Non-α-helical elements modulate polytopic membrane protein architecture

doi:10.1006/jmbi.2000.4402 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 306, 349±362

Non-aaa-Helical Elements Modulate Polytopic MembraneProtein Architecture

R. Peter Riek1, Isidore Rigoutsos4, Jiri Novotny1,3* andRobert M. Graham1,2*

1Molecular Cardiology UnitVictor Chang Cardiac ResearchInstitute, St Vincent's HospitalDarlinghurst, 2010, New SouthWales, Australia2School of Biochemistry andMolecular Genetics, Universityof New South WalesKensington, 2052, New SouthWales, Australia3Department of MolecularBiology, Princeton UniversityPrinceton, New Jersey08544, USA4Bioinformatics and PatternDiscovery Research GroupIBM Research Division, T. J.Watson Research CenterP.O. Box 218, YorktownHeightsNY 10598, USA

E-mail addresses of the [email protected]@victorchang.unsw.edu.au

Abbreviations used: GPCR, G-proreceptor; PDB, Protein Data Bank.

0022-2836/01/020349±14 $35.00/0

In ``all a-fold'' transmembrane proteins, including ion channels, G-pro-tein-coupled receptors (GPCRs), bacterial rhodopsins and photosyntheticreaction centers, relatively long a-helices, straight, curved or kinked, packinto compact elliptical or circular domains. Using both existing andnewly developed tools to analyze transmembrane segments of all avail-able membrane protein three-dimensional structures, including that veryrecently elucidated for the GPCR, rhodopsin, we report here the ®ndingof frequent non-a-helical components, i.e. 310-helices (``tight turns''), p-helices (``wide turns'') and intrahelical kinks (often due to residues otherthan proline). Often, diverse helical types and kinks concatenate overlong segments and produce complex inclinations of helical axis, and/ordiverse frame shifts in the ``canonical'', a-helical side-chain pattern.Marked differences in transmembrane architecture exist even betweenseemingly structurally related proteins, such as bacteriorhodopsin andrhodopsin. Deconvolution of these non-canonical features into their com-posite elements is essential for understanding the pleiotropy of polytopicprotein structure and function, and must be considered in developingvalid macromolecular models.

# 2001 Academic Press

Keywords: proline; transmembrane helices; helical ®ne-structure; kink;p bulge
*Corresponding authors
Introduction

Several biologically important protein familiesadopt an ``all a-fold'' structure, i.e. their structuresconsist solely of a-helices interconnected by loops.In transmembrane proteins, such as ion channels,G-protein-coupled receptors, rhodopsins of bac-teria, and photosynthetic reaction centers, rela-tively long a-helices, straight, curved or kinked,pack into compact elliptical or circular domains.The origin, stability and biological signi®cance ofthese structures has attracted much attention andbeen the subject of intense study.1 ± 7 To date, theseanalyses have focused largely on interhelical inter-

ding authors:;

tein-coupled

actions, although recently, analyses of intrahelicalinteractions have also been reported.8,9

It has been noted that transmembrane, helicalproteins possess unique structural features absentfrom helices in globular proteins. In particular, pro-lyl residues, very rare in a-helices of globular pro-teins (excepting at their N termini),10,11 andthought to be incompatible with a-helicalstructures,12,13 have been found to be abundant intransmembrane helices. In some cases, the presenceof these prolyl residues is known to induce kinks,i.e. changes in direction of the helical axis.11,14

Here we focus on the intrahelical factors (residuetorsional angles, helical pitch, angular periodicityof residues, and backbone hydrogen bonding pat-terns) associated with the diversity of helical habits(e.g. a, p, 310, kinks) found in transmembrane seg-ments which doubtless affect interhelical relations,i.e. the architecture of transmembrane helical struc-tures. We extend the existing tools of a-helicalstructural research by introducing diagnostic

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350 �-Helical Fine-structure

measures (atomic distance plots, ``spoke angle''and ``rise per residue'' plots) allowing for betteranalysis of helical ®ne-structure (Figure 1). Weshow that (i) helical axis bendings similar to pro-line kinks can be induced by other residues, (ii) themagnitude of kink angles differs according to thehelical boundaries (a$ a, a$ 310, a$ p) at

