Four helix bundle diversity in globular proteins

J. Mol. Bzw. \1394) 236, 1356-1368

Four Helix Bundle Diversity Nomi

L. Harrislf-,

in Globular

Proteins

Scott R. Presnell’ and Fred E. Cohen112*3$

Departments of 1Pharmaceutical Chemistry ‘Medicine and 3Biochemistry and Biophysics University of California, San Francisco San Francisco, CA 94143-0446, U.S.A. Four helix.bundles are a common structural motif that can be observed both independently and as components of larger folding units. We examined 221 globular proteins of known structure for possible four helix bundles. Previous computational studies of four helix bundles have placed arbitrary restrictions on interhelical packing angles. In this study we develop a geometric definition of four helix bundles based in part on solvent accessibility criteria that permits the removal of constraints on interhelical packing. Based on the observed pattern of interhelical angles, a bundle taxonomy is presented. This formalism should provide a useful categorization method for future structural studies of proteins rich in a-helices. The helix-helix interactions within bundles were studied in detail. Central residues. contact normals, and skew angles all were observed to have non-random distributions. A simple geometric model was developed for the helix-helix interface to explain these findings. Analysis of the helix-helix interaction data collected in this work confirms t,he importance of including skew angles in models of helix packing, and should improve t,he accuracy of combinatorial strategies for the prediction of t,he tertiary structure of all-helical proteins. Additionally, the geometric properties observed in globular proteins provide insight into the structural organization of membrane spanning proteins. Keywords:

protein conformation; four helix bundle; structural supersecondary structure

motif;

1. Introduction

The four helix bundle is a simple protein structural motif found in many globular proteins. For Proteins fold to optimize the conformational this reason, several groups interested in protein preferences of amino acids subject to local and design have synthesized sequences in hopes of global constraints. The result is a compact polycreating a new four helix bundle protein (DeGrado peptide chain dominated by secondary structure et al., 1989; Hecht et al., 1990; Schafmeister et al., elements. X-ray crystallography and multi1993). Early studies of this motif suggested that dimensional NMR spectroscopy have been used to geometric and energetic constraints would favor a decipher the structures of more than 500 proteins. serpentine right-handed organization (Chou et al., While many proteins have unique structural 1988; Weber & Salemme, 1980). A more recent features, a-helices and/or /?-sheets commonly cluster analysis of proteins of known structure revealed into familiar assemblies called motifs. These include substantially more topological diversity within this the a/p barrel of triose-phosphate isomerase, family, with left-handed structures occurring mandelate racemase and approximately 20 other slightly more commonly than their right-handed proteins (Farber BEPetsko, 1990) and the jelly roll counterparts (Presnell & Cohen, 1989). In an topology observed in viral coat proteins and tumor attempt to follow the lead of Weber & Salemme necrosis factor. Extensive reviews of this subject are (1980), the survey by Presnell & Cohen (1989) was available (Chothia & Finkelstein, 1990; Richardson, limited to four helix bundles where the absolute 1981). value of the packing angle between interacting helices was <40”. However, it was clear that there was no specific preference for approximately parallel t Current address: Arris Pharmaceutical Corp., helix-helix interactions. Instead, a continuum of 385 Oyster Point Blvd, Suite 12, South San Francisco, four helix bundle structures was observed. CA 94080, U.S.A. We have completed a study of globular proteins $ Author to whom all correspondence should be addressed. of known structure that contain four or more 1356 0022-2836/94/101356-13

$08.00/O

0

1994 Academic Press Limited

Four

Helix

1357

Bundle Diversity

cc-helices

and identified those containing four mutually interacting helices. Bundles are found that bury substantial surface area through helix-helix interactions and exploit the known geometries for interhelical packing. The precise details of the interhelical interface are also explored. Contact normals are calculated

to

define

the

central

residue

from

each interacting helical face, and the deviation of the central residue from the contact normal is used to define a skew angle that further characterizes the relative geometry in helix-helix interactions (Cohen et al., 1979; Richmond & Richards, 1978). A simple geometric model is developed for the interface to explain the observed trends in contact normal lengths and skew angles. Finally, a more generalized taxonomy of four helix bundles is developed that should provide a useful categorization method for future structural studies of proteins rich in cr-helices. We find that the all anti-parallel arrangement of helices favored by most energy calculations (Carlacci & Chou, 1990a,b; Chou et al., 1988; Presnell & Cohen, 1989) and attributed to the interaction of neighboring helix dipoles (HOI, 1985; Sheridan et al., 1982) becomes less common as the packing angle between helices is allowed to widen. 2. Methods (a) Dahsel The Brookhaven Protein Databank (Bernstein el al.. 1977) was examined (January 1993 release). The subset of unique high resolution globular protein structures determined by X-ray crystallography (<2.5 A resolution) was st,udied. After the elimination of extremely similar struc-

tures (r.m.s.d.t <@5 A) and oligomeric structures beyond the structure

of a single monomer

chain, 221 structures

remained.

