence determines the electrostatic structure and interactions with other proteins and nucleic acids. The pitch is especially important in the fibrous proteins of muscle because it sets the side-to-side packing in selfassembled, higher order aggregates and, in the case of tropomyosin, its regular, periodic interactions with actin. Fraser and MacRae” have mathematically described the relationship between the pitch and radius of the coiled coil, P and ro, and the a-helical versus sequence-derived twist differential and the axial rise per amino acid, At and h. This relationship is given by

P = (2.rr/At)[h2- (roAt)2]1’2. The twist differential can be related directly to the number of residues per turn of the a-helix by At=2.rr(lla-1/3.5), where a is the number of residues per turn of the a-helix. The qualitative aspects of this relationship are illustrated in Figure 1. (The fact that the twist differential, and hence the pitch, turn out to be negative means simply that the coiled coil is left handed.) Pauling and Corey have already noted the steep dependence of P on At, because of the requirement to maintain the meshing of hydrophobic residues. Thus the number of residues per turn in the a-helix is a critical determinant of the pitch of the coiled coil. It is important to point out that there is no reason why the value of a must be exactly 3.6, as commonly assumed throughout the biochemistry community. In fact, Chothia and co-workers’ measured the number of residues per turn from a num-

Received April 13, 1992; revision accepted July 23, 1992.

426

G.N. PHILLIPS, JR.

Fig. 1. (Upper left) The pitch of the coiled coil is a measure of the rate of winding of the a-helices around the central molecular axis, and is defined as the distance required to complete one full turn of the a-helices around one another. One repeat length is shown along the coiled coil of the muscle protein, tropomyosin.” The pitch determines the ultimate arrangement of surface side chains, and hence sets the kinds of interactions the coiled coil can have with other molecules. (Lower left) Illustration of the relationship between the number of residues per turn in the a-helix and the disposition of hydrophobic residues within the coiled-coil heptad repeat. a-Helices have about 3.64 residues per turn, on average. The heptad repeat alternates with a hydrophobic residue every three, then every four residues. As illustrated, this threefour-threefour repeat produces a zig-zag pattern of hydrophobic

residues along the a-helix. But because the average recurrence of hydrophobic residues is 3.5 and not 3.64, there is a net lag of the hydrophobic residues with respect to the a-helical geometry. This “twist differential” is a strong determinate of the meshing of hydrophobic residues at the interface of a-helices in the coiled coil, and hence is a critical determinate of the pitch of the coiled coil. (Right) Illustration of the method used to determine the pitch of the GCN4 structure. First, the angular change between equivalent heptad positions, AA, is determined by a set of vectors that is perpendicular to the coiled coil axis. Then, the axial change in distance, AP, corresponding to this angular change is calculated. These two numbers can be used to extrapolate the distance required to make a full turn of the a-helices around one another according, to the equation in the text.

ber of high resolution X-ray structures of globular proteins and determined that there is a wide distribution of values of a from 3.45 to 3.85 with a mean value of 3.64 2 0.09. Substitution of this mean value into the above formula with h = 1.5 and r, = 4.6512 gives a coiled-coil pitch of about 130 A, quite different from the original estimate of 180-190 A first given by Crick for two-stranded coiled coils7 and Pauling and Corey,13 assuming a value of 3.6 for a. As a historical note, the original paper by Pauling et al. describing the parameters of the a-helix used a figure of 3.7 for a. Both Pauling et al.14 and Crick‘ later used a = 1815, just because it was the ratio of two integers and hence simplified the layer line analysis of the diffraction of a-helices and coiled coils. (I hate to appear overly critical of their semi-

nal work on this topic, but Crick and Pauling and Corey also got the handedness of the coiled coil wrong, a natural consequence of wrongly thinking that a-helices were not right-handed, but lefthanded.) There have been several experimental studies aimed at the elucidation of the pitch of a-helical coiled coils. Early estimates by Cohen and Holmes appeared to confirm Crick’s original postulated value of 180 A for the coiled coil pitch of paramyosin, but this now seems unlikely in light of Elliott’s later workg on hydrated oyster adductor muscle (giving a value of about 140 A), results from tropomyosin, and the GCN4 structure described below. Phillips et a1.18-20 determined that the average pitch of the parallel, two-stranded tropomyosin molecule is 140

427

PITCH OF THE a-HELICAL COILED COIL

TABLE I. Coil Coil Pitches for Every Possible Heptad-Equivalent Pair in the 31 Residue GCN4 Fragment* 120 10 -

_

-

149 - 150

-

15 137

_ -

20

140 - 143

_ _ _ _ _

-

c

_ _ _ _

-

138 -

-

25 -

_ _

135 30 -

_ _ _ _ _

_ _ _ _ _ _ _ _ _

147 - 146

_ _ _ _ _

_ _ _ _ -

133 -

1 ~~~

~

*The C-terminal amino acid number of the heptad pair is given on the ordinate and the N-terminal amino acid number on the abscissa. The numbering scheme is as defined by Alber and co-workers.” “Stripes”of numbers come from averaging over 1 , 2 , 3 , or 4 heptad repeats, with 4 heptad repeats being at the lower left of the table and 1 heptad repeat running from the upper left to the lower right. Considerable variation can be seen in the single heptad-derivedvalues, but the numbers become more constant as the average pitch is determined over more heptads. The numbers shown in bold italic type should be excluded from the average because of special circumstances.Glycine 31 is not in an a-helical conformation and asparagine 16 is in a core position but is not hydrophobic.