Figure 1. Helical geometry. Six Ca atoms (CAs) inhelical conformation are shown as orange spheres, pro-ceeding from bottom to top. Bonds connecting backboneatoms in residues containing the CA-3 through CA0atoms are highlighted and color-coded (carbon, green;nitrogen, blue). The helical path of the backbone istraced as a blue ribbon and Ca-Ca distances are indi-cated by dotted lines (Ca[i]-Ca[i ÿ 2] blue, Ca[i]-Ca[iÿ3]

green, Ca[i]-Ca[iÿ4] red, Ca[i]-Ca[iÿ5] yellow, scale in aÊng-strom units). Helical rise per residue and spoke angleswere determined from the coordinates of the helical resi-dues A, B, C, and D as follows. For any residue pair, ni

and ni ÿ 1 (such as, e.g. the atoms A and B, respectively),their c, o and f dihedral angles, bond angles and bondlengths were determined from the crystal structure coor-dinates and used to construct a regular helix containingthree repeats of the ni and ni ÿ 1 residue pairs with iden-tical geometries. The points E and F were found as thepoints of bisection of the lines AC and BD, respectively.Lines BE and CF, then, reside in parallel planes. The riseper residue is the shortest distance between BE and CF,indicated by the distance between points E' and F'. Thespoke angle is the projected angle between BE0 and CF0.

which kinks occur, (iii) helical ``perturbations'' arenot con®ned to kinks but involve runs of 310 and phelices (appearing as ``tight turns'' and ``wideturns'', respectively), (iv) fuzzy but speci®csequence ``signatures'' are associated with thedifferent helical habits, indicating that (v) inductionof these diverse conformations involves the coop-erative participation of several residues in up totwo helical turns, N-terminal to the signature pro-line or other perturbing residue. Taken together,these ®ndings reveal that non-a-helical confor-mations occur frequently and are critical determi-nants of polytopic protein structure.

Results

Helical conformations

The ``Pauling'' a-helical conformation15, charac-terized by f and c backbone torsional angles ofÿ57 � and ÿ47 �, respectively, and giving rise to astraight a-helix, rarely occurs in nature. Instead,natural helices are slightly curved towards thesolvent,16 with backbone torsions approachingÿ62 � (f) and ÿ41 � (c). Here, we refer to such heli-cal conformations as ``canonical''.10,16 A canonical aor 3.613-helix (Figure 2) carries an angular shift ofthe successive residues of 100 �, with 3.6 residuesper turn, and has the ith and n � 4th residues (con-veniently referred to by their Ca atoms) approxi-mately eclipsed along the helical axis. Its rise perresidue is 1.56 AÊ . In contrast, a 310-helix,10,17

(Figure 2) has three residues per turn, with a riseper residue of 2.0 AÊ . A p-helix18 (Figure 2), con-tains 4.4 residues per turn, with a rise per residueof 1.0 AÊ . The radius of a canonical a-helix is 2.3 AÊ ,that of a 310-helix is 1.9 AÊ , and that of a p-helix is2.8 AÊ . The backbone torsions quoted as character-istic of a p-helix18,19 are approximately ÿ57 � (f)and ÿ70 � (c), and for a 310-helix are ÿ71 � andÿ18 �, respectively.10 Since the relationshipbetween backbone torsions and helical geometriesis complex and degenerate (i.e. many differentcombinations of f,c,o angles are compatible witha single Ca trace), we sought more discriminategeometric descriptors for analyzing different heli-cal habits. The most discriminant included Ca-Ca

distances (plotted as differences from the corre-sponding values in a canonical a-helix), spokeangle (helical wheel20) plots, rise per residue plots,and H-bond connectivity plots (Figure 3).

Crystallographic, transmembrane,helical structures

We inspected all the transmembrane helicalstructures present in the RCSB (``Brookhaven'')Protein Data Bank21,22 as well as that just reportedfor bovine rhodopsin,23 and identi®ed 73 helicalsegments (contained within 53 helices out of a totalof 119 transmembrane helices), containing conspic-uous non-canonical elements (Table 1). In approxi-mately 50 % of cases, ``true'' proline kinks could be