Figure 1. Illustration of the “waist constraint.” A plane is constructed through the center of mass of the bundle, and all 4 helices must pass through the plane.

with another from the bundle must lose at least 3% of its solvent-accessible surface area. This constraint must be fulfilled for at least 2 of the 3 possible pairs of helices in each bundle. Moreover, the sum of all 3 pairwise interactions must result in a 20% or greater loss of surface area when compared to the free-standing helices. These constraints yield a group of structures with an extensive network of interactions similar to that seen in the previously described “classical” 4 helix bundles. (d) Waist cmstraint When only (anti) parallel helix-helix interaction angles are considered, the surface area constraints suffice to define a set of 4 helix bundles. When the angular constraint is relaxed, some of the putative bundles found

appear to have little structural integrity. For these struc(b) Helix boundaries

To obtain a self-consistent set of secondary structure definitions, the automatic assignment program DEFINE (Richards & Kundrot. 1988) was used to assign the N and c’ termini of the a-helices. Helices less than 5 residues in lengt,h were not considered. It was our impression that DEFIKE, rather than the DSSP algorithm (Kabsch & an assignment of a-helical Sander, 1983), provided secondary structure most closely resembling crystallographers’ assignments. (c) Identijcation

of four helix bundles

In a protein with /c helices, there are k!/(k-4)! x 4! potential four helix bundles. Our dataset of 221 proteins contained 44,000 potential bundles. Following the lead of Richmond 8r. Richards (1978) and Presnell & Cohen (1989), the change in solvent-accessible surface area on forming the bundle from the isolated helices was used to define a cluster with potential intrinsic stability. When considering pairwise interactions, each helix, when paired t Abbreviations used: r.m.s.d., root-mean-square deviation; MHC, major histocompatibility complex; GM-CSF, granulocyte-macrophage colony-stimulating factor.

tures, a hypothetical band tightened around the waist of the molecule would show that at least 1 helix is not strongly associated with the other 3. In many cases, these putative bundles passed the surface area constraints largely because of interactions between the end of one helix and the end or middle of another helix. The waist constraint was developed to reject some of the lesscohesive putative bundles in a consistent manner (Figure 1). First, the average helix axis vector (unsigned) was computed from the 4 helix axis direction vectors and a plane normal to this vector (representing the “waist” of the bundle) was constructed through the center of mass of the bundle. For a bundle to pass the waist constraint, the termini of each helix must be located on opposite sides of this plane (i.e. each helix must pass through the plane). In practice, t,he waist constraint is less C.P.U. intensive than the solvent accessible surface area constraint and is applied first when searching for bundles. (e) Dejlnitions

of helix, helix-helix

and bundle geometries

Helix axis (Hi). This is a vector describing the direction of the N-G direction of a helix computed using the adaptive helix parameter method of Kneller (1988). angle (a,). IQ,1 = cos-’ Helix-helix interaction

[(Hi.Hj)]/[(llHi II II Hjll)]. The sign of Rij is positive if and only if Hi. (a, x Hj)>O.

Four

1358

Helix

Bundle

Diversity

Skew angle

Ca

Centralresidues

01centralresidue

Contactnormal

Figure 2. Illustration of central residues, contact normals and skew angles. Cylindrical helices are shown as large circles from an axial projection. Individual residues are approximated by spheres and shown as smaller circles in projection. The contact normal is the line between the c” atoms of the central residues. The skew angle is the angular displacement of the central residues wit,h respect to the contact normal.

Central residues. If {CTk},.. , n are the residues in helix i and {CJJI= 1.m are the residues’in helix j. then k’ and 1’are the central residues for the interaction between helices i and j when d(C$, C;r) 0 (Figure 2). Handedness of 4 helix bundles. Following the conventions of Presnell & Cohen (1989), a 4 helix bundle is right handed if (C,, x C,,)*H, >0 and left handed otherwise. Overhand connections. These are connections between consecutive helices that traverse the diameter of the bundle. In general, overhand connections are present between helices i and i+ 1 when Hi.Hi+, >0 (Figure 3).

Figure 3. Overhand (long) and underhand (short,) connections in 4 helix bundles. Overhand connections bet.ween adjacent helixes traverse the length of the bundle, while underhand connections stay at one end of the bundle. A. Four helix bundle with all underhand connections. B. Four helix bundle with one overhand connection.

3. Results and Discussion The original work on helix-helix packing by Crick (1953) suggested that. two helix-helix interaction angles were likely, a parallel arrangement (II) with interhelical angle around +20” and a perpendicular geometry (I) with interhelical angle around -70”. Although subsequent studies of proteins of known structure have subdivided these groups (Chothia et al., 1977) and extended the allowed angles fol parallel interactions (Presnell & Cohen, 1989), the essential features of Crick’s original argument hold. For an acute interhelical angle Q, IQ1 =40” provides a useful separation between the parallel and perpendicular groups. If four helix bundles are constructed from four overlapping sets of helix pairs, then six classes of four helix bundles (based on patterns of interhelical angles) are expected (see Table I and Figure 4): (1) Square. All four helix-helix interaction angles

are parallel (II, II, II, II). (2) Splinter. All but one of the four helix-helix interaction angles is parallel (II, (I, )I, I). (3) X. Alternating pattern of parallel and perpendicular helix-helix interactions around the perimeter of the bundle (II, I, 11, I). (4) Unicornate. A pair of parallel helix-helix interactions followed by a pair of perpendicular helix-