*

5 A from low resolution X-ray crystallographic studies, and this value has been confirmed in a more recent 9 hl resolution study of tropomyosin in a new crystal form.23 The recent high resolution structure of a coiledcoil fragment of GCN4 from yeast allows a n accurate analysis of the coiled-coil pitch and its sequence specific variations. Unfortunately, the original analysis by Alber and co-workers” was not described in detail, nor does their result of 180 hl for the pitch agree with the recent experimental measurements of the pitch in fibrous proteins described above. Here a reexamination is reported of the pitch of the GCN4 leucine zipper fragment. The pitch of the coiled coil as seen in the GCN4 structure from X-ray crystallography has been determined using a more straightforward method than that reported by Albert et a1.” and the results are also considerably different from what they reported. Here, the pitch is derived from pairs of points separated by multiples of seven residues, corresponding to heptad repeats of the coiled coil. By using points separated by heptad multiples, the pitch can be obtained directly from the atomic coordinates, as described below and illustrated in Figure 1. The pitch

is calculated from the separation of the points as projected along the axis of the coiled coil, multiplied by 2 a and divided by the azimuthal angle (in radians) between lines drawn from these points to the axis of the coiled coil, according to the formulas described below:

AA

A,

P = 2a = (a,+b,)/2;

AP

=

lAPl / lAA B, = (a,+b,)/2

A, - B,; AP,

=

A P i I AP

1

COS-~([AP,x (A, x APJ1 . [AP,X (B,X APJ1 llW, x (A, x AP,)11 ([Mux (B, x AP,)lIl.

=

The coiled coil axis, AP,is thus defined as the line connecting the midpoints of lines drawn from an atom on one chain of the coiled coil across to the corresponding atom on the other chain of the coiled coil. The parameters, a,, b,, a,, and b, are the cartesian vector coordinates of the average atomic positions of the backbone atoms for the N-terminal amino acid in the a chain, the N-terminal amino acid in the b chain, the C-terminal amino acid in the a chain, and the C-terminal amino acid of the b chain, respectively. The values of the pitch of GCN4, as determined

428

G.N. PHILLIPS, JR.

from every possible pair of atoms separated by seven residues, are given in Table I. By averaging the atomic locations for the N, C,, C, and 0 backbone atoms for each residue, small errors associated with individual atomic locations have been reduced. Of course, the most accurate measures of the average pitch come from the measurements over the maximum number of complete heptads, as given in the lower left of the table. The overall average pitch of the coiled coil has also been calculated from the values in the table, both unweighted and weighted by the number of heptad repeats included in the calculation. The unweighted results, including all residues, are 143.0 A with a standard deviation of 13.3 A, and a standard error of the mean of 1.8 A; the heptad weighted results are 142.5 A with a standard deviation of 11.1A, and a standard error of the mean of 1.5 A. By excluding glycine 31 from the analysis, which is not in an a-helical conformation, and excluding values calculated from asparagine 16, which is in a core position but not hydrophobic, the weighted results are 144.0 A with a standard deviation of 6.9 and standard error of the mean of 1.0 with unweighted results of 144.3 for the mean, 8.1 for the standard deviation, and 1.2 for the standard error of the mean. Thus the average pitch of the coiled coil is precisely determined to within an A or so, but the pitch for any individual heptad varies from 120 to 160 A with a standard deviation of slightly less than 10 A. Other coiled-coil structures are also currently under structural investigation and further evaluations of the pitch of these molecules will be interesting. A structure for a similar fragment of GCN4 and a much longer 62 residue fragment has been reported by Pastore and c o - ~ o r k e r s ’using ~ 2D-NMR techniques.l 5 3 l 6 However, coordinates are not yet available for a similar analysis of the pitch from those data. It will also be interesting to see how the pitch of the coiled coil varies along the 400 A long tropomyosin structure (work in progress a t 3.5 A), but it already seems clear from low resolution studies that the average pitch will be close to that seen in the GCN4 s t r ~ c t u r e . ’It~ will also be interesting to see if molecular dynamics refinements and model building studies produce the right pitch based simply on physical chemical principles.’ There are numerous other measurements of the pitch of two stranded coiled coils under various degrees of dehydration,” but their relevance to the biologically functional states is not clear. The recurrence of measured values for the pitch of hydrated coiled coils around 140 A suggests that although there can be local distortions, for any coiled coil of at least several heptads the mean value of the pitch is conserved. This value for the pitch implies that the average number of residues per turn of the a-helix will be 3.63 in coiled coils, very similar to the value of 3.64 seen for globular proteins. Thus the

hydrogen bonding in the backbone of each helix appears to set the geometry of the coiled coil. Key questions now are to what extent the amino acid composition or sequence or degree of exposure to solvent can influence the number of residues per turn in the a-helix, and hence cause variations in the pitch of the coiled coil. Some answers to these questions are starting to become clear. For example, since paramyosin, tropomyosin, and GCN4 seem to have similar average pitches, the increased regularity of leucine in the bZIP proteins seems not to matter in defining the pitch of the parallel two-stranded coiled coils. Other factors may also affect the pitch to some degree, and as more coiled coil structures are examined, it will be interesting to ascertain the structural determinates of pitch variation. It will also be interesting to see how nature has made use of the periodicity that the coiled coil provides, in terms of tertiary and quaternary interactions. The fact is that it now seems time to graduate from the perfectly regular, “platonic” models of coiled coils of Crick, and Pauling and Corey to “real” structures of this widespread and important protein motif.