Figure 2. 310, a and p confor-mations of polyalanine helices.Views along the helical axis andperpendicular to the axis are dis-played in the top two rows andmolecular surface representation isshown in the bottom row. Methylside-chains of residues 3 and 6have been color-coded, yellow andmagenta, respectively, to illustratespatial and surface relationships inthe three different conformations.(310) Using the program ICM,47,48 apolyalanine helix was built withuniform backbone torsional anglesf, c ÿ71 �, ÿ18 �, respectively(these values correspond to thestaggered, ``3.210`` helix observedby Barlow & Thornton10 ratherthan the original 310 helix, witheclipsed side-chains, as originallydescribed by Pauling et al.15 Rise/residue was 1.8 AÊ , spoke angle112 �, helical radius 1.9 AÊ . (a) Cano-nical helix, (f, c ÿ62 �, ÿ41 �,respectively), with rise/residue1.5 AÊ , spoke angle 102 �, and radius2.3 AÊ . (p) Helical backbone torsionangles were those quoted byCreighton,19 i.e. f, c ÿ57 �, ÿ70 �,respectively (rise/residue 0.9 AÊ ,spoke angle 86 �, radius 2.8 AÊ ).Note the large and small radii andunique surface side-chain disposi-tion of the p and 310-helices,respectively.

�-Helical Fine-structure 351

identi®ed as short local disturbances (approxi-mately one turn) in helical conformation, whichbent the helical axis at a position N-terminal to theproline. Additional (non-proline) kinks wereclearly visible at interconnections of different heli-cal habits. The remaining instances of non-canoni-cal behaviour manifested themselves not as kinks,but as short runs of p (about 30 % of cases) or 310

(20 % of cases) helices. p-Bulges,30,34 apparent assingle residue insertions in helical turns precedingthe kink-inducing proline, were also observed.

Detailed geometries of helices and kinks

As shown by the plots in Figure 3, the non-aconformations are apparent as deviations in theCa-Ca distances, spoke angles and rise per residuedistances from those found in a canonical a-helix,

as well as changes in backbone H-bond register. Inmany cases, the residues involved were located N-terminal to the signature proline or other perturb-ing residue. For p conformations (wide turns),seven to nine residues (2 to 2.5 helical turns)showed deviations from canonical values.310-helices (tight turns) also involved seven to nineresidues and contained a proline towards the Cterminus, but never at the C terminus. About one-half of these non-canonical conformations, how-ever, involved residues other than proline. Forkinks, fewer residues were involved and typicallya proline was at the C terminus of the pertur-bation, although residues other than proline alsoproduced kinks (nine instances altogether).

The major diagnostic features of p helices(Figure 3(a)) are: a conspicuous shortening of theCa

i-Ca

i ÿ 4 and Cai-C

ai ÿ 5 distances; rise per residue

Figure 3 (legend shown on page 354)


distances of less than 1.0 AÊ /residue; and smallerspoke angle shifts between the residues participat-ing in these wide turns (up to about ÿ25 � differ-ence compared to the a-helices). 310-helices, incontrast, have markedly increased Ca

i-Ca

i ÿ 4 andCa

i-Ca

i ÿ 5 distances (Figure 3(b)). Their spokeangles are also increased (by a maximum of10-15 �), as are the rise per residue distances (toabout 2.5 AÊ /residue); changes that are particularlyevident in the most N-terminal residues. Kinks arecharacterized by abrupt increases and decreases inboth the Ca

i-Ca

i ÿ 3 and Cai-C

ai ÿ 4 distances

(Figure 3(c)). Compared to wide turns and tightturns, kinks also show more varied pro®les. The

largest changes usually occur one helical turnbefore the perturbing proline which, because of itspyrrolidine ring secondary amide, is unable to H-bond with the oxygen carbonyl of the i ÿ 4th resi-due. In a number of cases, the geometry of thekink also prohibits H-bonding between the i ÿ 3carbonyl oxygen and the i � 1 amide hydrogen. Incontrast, tight turns often restore the 310-like, i toi ÿ 3 and i to i ÿ 4 backbone H-bond connectivity.It is possible that, in the kinked a and p confor-mations, partial stabilization is achieved by weak,CH � � �O hydrogen bonding to the H-bondde®cient i ÿ 3 carbonyl.35,36

Figure 3 (legend shown on page 354)


Changes in axial directions at kinks andhelical boundaries

In many instances, transmembrane helices dis-played diverse, often complex morphologies. Somewere mostly a-helical with straight, linear axes, orcontaining a single abrupt kink, others containedtwo or more kinks of unequal magnitude impart-ing an apparent curvature to the helix.