Table 1 Classes of four helix bundles

Bundle type Square Splinter X Unicornate Bicornate Splayed

Helix-helix packing

Observed frequency

II II II II II II II 1 II 1 II 1

13 11 26

II II 1 1

29

1111

30 9

II -L 1 J-

Expected frequency (binomial distribution) p=O48) 6 27 22 22 32 9

[(P? x 1181 [4p’(l-p)x 1181 [3&l -P)~ x 1181 [3pz(1-p)‘x 1181 [4p(l-~)~x 1181 [(l-p)4X 1181

All anti-parallel helix packing 8 2 10 6 9 4

Other bundles 5 91 I6 23 21 5

1359

Four Helix Bundle Diversity SQUARE

UNICORNATE

SPLINTER

BICORNATE

Figure 4. There are 6 classes of bundles as defined by the pattern of interhelical angles. Prototypes and representative bundles of each type are shown. helix interactions resulting in one helix poking out of the barrel (II, II, 1, I). (5) Bicornate. All but one of the four helix-helix interactions is perpendicular. This creates a structure with two protruding helical horns (II, I, I, I). (6) Splayed. All four helix-helix interactions are perpendicular. The result is a bundle with one very open end (I, I, I, I). From an analysis of 221 moderate to high resolution unique crystal structures, the result was a set of 138 four helix bundles. However, 20 of the algorithmically “acceptable” bundles contained geometric features inconsistent with our visual expectation of a four helix bundle (Figure 5). These four helix “bungles” frequently included three coplanar helices or two nearly colliding helices and it appeared that a substantial part of the helix-helix interactions could be attributed to the terminus of one or more helices. In the case of glucoamylase (Aleshin et al., 1992), a fifth helix (lgly: 126-140) seemed to occupy the center of the bundle (lgly: 52-70, 74-86, 154-170, 185-207); the bundle managed to pass the area-loss criteria because of terminus interactions. Helix pairs that form aa corners with extensive interactions between the termini have been studied in detail by Efimov (1984). Although these bungles failed to conform to our expectations, the geometric conclusions of our analysis are not altered by the inclusion or exclusion of these structures. The bungles were not included in further analyses.

A) Glucoamylase (1glY)

B) Glycogen Phosphorylase (Wb)

Cy?ochryt;y;j

Peroxidase

Figure 5. Bungles are groups of four helioes that pa& the bundle constraints but lack the structural integrity of bundles. This Figure shows several examples of bungles. A. The helix in the middle of the bundle does not belong to the bundle. B. Three of the helices are coplanar, with the 4th roughly perpendicular to the others. C. Several of the helices are about to collide; this is is not an appropriate type of interaction in bundles.

1360

Four Helix Bundle Diversity

3-Isoproylmalate

Dehydrogenase

(lipd)

Bovine

Calbindin

D9K

(Minor

A form)

(4icb)

B A Figure 6. Two examples of bundles complemented by the rest of the protein. A. An X bundle that appears on the surface of 3-isopropylmalate dehydrogenase (lipd). (B) Bovine calbindin (4icb) is a stand-alone splayed bundle.

The remaining 118 four helix bundles (Table 2) can be grouped into the six classes described above. Parallel (48%) and perpendicular (52%) helix-helix packing are almost equally likely. If there were no energetic bias favoring one arrangement over another, we would expect to find six square bundles (118. (0.48)4) a,nd the other five classes of bundles would follow a binomial distribution. The expected and observed frequencies of each class of bundle are shown in Table 1. Although reasonable agreement is observed between the values expected from this simple binomial model and the actual frequencies of the different classes of bundles, it seems clear that the square bundles must be more stable than their splintered relatives. Figure 4 shows examples of each of the six classes of bundles. Square bundles are typified by three bundles found in this study. Ferritin (Lawson et al., 1991; Ifha: 1343, 48-77, 95-125, l26-l59), a lefthanded bundle with one overhand connection, and cytochrome c’ (McRee et al., 1990; lbbh: 4-28, 35-52,81-103, 106123) a right-handed bundle with no overhand connections, are examples of bundles which encompass the entire protein structure (“stand-alone” bundles). These structures are probably sufficiently stable in isolation because of an extensive coverage of the hydrophobic core created by the relatively parallel helical interactions. Another square bundle found in cytochrome P450-camphor monooxygenase (Poulos et al., 1987; Bcpp: 128-143, 154-168, 234-267, 359-378) makes up only part of a larger protein structure. Splinter bundles might best be characterized as a packing variant of square bundles. Once again,

there are examples of stand-alone bundles, such as GM-CSF (Diederichs et al., 1991; lgmf: 12-29, 54-66,73-87, 102-l 15), and bundles that are part of a larger structure, such as a bundle found in glucoamylase (Aleshin et al., 1992; lgly: 52-70, 74-86, 126-140, 154-170). These structures are not as geometrically regular as the square bundles, but seem to be similarly stable in isolation. X bundles are named for their two pairs of crossing helices. Within this study, X bundles were only found as part of a larger structure: for example, 3-isopropylmalate dehydrogenase (Imada et al., 1991; lipd: 11-30, 287-301, 304-322, 332345), and the A-chain of oxygenated hemoglobin (Shaanan, 1983; lhho: 3-18, 52-73, 94-114, 118138) both contain examples of X bundles. In terms of Crick’s knobs in holes idea, this structure would leave many open knobs and holes, and therefore invite interactions from other helices. For example, Figure 6A shows the 3-isopropylmalate dehydrogenase (lipd) X bundle complemented by the rest of the structure. Unicornate bundles contain one helix that forms a large angle with the other members of the bundle: examples are found in glucoamylase (Aleshin et al., 1992; lgly: l-22, 317-339, 367-387, 415431), isoenzyme 3-3 of glutathione S-transferase (Liu et al., 1992), Igst; 89-116, 123-142, 153-170, 177-190), and T4-lysozyme (Rose et al., 1988; llyd: 2-12, 5981, 92-106, 142-156). Bicornate bundles contain two helices at large angles, but opposed across the bundle rather than adjacent as with the X bundle type. Examples include neutral protease (Stark et al., 1992; lnpc:

Four Helix Bundle Diversity

Table 2 Four helix bundles protein (chain) I ald lbbh (A) IeWI I tV:* lwa lfba 1RlY lf4Y 1PlY IdY lf4Y lgly lgly IdY ‘dY Ids I.& 4-N (A) I gox kpb kpb ‘gp’, ‘gpb lgst (A) I@ (A) lgst (A) lgst (A) I hds (A) lhds (A) I hds (A) lhho (A) lhho (A) lhho (A) I i pd lb 1lyd I Iyd I lycl I lyd 1lyd I lyd I lyd I Iyd lmbd I mhd Inpc I npc lnpc I np< I npc lnpc Inpc lphh I phh Iphc (A) lphc (A) lphc (A) lphc (-4) I r69 lthb (A) lthh (A) lthb (A) lthb (A) lutg 256b (A) zcpp “CPQ

2CPP 2cro

Helices in bundle 35-45,51-65, 306-314, 319-340 4-28, 35-52, 81-103, 106-123 2-14, 52-73, 93-l 12, 117-136 19-32, 52-73, 93-112, 117-136 52-73. 7688, 93-l 12, 117-136 1343.48-77,95-125. 126-159 l-22, 317-339, 367-387, 415-431 52-70. 74-86, 126-140, 154170 52-70, 74-86, 126-140, 185-207 52-70. 126-140, 154-170, 18.5207 52-70. 185-207, 24.5256, 41.5431 12&140. 154-170, 185-207. 210-225 245-256, 271-284. 317-339, 347-355 245-156. 27 l-284, 3 17-339. 367-387 245-256. 271-284, 317-339. 41.5431 2-K&256. 317-339. 367-387, 415-431 271-284. 317-339, 347-355. 367-387 12-29. 5446. 73-87, 102-l 15 5-l7,88-100.308-318,319-342 21-37, 47-78, 94-103, 104-116 289-314. 328-333, 344-355, 36&372 496-507. 517-524, 527-553, 793-808 575-593, 735-748, 758-767, 777-792 13-24, 71-84.89-116. 153-170 13-24.71-84. 153-170, 177-190 13-24.71-84. 153-170, 190-199 89-116, 123-142, 153-170. 177-190 3-15. 52-72. 9&I 14. 118-137 20-28. 52-72. 94-114, 118-137 s-72, 75-81. 94-114, 118-137 3-18. s-73. 94-l 14. 118-138 20-37. 52-73, 94-114, 118-138 M-73. 75-81, 94-114, 118-138 I I-30. 287-301. 304-322, 332-345 244. 54-79. 86-125, 130-163 2-12. 59-81. W-106, 107-113 “-12, 59-81. W-106, 142-156 P-12. W-106. 12.5-135, 142-156 2-12, 92-106. 142-156. 158-162 8491, W-106, 114-124, 125-135 84-91. X-106, 114-124, 14e-156 s-106. 114-124. 125-135, 142-156 W~lO6. 12.5-135. 142-156, 158-162 3-l5,58-77. 100-120, 124-150 20-36, 58-77, 100-120, 124-150 137-153. 16&182. 231-247, 260-274 137-153, 160-182, 260-274. 281-297 16O-182,231-247. 260-274, 281-297 160-182. 231-247, 260-274. 301-313 16Cl82. 231-247. 281-297. 301-313 160-182, 26G274. 281-297, 301-313 231-247. 26C274, 281-297, 301-313 II-25.50-60. 61-68, 101-116 I l-25, 50-60. 101-l 16. 297-314 12-27.44-54. 106-125. 217-242 12-27. 86-i-104, lOGi-125. 217-242 12-27, 106-125. 171-187, 217-242 86-104. 106-125, 171-187.217-242 l-14, 16-25, 44-53, 55-61 3-18.20-36. 95-114, 118-138 3-18, 52-73, g&114. 118-138 20-36.52-73.95-114, 118-138 52-73, 75-81, 95-114, 118-138 3-15, 17-26, 31-46, 49-66 2-20. 2231, 5&82. 83-105 128-143. 154-168, 234-267, 359-378 154-168. 173-186. 192-214. 234-267 154-168. 173-186, 234-267, 359-378 l-14, 16-25, 27-37, 44-53 I-14, 27-37, 44-53, 55-61

Bundle type Bicornate Square7 X X Squaret Unicornate Splinter X Unicornate X Splinter &ornate X Splinter Unicornate Unicornate Splint& Unicornate Splayed Unicornate X Uicornate Splinter l&ornate Bicornate Unicornate X Bicornate X X Bicornate X X Squaret Bicornate Unicornate Square Unicornate Square $L quare Square Unicornate X Ncornate Bicornate Bicornate Bicornate Bicornate Unicornate Unicornate Unicornate Splinter X Unicornate Unicornate Square Splayed Splayed X Bicornate X Splint&f Square? S&are Unicornate Uicornate Uicornate Splayed

Antiparallel?