ACKNOWLEDGMENTS This work was supported by NIH Grant AR30274, the W.M. Keck Center for Computational Biology, and Grant C-1142 from the Robert A. Welch Foundation. I thank Prof. Tom Alber for providing GCN4 coordinates.

REFERENCES 1. Astbury, W.T. Croonian Lecture on the structure of biological fibers and the problem of muscle. Proc. Roy. SOC.134B: 303-328, 1947. 2. Nilges, M., Brunger, A.T. Automated modeling of coiled coils: Application to the GCN4 dimerization region. Prot. Eng. 4:649-659, 1991. 3. Cohen, C., Parry, D.A.D. a-Helical coiled coils-a widespread motif in proteins. Trends Biochem. Sci. 11:245-248, 1986. 4. Cohen, C., Parry D.A.D. a-Helical coiled coils and bundles: How to design a a-helical protein. Proteins 7:l-9, 1990. 5. Crick, F.H.C. Is a-keratin a coiled coil? Nature (London) 170:882-883, 1952. 6. Crick, F.H.C. The Fourier transform of a coiled coil. Acta Crystallogr. 6:685-699, 1953. 7. Crick, F.H.C. The packing of a-helices: Simple coiled coils. Acta Crystallogr. 6:689-697, 1953. 8. Chothia, C., Levitt, M., Richardson, D. Helix to helix packing in proteins. J . Mol. Biol. 145215-250, 1981. 9. Elliott, A,, Lowy, J., Parry, D.A.D., Vibert, P.J. Puzzle of the coiled coils in the a-helical protein paramyosin. Nature (London) 218:656-659, 1968. 10. Fraser, R.D.B., MacRae, T.P. “Conformation in Fibrous Proteins and Related Synthetic Polypeptides.” London: Academic Press, 1973. 1. Lupas, A,, Van Dyke, M., Stock, J. Predicting coiled coils from Drotein seauences. Science 252:1162-1164, 1991. 2. OShea, E.K., Klemm, J.D., Kim, P.S., Alber, T. X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science 254539444, 1991. 3. Pauling, L., Corey, R.B. Compound helical configurations of polypeptide chains: Structure of proteins of the a-keratin type. Nature (London) 171:59-61, 1953. 4. Pauling, L., Corey, R.B., Yakel, H.L., Jr., Marsh, R.E. Calculated form factors for the 18-residue 5-turn a-helix. Acta Crystallogr. 8:853-855, 1955.

PITCH OF THE a-HELICAL COILED COIL 15. Saudek, V., Pastore, A. Morelli, M.A.C., Frank, R., Gausepohl, H., Gibson, T., Weih, F., Roesch, P. Solution structure of the DNA-binding protein domain of the yeast transcriptional activator protein GCN4. Protein Eng. 4:3-10, 1991. 16. Saudek, V., Pastore, A., Morelli, M.A.C., Frank, R., Gausepohl, H., Gibson, T.The solution structure of a leucinezipper motif peptide. Protein Eng. 4:519-529, 1991. 17. Phillips, G.N., Jr., Lattman, E.E., Cummins, P., Lee, K.Y., Cohen, C. Crystal structure and molecular interactions of tropomyosin. Nature (London) 278:413-417, 1979. 18. Phillips, G.N., Jr., Fillers, J.P., Cohen, C. Motions oftropomyosin: Crystal as metaphor. Biophys. J . 10:485-502, 1980.

429

19. Phillips, G.N., Jr., Fillers, J.P., and Cohen, C. Tropomyosin crystal structure and muscle regulation. J . Mol. Biol. 192:lll-131, 1986. 20. Phillips, G.N., Jr., Cohen, C. Entry 2TMA in the Brookhaven Protein Data Bank, 1987. 21. Weber, I.T., Steitz, T.A. Structure of a complex of catabolite gene activator protein and cyclic AMP refined a t 2.5 A resolution. J . Mol. Biol. 198:311-326, 1987. 22. Whitby, F.G., Kent, H., Stewart, F., Stewart, M., Xie, X., Hatch, V., Cohen, C., Phillips, G.N., J r . Structure of tropomyosin at 9 Angstroms resolution. J. Mol. Biol. 227:441452,1992.

What is the pitch of the alpha-helical coiled coil?

The alpha-helical, coiled-coil protein motif is increasingly recognized in a variety of functional classes of proteins. The pitch of a coiled coil, or...
443KB Sizes 0 Downloads 0 Views