Least-squares ®tting of axes into the straight (a,p or 310) helical segments allowed the magnitudeof the kinks to be measured. Directions of theincoming and outgoing backbone axes were alsoestimated, from their respective dihedral angles,

de®ned relative to a common reference point (theplane of the prolyl pyrrolidine ring at the break-points, where present). Angular magnitudes of thekinks depended on the length of the segments cho-sen for least-squares ®tting. In what follows, wediscuss the ``local'' magnitudes obtained with axes®t into single helical turns (®ve residues) that pre-ceded and followed the kink points. In summary:(i) the magnitude of proline-induced kinks in a-helices was 23(�4) �, i.e. not signi®cantly differentfrom the �26 � value reported by Barlow andThornton10 for a-helical kinks in globular proteins,the maximum bend always occurring in the turn

Figure 3. Backbone geometry plots diagnostic of the p and 310-helices and of proline kinks. (a), p-Helix (twosegments in the A2 helix of bovine cytochrome c oxidase, PDB code 2OCC, are boxed in the top panel). From top tobottom, Ca-Ca distance differences (Ca[i]-Ca[iÿ2] blue, Ca[i]-Ca[iÿ3] green, Ca[i]-Ca[iÿ4] red, Ca[i]-Ca[i ÿ 5] yellow, (scale inaÊngstrom units); rise per residue distance (in aÊngstrom units); spoke angles (inter-residue angular shifts, in degrees);and backbone hydrogen bonding (continuous lines showing the regular a-helical pattern (i! i ÿ 4), dotted linesshowing the i! i ÿ 3 and broken lines the i! i ÿ 5 CO-NH distances within hydrogen bonding range. Ranges:brown, <2.0 AÊ ; red, 2.0 AÊ ! 2.2 AÊ ; orange, 2.2 AÊ ! 2.5 AÊ ; green, 2.5 AÊ ! 2.6 AÊ ; light blue, 2.6 AÊ ! 2.7 AÊ ). (b), 310-Helix (the 310 segment in the A8 helix of bovine cytochrome c oxidase, PDB code 2OCC, is boxed in the top panel).Details as for (a), above. (c) Two consecutive Pro-kinks (two kinked segments in the A6 helix of bovine cytochrome coxidase, PDB code 2OCC, are boxed in the top panel). Details as for (a), above.


preceding the proline; (ii) in four cases (2OCC,A11/Pro427; 1AR1, A11/Pro462; 1C3W, A2/Pro50and A3/Pro91) the ``kink'' angles at proline resi-dues were smaller (15 �-18 �), and in 1AR1 B2/Pro85 it was essentially negligible; (iii) in caseswhere kinks occurred at the C-terminal boundary

of 310-helices with a-helices, the average angle was16(�4) �, excepting three larger angles (20 �-23 �)and one straight connection (2OCC D1/Leu95). Sixof these kinks were associated with proline resi-dues, the rest occurred at hydrophobic residues,i.e. Leu, Val and Ile; (iv) similarly, in 11 cases of

Table 1. Non-canonical helical conformations in transmembrane proteins

PDB codea Chain/Position Typeb PDB code Chain/Position Type

A. Proline20CC A2/P72 W 1C8S A2/P50 K

A2/P84 K A3/P91 KA3/P106 K A6/P186 TA3/P107 T 1E12 A2/P70 KA5/P200 K A3/P117 KA6/P241 K A6/P211 TA6/P249 K 1EUL A7/P803 KA8/P315 T A10/P976 KA10/P398 K 1F88 A1/P53 KA11/P427 K A4/P170 KB2/P69 K A5/P215 WB2/P79 K A6/P267 KG1/P26 W A7/P291 KL1/P36 K A7/P303 KM1/P29 K

1AR1 A2/P105 W B. Non-prolineA2/P117 KA5/P236 K 20CC A2/N80 WA6/P277 K A10/F387 WA6/P285 K A11/F430 TA8/P350 T D1/L96 TA11/P462 K I1/K42 WB2/P85 K K1/N15 KB2/P98 K 1AR1 A2/N113 W