Number overhand

Four Helix Bundle Diversity

1362

Table 2 (continued) Helices in bundle

Protein (chain) 2CYP 2hmq (A) .Zlhl 2lhl 21hb 2scp (A) 2scp (A) 2scp (A) 2ser (E) etec (E) 2tsl 2te.l 3grs 3lad (A) 3tln 3tln 3t1n 3tln 3tln 3tln 3tln 4blm (A) 4blm (A) 4blm (A) 4enl 4enl 4icb

,

5CtS 5CtS

5cts 5cts 5cts 5cts 5cts 5cts 5ct.s 5cts 5cts 5cts 5CtS 5Cts

5cts 5cts 5cts 5cts 5pal 5tnc 7icd

15-34. 4235. 85-93, 103-120 21-38, 40-64. 69-87, 90-108 4-18, 22-37, 57-74, 107-123 22-37, 57-74, 107-123. 127-147 30-46, 67-83, 11.%128, 131-147 I-16, 71-84. 89-94, 146-160 24-39, 44-56, 95-104. 112-124 9.5-104, 112-124. 130-138, 146-160 12-20, 219-238. 242-254. 269-275 19-24, 223-240.244-256, 272-279 247-257, 262-27 1. 274-288, 292-307 247X257.262-271, 292-307, 308-319 63-80. 96-122. 176-183, 196-210 52-71. 86-l 13, 169-175, 189-903 67-88, 136-146, 159-181,259-273 67-88, 159-1811 230-246. 259-573 159-181. 230-246, 25S273. 280-296 159-181. 230-246. 259-273, 30&314 159-181. 230-246. 280-296. 300-31-I 159-181. 259-273, 280-296, 300-314 23C246, 259-273, 280496, 300-314 71-83. 118-129. 131-143. SO&flS 71-83. 118-129, 182-195. 20&214 71-83, 144-156. 182-195. 200-214 85-98, 106-126, 351-366, 381-391 128-138. 351-366, 381-391,402420 2-15. 24-35, 4.5-54, 62-67 70-78. 88-99.221-236.327-340 88-99, 103-118, 166-182, 402416 88-99. 166-182. 257-270, 402416 88-99. 166-182, 257-270.402-416 121-131, 13&148, 182-194, 392402 136-148. 166-182. 257-270, 402-416 136-148. 182-194.208-218, 392402 136-148. 182-194, 257-270, 392402 136-i-148, 24%255, 257-270, 392-402 166-182. 242-255, 257-270, 402416 182-194, 208-218. 221-236, 392402 “08-218. 22-236. 373-386, 392402 VI-236 1 276-291, 373-386, 392-W 221-236. 327-340.344-365, 373-386 221-236, 327-340, 373-386. 392-402 276-291.297-311, 344-365, 373-386 276-291, X7-340, 344365, 373-386 276-291, 344365, 373-386, 392402 25-34, 39-5 1, 66-7 I, 98-l 09 2-14, 15-28, 41-50, 74-102 37-58. 353-368, 369-387, 404416

I3undlr type

Antiparallel !

Splayed Squaret Bicornate Bicornate Noornate Splayed Unicornate Splayed Ncornate Unicornate Bicornate &ornate )I Splinter Bioornate Ncornate x

.\. .

Unicornate s llnicornate Unicornate I’nicornate Bicornate x Bicornate Bplayedt I’nicornste Unicorn&e llnicornate Unicornate Square >I Splinter x Splinter Splinter Unicornate x s Square Llnicornate Bicornate Unicornate s Bicornate Splayed x

Y s Y s Y s s s 1’ s T s s uI Y s s s

Number ovrrllurltl

1 0 1 2 1 0 0 0 1 I 0 I 1 1 I I I 2 2 0 0 0 0 2 2 3 0 I I I I 0 I 1 I I 0 2 0 I 0 I I 1 0 1 1 I

t Stand-alone bundle

137-153, 160-182, 260-274, 281-297), isoenzyme 3-3 of glutathione S-transferase (Liu et al., 1992; lgst; 13-24, 71-84, 153-170, 190-1991, and cytochrome P450-camphor monooxygenase (Poulos et al., 1987; Bcpp: 128-143, 154-168, 234-267, 359-378). Splayed bundles show little geometric symmetry, yet create a substantial hydrophobic core owing to helix packing as quantified by accessible surface area loss. Examples of this bundle type include sarcoplasmic calcium binding protein (Vijay-Kumar & Cook, 1992; 2scp: 1-16, 71-84, 89-94, 146-160), and bovine calbinden (Svensson et al., 1992; 4icb: 2-15, 24-35, 45-54, 62-67). Bovine calbindin may be considered an example of a stand-alone bundle (Figure 6B).

The original observations of Weber & Salemme (1980) suggested that square four helix bundles should be right-handed. Subsequent work by Presnell & Cohen (1989) on a larger set of square four helix bundles revealed that left and righthanded structures were equally likely. Within the expanded set of 118 four helix bundles, 67 are lefthanded and 51 right-handed. Weber & Salemme (1980) asserted that sequentially consecutive or-helices in a four helix bundle would pack in an antiparallel fashion to create an entropically favored short loop structure. Presnell & Cohen (1989) found a bias towards these serpentine structures with short loops but noted several examples of long (overhand) connections that traversed the entire long axis of the molecule. For the 118 general-

Four Helix Bundle Diversity ized four helix bundles, short connecting loops (256/ 354) are 2.6 times more common than their overhand (98/354) counterparts. This could be due to the relative entropic penalty for creating an overhand loop connection or reflect sampling bias toward smaller structures in the current version of the Protein Databank. While some researchers have suggested that helix dipoles play little or no role in dictating bundle geometry (Gilson & Honig, 1989), there is evidence that electrostatic interactions, including helix dipole-helix dipole interactions, could help account for the topological preferences of square four helix bundles (HOI et al., 1978: Presnell & Cohen, 1989). These interactions would favor antiparallel packing between neighboring helices. The experimental work of Robinson & Sligar (1993) on cytochrome P 562 suggested that the strength of the dipoledipole interaction is 0.6 kcal/mol per helix pair. For these initial observations are square bundles, confirmed in this work. Eight of 13 square bundles contain this rest,ricted packing arrangement. If there were no energetic preference for an antipa.rallel helix-helix interaction, one would expect only one in eight bundles to exhibit the all antiparallel geometry. Although stabilization is associated with antiparallel dipole-dipole interactions, some parallel helix pairs are expected as well. For example, parallel helix pairing is central to the dimerization of leucine zippers (O’Shea et al., 1991). The strength of a dipole-dipole interaction tends to fall off in proportion to the cosine of the angle between the dipoles. Since the helix dipole is coincident with the helix axis, it would be expected to play a much smaller role in bundles containing perpendicular helix packing. In fact, only 39 of the 105 non-square bundles show the all antiparallel arrangement. If the helix dipole had no impact on these structures, only 13 of the 105 non-square bundles would be expected to adopt the all antiparallel structure (Figure 7). From this extended set of all-helical assemblies, it is possible to re-examine the geometric features of helix~helix packing. Nuch has been written about the abilit,y of two cc-helices to fit together to form a close packed constellation of side-chains that exclude solvent (Chothia et al., 1977; Crick, 1953; Richmond & Richards, 1978). A histogram of the interhelical angles seen in the 472 helix pairs that comprise the 118 four helix bundles is presented in Figure 8. The distribution is bimodal with a peak at + 24” and a second peak at. -46” with a shoulder at - 65”. The + 20” interaction angle was anticipated by Crick (1953) and reinforced by the efforts of subsequent investigat,ors. Crick anticipated a second favorable interaction angle of -70”. Chothia et al. analysis of ridge-into-groove (1977), in their packing, suggested a favorable interaction at + 19” and predicted that -60” and -82” would form the centers of distinct angular subsets for helix-helix interactions. This simple ridge-into-groove model for helix packing continues to be relevant. However, the Efimov (1979) layer model of the packing of

1363

Figure 7. The distribution of all-antiparallel bundles IJUYU.Sother bundles, for each bundle type. Allantiparallel bundles are shown as black bars, and all other bundles are shown as gray bars.

a-helices that attempts to include side-chain dihedral angle preferences is less successful. This work also anticipated three classes of helix-helix packing, but the most likely interaction angles were supposed to be 90”, 30” and -30”. Weber & Salemme (1980) analyzed the small set of square four helix bundles available at that time and demonstrated that the helix packing was “quite intimate (with) the mean adjacent interhelix axis distance of closest approach being 968, with a standard deviation of 1.4 A”. The contact normal length for the expanded set of four helix bundles is 10.2( k2.1) A. (Our definition of contact normals is slightly different from that of Weber & Salemme (1980); recalculation of the average contact normal length using their definition confirms their findings.) Little variation of the average contact normal length is seen between the subtypes of four helix bundles. The average contact normal length is smallest for the splayed bundles (190 A) and largest for the X bundles (105 A). Consistent with this result, little variation is observed in the average

.m 80 -70 .a -54 4

Jo ..m .,o 0

10 20 30 40 YI Bo 10 m

hmmo81 ug!a (a.ww,

Figure 8. The distribution of interhelical angles. Peaks are at -65”, -46” and +24”.

Four Helix Bundle Diversity

1364

Figure 9. The predicted half contact normal (by residue type) is plotted against the cube root of residue volume. Half contact normals were predicted by linear regression. based on the observed contact normals between pairs of residues. The residue volumes are from Gregoret & Cohen (1990).

contact normal length as a function of interhelical packing angle. However, half contact normal length (as predicted by linear regression) does appear to increase as a function of central residue volume (as determined by Gregoret BE Cohen (1990; Figure 9, Table 3). Richmond & Richards (1978) defined the central residues for an interacting pair of helices as the contact residues between the two helices. These residues are usually close to the contact normal or occasionally closest to the middle of the interaction site. Richmond & Richards (1978) were able to predict the location of plausible central residues Table 3 Half contact nflnnals Amino acid type A R x D (’ : C l-i I I, K M F P s T W Y V

Predicted Half contact normal? $3 -.I 1.7 52 43 43 43 43 69 58 62 49 5.1 57 52 48 56 54 51 53

t These half contact normals were predicted by linear regression, based on the observed contact normals between pairs of residues.