1FUM C1/P334 T A10/F422 WD1/P29 K A10/G433 KD2/P74 K A11/F465 T

1BE3 C6/P305 T 1BE3 C6/L301 TC18/P367 W 1C3W A7/F219 WD9/P217 K 1C8S A7/F219 W

1PRC L3/P124 T 1E12 A7/F245 WL3/P136 W 1PRC M3/A151 TM3/P163 W 1AIJ L3/A124 T

1AIJ L3/P136 W M3/A153 TM3/P165 W 1EUL A1/F57 W

1C3W A2/P50 K A11/G979 TA3/P91 K 1F88 A2/T92 WA6/P186 T A7/V300 T

a 2OCC, bovine cytochrome C oxidase;24 1AR1, cytochrome C oxidase(P. denitri®cans);25 1FUM, fumarate reductase respiratorycomplex;26 1BE3, bovine cytochrome BC1 complex;27 1PRC, photosynthetic reaction center (R. viridis);28 1AIJ, photosynthetic reactioncenter (R. sphaeroides);29 1C3W, bacteriorhodopsin;30 1C8S, bacteriorhodopsin (M state);31 1E12, halorhodopsin;32 1EUL, rabbit muscleCa2 � ATPase;33 1F88, bovine rhodopsin.23

b W, wide turn; T, tight turn; K, kink.


p! a connectivity, mostly across a proline residue(seven occurrences), a variety of angles wereobserved (12 �-16 �, four times; 21 �-24 �, ®ve times;29 � and 35 �); (v) 20 cases were observed where a-helices connected, via a kink, into either a p or a310-helix. None of these connections contained pro-line and the majority of them (16 cases) formed anangle of 14(�4) �. However, some a! p connec-tions (1AIJ, L3/Val132, M3/Gly161; 1PRC, M3/Gly159; 1BE3, C18/Leu363) produced a largerangle, 35(�2) �.

In proline-induced kinks embedded in a or 310-helices, dihedral angles between the incoming andoutgoing helical axes were measured relative to theplane of the prolyl pyrrolidine ring. As Figure 4shows, these kinks were left-handed, with thedirections of incoming segments clustered withinthe lower-left quadrant of the dihedral space(between ÿ90 � and 180 �, rarely up to 160 �). The

same left-handed trend was apparent in the fouracute (35 �) a! p connections discussed above.Interhelical connections with more obtuse angles(12 �-16 �) did not have a preferred directionality.

Sequence patterns in ppp and 310-helices,and kinks

Although no de®nite sequence motifs could bediscerned, the following ``fuzzy'' sequence featuresappeared to be associated with the non-canonicalstructures. In the p-helices, residues with large aro-matic or hydrophobic side-chains often precededthe proline residues. The 310-helices containedb-branched side-chains N-terminal to proline resi-dues and, in proper kinks, aromatic residues werefrequent with glycine near, and sometimes outside,the kinks. Using the program Teiresias,37 it hasbeen possible to construct complex, degeneratesequence motif descriptors that correctly classi®ed

Figure 4. Relative directions of the helical axes incom-ing to and outgoing from kink breakpoints (prolyl orother disturbing residues), determined after least-squares superimposition of backbone and Cb atoms. (a)Fourteen a-helices with proline-induced kinks; (b) eight310-helices with ®ve proline and three non-proline-induced kinks. Helical axes of the incoming and out-going segments were calculated as described inMethods. They are displayed as grey and red arrows,respectively, pointing from the C to the N termini of the®tted backbones. A prolyl side-chain is also shown forreference, with carbon atoms color-coded green andnitrogen blue. As a result of residue superpositions, theoutgoing (red) axes are seen better clustered than theincoming (grey) axes. Directions of the pairs of arrowsand the plane of the prolyl ring de®ne a dihedral angleand reveal the left-handed tendency of the helical axesat kink breakpoints.


training-set sequences into the three classes of non-canonicity. The possibility of developing motifdescriptors predictive of all the helical habits isbeing actively pursued.

Concatenation and stability ofnon-canonical helices

Local canonical features described above oftenfollowed one another over long stretches of thehelix (more than three to four turns). In theseinstances, complex inclinations of the helical axes,and/or diverse frame-shifts in helical periodicityresult, giving rise to contiguous regions highlyvariable in terms of helical path and helical surfacefeatures (Figure 5). While detailed analyses of allthese non-canonical regions is beyond the scope ofthis paper, deconvolution of these complex non-canonical features into their composite localelements (wide turns, tight turns and kinks) is animportant ®rst step towards our understanding,and realistic modeling, of the ®ne structural detailsof transmembrane helices.