Figure

10. The dist,ribution

are at -24”

and

of skew angles.

The peaks

+Z3”.

from an ana.lysis of amino acid sequence and the location of the a-helices. Cohen and co-workers (Boissel et al., 1993; Cohen et al., 1986, 1979; Curtis et al., 1991) exploited this information to predirt t,he tertiary structures of several all-helical proteins. By our definition, the central residues that mediate t,he interaction between a pair of helices are the residues (one on each helix) whose (1’ atoms are closest together. The contact normal is defined as the distance bet,ween the projections of the central residues onto their respective helix axes. Although the central residues are adjacent to the contact normal, packing constraints force the side-chains of the central residues to lie on one side or the other of the contact normal (Figure 2). Following (‘ohen ef al. (1979), we define t.he angular deviation of the central residue from the contact normal as the skew angle. A bimodal distribution of skew a.ngles is observed with peaks at -SO” to - 30” and + 20” to +30” (Figure 10). A geometric analysis of helix packing corroborates these values. Figure 11 is a schematic of two parallel helices examined from the X-terminal ends separated by a IO.2 ,A contact normal. Tn the close packed spheres approximation of an a-helix. the residue center lies +I A from the helix axis (Richmond & Richards, 1978). \Vhen residues are modeled as single spheres. the average radius is 2.4 A (Gregoret & Cohen, 1990). Using the 1a.w of cosines, it can be shown that the skew angle associated with these values is 18”. However. residues are not spherical. Elliptical representations would decrease the skew angle somewhat. In the limit of glycine, where a smaller sphere is a sensible model of the peptide side-chain, the skew angle could be as small as 20”. While the average absolute value of the skew angle is 26” for all residue types, it is observed to be only 22” for glycine. Given the intrinsic symmetry of the helix-helix interactions shown in Figures 2 and 11, positive and negative skew angles should be equally likely. Figure 10 suggests a slight preponderance of negative skew angles. For parallel helix-helix interactions, the skew angles should be correlated, (+,

Four Helix Bundle Diversity

I365

Skew angle -28’

Figure 11. This Figure is a schematic of 2 parallel hrlicos rxaminetl from the N-terminal ends separated hy a 109 A contact normal. In the close-packed spheres approximation of an a-helix, the residue center lies 4.1 A from the helix axis. When residues are modeleti as single spheres, the average radius is 24 A. Using the law of cosines. it ~a.11he shown that the skew angle associated with thrsr values is 28”.

-). For anti-parallel interactions, the +) or (-. ( + - ) or (-. + ) skew angle pairs should prevent van der \Vaals overlaps and facilitate close packing. However. a review of skew angle pairs observed in helix-helix

interactions

fails

to show

the expected

angle correlation, even if this analysis is restricted to relativeI> parallel ( lQil < 40”) interactions. Presumably. side-chains possess sufficient flexibility to solve the local helix packing problems that may arise from the formatsion of more efficient, interfaces elsewhere in the structure. \4’it.h a precise geometric

definition

of

central

residues. it is possible to st,ucly the characteristics of these residues from a large number of helix-helix interactions (Figure 12). Tt is not, surprising that the most common central residues are hydrophobic (Ala > Leu > Val > Ile > Phe > Ser). Among the other common central residues. t,he helix breakers

glycine and threonine are observed more commonly than would be expected in the core of an a-helix. For glycine. presumably this reflects the potential for rstensive side-chain interdigitation allowed by the a.bsence of a side-chain. In fact, glycine central residues frequently pair wit,h another glycine to creat,e a. dovetail joint bet,ween the two helices, 01 the glycine interacts with an aromatic residue (His, Trp. Pbe) or an aliphatic side-chain (Leu, Ile, Val) to form a finger into notch structure. This packing pattern may be particularly relevant for molecular recognition between transmembrane helices where glycine residues seem to play an important role (Cosson 8: Bonifacino, 1992; Lemmon et al., 1992:

Figure 12. The observed frequency of central residues is compared with the expected freyuency (calculated from the overall residue distribution in all helices). Residues are shown in log-odds order: those that appeared less frequently than expected are towards the left side of the graph. and those that appeared more frequently than expected are towards the right.

Sternberg & Gullick, 1990). For example, the transmembrane helices of class II MHC a and p-chains and contain the sequences Ai: Gzz5L”G,,,L.G,,,I A;: G22sI..G231C..G235V. Site-directed mutagenesis has documented the importance of the glycine residues (Cosson & Bonifacino, 1992) and the glycine spacing is compatible with a consecutive series of interacting notched rungs of two a-helices. Lemmon et al. (1992) have demonstrated the relevance of Gly79, Gly83 and Thr87 in homodimer formation of the transmembrane helices in glycophorin A. The sequence pattern G,,V.*Gs,V..T,,I is similar to the class II MHC sequences and clearly compatible with the notched packing pattern. Energy calculations by Treutlein et al. (1992) on the glycophorin A dimer suggest that the helix may form a right-handed supercoil that would facilitate the efficient stacking of notched interactions. Glycine or perhaps another residue containing a very small side-chain could form these notched helices. Thus, it is not surprising that Lemmon et al. (1992) found only GIy74 or Ala, only Gly83, and Thr87 or CIy were compatible with the efficient formation of homodimers. Finally, Sternberg & Gullick (1990) in their study of the transmembrane helices of tyrosine kinase containing growth factor receptors identified a putative helix dimerization sequence motif. P,, P,, P4 in 18 of 20 sequences, where I’,, was a small side-chain (Gly, Ala, Ser, Thr or Pro). P, was an aliphatic side-chain (Ala, Val, Leu or Ile) and P, was Gly or Ala. A review of the 20 sequences presented in that work shows that 14 of these contain the sequence pattern X,,Z,.. X,Z, where X is Gly, Ala, Ser or Thr and Z is an aliphatic hydrophobic amino acid or Phe. The 1 l&bundle dataset contains only eight standalone bundles (starred bundles in Table 2); the other 110 belong to larger protein structures. Of these