Analysis of bacteriorhodopsin andbovine rhodopsin

In bacteriorhodopsin our analysis (Figure 6)revealed three canonical, straight, a-helices contain-ing no proline residues (A, D and E); one proline-less helix bent by a run of a p-helix (G, with a localbend of 24 � at Lys216); two proline-induced kinksin helices B (19 � at Val49) and C (28 � at Phe88);and a turn of 310-helix, following the proline kinkin helix F (20 � at Tyr185) (Figure 6). The bacterior-hodopsin transmembrane helical bundle involving163 residues is, therefore, composed largely ofcanonical a-helices, with only about 14 % (23 resi-dues) of non-a-helical habit. Contrariwise, inbovine rhodopsin (Figure 6), only one helix (helixC, with no proline) is apparently straight,although, it contains several subtle bends (12 � atVal130, 11 � at Trp-Ser 126-127, 11 � at Glu113) thatimpart a certain amount of supercoil to the helix.The only other proline-devoid helix, B, is stronglybent by a p-helical segment (30 � at Gly89). In theremaining helices, there are three relatively simpleproline-induced kinks (16 � at Leu50 in A, 24 � atAla166 in D and 33 � at Trp265 in F) and two morecomplex proline-associated motifs. In helix E, thereis a p-helical segment with a bend of 18 � atHis211, occuring before Pro215. The second morecomplex proline-associated motif occurs in helix Gwhere a most unusual, relatively long run of an``eclipsed'', or ``Pauling'', 310-helix (as opposed tothe prevailing 3.210-helix described by Barlow andThornton, cf. legend to Figure 2) is presentbetween Pro291 and Val300, with an exceptionallyacute bend of 41 � at Ala-Val 299-300. The Ca-Ca

pro®le for helix G is indicative of a proline-induced/-associated kink (Pro303) preceded by arun of 310-helix. The complexity of this pro®le,including the kink associated with Pro291, is insharp contrast to the relatively simple a-helical andwide turn pro®le of helix G of bacteriorhodopsin.Thus, the bovine rhodopsin transmembrane helicalbundle, involving 188 residues, is conspicuously

Figure 5. Examples of non-cano-nical helical conformations. Back-bones of selected polypeptidesegments are shown in the variouspanels. Atoms are color-coded asfollows: carbon, white, green oryellow; nitrogen, blue; oxygen, red.Proline side-chains, where present,are highlighted in magenta.(a) Helix B2 from P. denitri®canscytochrome C oxidase with Pro85embedded in a straight segment(measured ``kink'' angle 6 �), andPro95 (bottom) at which the helixkinks at 22 �. (b) Helix M3 from theR. sphaeroides photosynthetic reac-tion center where a p-helix joins ana-helix at an angle of 40 �. Arrowspoint in the directions of least-squares-®tted axes in the p segment(top; note the wide radius of thep-helix) and the a segment.(c) Comparison of two apparentlystraight helices from bacteriorho-dopsin (helix A, white) and bovinerhodopsin (helix C, yellow). Ca

atoms on ``upper'' face of eachhelix are emphasized as magentaand cyan balls, respectively. TheN-terminal turns (four residues) ineach helix were least-squares super-imposed. The A helix, the straight-est one we have encountered, isseen to follow a direct path whilethe C helix, seemingly straight, too,actually diverges from linearityunder the in¯uence of three subtlekinks (�11 �) present at residues113, 126-127 and 130. At theirC-terminal ends, the helices havediverged by 7.8 AÊ , while residuesin their upper faces kept their rela-

tive, ``upper'', orientations. (d) As in (c) but viewed down the axis of helix A, emphasizing its straightness. (e) HelicesG of bacteriorhodopsin (white) and bovine rhodopsin (yellow) least-squares superimposed at their N-terminal turns(four residues). The bacteriorhodopsin helix has no proline and contains a p-bulge in the middle, the rhodopsin helixcontains three proline residues and a run of 310 helix in between proline 291 and valine 303. Despite the differentshapes in the middle both helices follow the same approximate direction. (f) Same helices as in (e) but superimposedon the backbones of lysine residues 296 and 216, respectively (cyan color). The lysine side-chains form a Schiff basewith the retinal ligand in both structures.


non-canonical, with as much as 25 % (47 residues)adopting various non-a-helical habits.

Discussion

The ®nding that transmembrane helices containa relative abundance of 310 and p conformationswas unexpected. The p-helix, in particular, is vir-tually absent from globular proteins10 except forshort, incomplete turns (p-bulges: the recent high-resolution structure of bacteriorhodopsin,30 con-tains a p-bulge that induces a kink in the middle ofthe G helix, a functionally important feature). Atde®ned temperatures (T), structures occur with fre-quencies de®ned by their stabilities (free energies,

E) and the Boltzmann factor, exp[E/kT]. Indeed,the abundant a-helix is also the most stable of thevarious helical conformations stereochemicallypossible in natural polypeptides.15 Perhaps fragilep-helices, with their large radii and empty holes intheir centers, survive better when supported byrelatively rigid, unsaturated lipid chains interactingwith the large aromatic and non-polar side-chainsin which these helices abound. In this regard, wenote that long chain, polyenoic fatty acids of dipo-lyunsaturated phosphatidylcholines are tightlyassociated with photoreceptor membranes.38,39

The prevalence of proline residues in transmem-brane helices, as compared to globular proteins,has been noted,14 and considered to have architec-

Figure 6. Comparison of the Ca-Ca distance differences (Ca[i]-Ca[iÿ2] blue, Ca[i]-Ca[iÿ3] green, Ca[i]-Ca[iÿ4] red,Ca[i]-Ca[iÿ5] yellow, scale in aÊngstrom units) for residues in the seven transmembrane helices of bovine rhodopsin(A1-G1) and bacteriorhodopsin (A2-G2). Note the disparate lengths and sequences of the synonymous transmem-brane domains of the two proteins, and the different content of non-a-helical habit in corresponding segments of thetwo proteins, e.g. helix E of bovine rhodopsin, but not that of bacteriorhodopsin, contains a conspicuous p-helicalsegment immediately N-terminal to residue 215.



tural importance. In support of this conjecture, wenote that a proline is present in up to ®ve of theseven transmembrane domains of G-protein-coupled receptors, where it is located at positionsapproximately in the middle of the helix. Giventhat some of these proline residues are invariablyconserved (e.g. that in helix VI of class I GPCRs ispresent in 100 % of 888 analyzed), and that theyoften impart non-canonical structures, they arelikely to contribute importantly to functional diver-sity of polytopic proteins, modifying both theirligand binding and G-protein activation properties.Thus, a proline is absolutely conserved in themiddle of transmembrane helix VI,40,41 whichundergoes major rigid body movement withreceptor activation,42 and is directly linked to theG-protein-activating third intracellular loop. Also,mutation of a cis-trans prolyl isomerase thatimpairs proline isomerization in the G-protein-coupled receptor rhodopsin, leads to the visualimpairment known as the Drosophila ninaAdefect.43

Although proline-induced kinks are readilyrationalized by the bulk of the pyrrolidine ring andthe inability of its amide to form a H-bond (as wasalready evident even with the low resolution struc-ture of the ®rst polytopic protein to be resolved),the ®nding here, that transmembrane helices canaccommodate proline residues by the immediateN-terminal residues adopting a wide-turn (p) con-

Figure 7. Comparison of ribbon traces of transmembrane(blue). The two proteins differ in the lengths and shapes osuperposition becomes dif®cult. In this picture, backbones odistal ends of the ellipsoidal cross-sections of their respectiveposed. Note the conspicuously different lengths of the ``A'' hwas superimposed on the whole of the bacteriorhodopsinhelices; a relative displacement of the two ``D'' helices; and t

®guration, was unanticipated. Equally unantici-pated was the extent to which residues other thanproline induce kinks or other types of non-canoni-cal behaviour (Figure 5); or that a-helices continueuninterrupted, with straight axes, through prolineresidues (Figure 5). Deviations from a-helicity can,as demonstrated, induce major changes in the pathof the helical axis and/or in helical periodicity and,thus, in side-chain orientation. Approximatelocations of ligand binding sites being known insome polytopic transmembrane receptors, we canassume that the above changes evolved to allowthe speci®city of the interactions that occur withthe diversity of compounds targeting these pro-teins. Similarly, subtle changes in helical habits areable to bring about large changes of moleculararchitecture that may modify existing functions, orevolve completely new functionalities.