1366

Four

Helix Bundle

eight,*five are square bundles, two are splinter, and one is splayed. Interestingly, all eight of the standalone bundles are antiparallel bundles. The other characteristics of the stand-alone geometrical bundles agree with the rest of the dataset. The structures of two integral membrane proteins .containing substantial a-helical structure have been determined at atomic resolution: the photosynthetic reaction center from Rhodopseudomonas zliridis (Deisenhofer & Michel, 1989), and bacteriorhodopsin frorp Halobacterium halobium (Henderson et al., 1990). Although the photosynthetic reaction center helices do not pack extensively enough with each other to meet our definition of a four helix bundle, the bacteriorhodopsin structure contains five four helix bundles: (1 (lO-32), 2 (3%62), 3 (SO-lOO), 7 (203-225)); (2, 3, 6 (167-191), 7): (3, 4 (10%127), 5 (137-157), 6); (3, 4, 6, 7); (3, 5, 6, 7). The average interhelical packing angle is 13( f 12)” with an average contact normal length of 10.6 A and an average skew angle (absolute value) of 23”. These values are very similar to t,he helix-helix interaction geometries observed in globular proteins. All five bundles are square with four left-handed bundles and one right-handed bundle. The central residues in the helix-helix interactions of bacteriorhodopsin have been analyzed. The amino acids Gly, Leu. Phe, Thr and Trp are found as central residues more often than would be expected from their observed frequency of occurrence in transmembrane helices. This is consistent with mutagenesis data on the packing preferences of other transmembrane helices that form helix-helix interactions (Sternberg & Gullick, 1990; Cosson &, Bonifacino, 1992; Lemmon et al., 1992). We anticipate that the results of our survey of globular proteins will be applicable to efforts to construct models of integral membrane proteins, including the large set of G-protein coupled seven transmembrane spanning receptors (Baldwin, 1993).

4. Conclusion Several groups have studied the four a-helix bundle as a structural motif (Chou et al., 1988: Weber & Salemme, 1980). Our previous efforts in this area described a series of bundles which fit a specific, narrow definition based in part on interhelical angle (a) limitation (Presnell & Cohen, 1989). In this study we have generalized the definition of a four a-helix bundle to include collections of four helices not restricted by interhelical packing geometry. Removing the angular constraint while still requiring a general cohesiveness revealed many new bundle structural forms. We were able to classify these structures into six types based on the pattern of interhelical angles. In three of these classes, we found stand-alone bundles. Myohemerythrin is an example of the square bundle variety and GM-CSF is of the splinter bundle class. Bovine calbinden is a stand-alone splayed bundle. Possible candidates for X-bundles as stand-alone

Diversity

structures also exist in the recent literature

(Dekkel

et al., 1993).

In our previous work on four a-helical bundles, careful examination of square bundle types disproved previous conject,ures about allowed bundle topologies. In this study, we have been able to more completely determine the geometric characteristics of helix-helix interactions over a broad range of bundle types. Certain values rega.rding interacting helices are critical to the correct modeling of all a-helical structures. Three different classes of helix-helix interactions had been determined in previous work (Chot’hia ef al.. 1977: Richmond B Richa.rds, 1978). Each helix interaction cla.ss has a specific set of residues that are allowed a.s t,he central or interaction residue. and specific values for interhelical distance and angle. With the additional data collected in this study, contact normal distances can be more accurate]) modeled using the specific information on interaction residue types (Table 3). Moreover, the information collected in this study has provided a det,ermination of likely skew angle values. Ideal skew angles have been calcula,ted from a geometric model of t,he helix-helix interaction site: these agree with t,he observed skew angles. Analysis of the helix-helix interaction data collect,ed in this work confirms the importance of including skew angles in models of helix-helix packing and will improve the accuracy of combinatorial strat)egies for the prediction of the tertiary structure of all-helical proteins. The geomet,ric properties observed in globular proteins suggest hypotheses concerning the sbructure of membrane spanning proteins. Specifically, we highlight the importance of glycine residues in creating a series of notched rungs in the homodimeric forms of transmembrane helical structure. On the basis of bacteriorhoclopsin, it appears that helical st,ructure parameters for transmembrane helices are very similar to those for helices in globular contexts. This further supports t.he observation of Rees et al. (1989) that the interiors of globular and integral membrane proteins are quite similar. Indeed, the geometric propensit,ies of helixhelix interactions may be independent of the external environment of the protein. This work was supported by a grant from thr Sational Institutes of Health (CM39900 to F.E.C.).

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Helix

Bundle Diversity

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(Received

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by B. Honig

1993; accepted 25 November

1993)