A case in point is the heptahelical proteins,bacteriorhodopsin30 and rhodopsin,23 whose crys-tallographic structures have only recently been elu-cidated. Their super®cial structural similarity, bothconsisting of antiparallel bundles of seven trans-membrane helices, does little justice to profoundfunctional differences that are, however, mirroredin dissimilarities of their ®ne-structure helicalhabits (Figures 6 and 7). The light-driven ionpump, bacteriorhodopsin, undergoes a functionalcycle whereby the isomerization of all-trans retinalto 13-cis retinal results in the cellular extrusion of a

helices in bacteriorhodopsin (red) and bovine rhodopsinf their helices so much that a meaningful least-squares

f helices A and E from both proteins, occupying the twoantiparallel helical bundles, were least-squares superim-

elices (only the C-terminal part of the rhodopsin A helixA helix); different shapes of the ``F'' and ``G'' pairs ofhe prominent kinks in the rhodopsin ``E'' helix.


proton. Re-isomerization to the ground state occurswhile the chromophore remains in the bindingpocket and is an intrinsic function of the apopro-tein. By contrast, light-activation of the rod cellphotoreceptor rhodopsin results in a sequence ofintramolecular rearrangements triggered by theisomerization of 11-cis retinal to all-trans retinal,which then act as a switch to relay the signal to thecognate protein, transducin. The photolyzed chro-mophore is rapidly hydrolyzed and dissociatesfrom the apoprotein. Rhodopsin is regenerated bythe binding of newly synthesized 11-cis-retinal.

In modeling polytopic proteins,44 ± 46 which is jus-ti®ed given the lack of structures for the bulk ofmembrane proteins, these non-canonical featuresare presently not considered or, alternatively,transmembrane proline residues are invariablyconsidered to induce helical kinks. Whether thenon-canonical behaviour of transmembrane helicesis due only to intrahelical factors or, alternatively,results from interactions with adjacent helices, ispresently not known. Preliminary evidencesuggests that the former may be largely respon-sible and, thus, that non-canonical behaviour canbe predicted from primary structure. This issue iscurrently being actively evaluated.

Methods

Database

The structures of the transmembrane proteins con-sidered in this study are shown in Table 1. Hydrogenatoms were added to the structures using Insight II(Molecular Simulations, Inc.). Helices investigated wereuniformly oriented in the Cartesian frame of reference,with the a-helical segment N-terminal to the signatureproline or other perturbing residue aligned parallel tothe Z-axis, and the direction of the peptide chain (N to Cterminus) running from ÿZ to �Z. Reference 22-residuepolyalanine right-handed a-helices were generated usingboth the Pauling,15 torsional angles and the canonicaltorsions.10

Caaa-Caaa difference determination

Distances between the Ca atoms of residues ni andni ÿ 2, ni ÿ 3 ni ÿ 4 and ni ÿ 5 were measured and the differ-ences between these values and the correspondingvalues in a canonical a-helix, were plotted as line tracesagainst the sequence.

Rise per residue and spoke angle

A single algorithm was developed to determine thesehelix descriptors, using only coordinates for the back-bone atoms of each residue as detailed in Figure 1.

Hydrogen bond connectivity

The distances between the carbonyl oxygen of residueni and the backbone amide hydrogen of residues ni ÿ 2,ni ÿ 3, ni ÿ 4 and ni ÿ 5 were determined and color-codedon an H-bond connectivity plot. Four residues of canoni-cal a-helix were modeled at the ends of each transmem-

brane segment to determine connectivity of the residuesat the N and C termini.

Interaxial angles and relative directions ofhelical axes

Transmembrane helices were built from their crystal-lographic coordinates using the program ICM47,48 (Mol-soft Inc., La Jolla) as described.49,50 The helical axes wereconstructed using the ``Mean'' and ``Rmsd'' functionsavailable in the program. Brie¯y, the center of mass wasfound for the selected backbone atoms and a root-mean-square, translational self-transformation of the helicalbackbone into itself de®ned the axis vector projectingfrom the mass center. The dot product of two such vec-tors equalled the magnitude of the kink subtended byhelical segments associated with these vectors. Angularmagnitudes measured depended on the length of thesegments chosen for least-squares ®tting and here, wediscuss the ``local'' magnitudes obtained with axes ®tinto ®ve residues that preceded and followed the kinkpoints. Relative directions of the helical axis, incoming toand outgoing from kink breakpoints (prolyl or other per-turbing residues) were estimated visually, from compu-ter graphics displays of the axes with the relevantbackbone and side-chain atoms least-squares superim-posed (cf. Figure 4).

Acknowledgments

We are grateful to D. Teller, Biomolecular StructureCenter, University of Washington, for the coordinates ofthe bovine rhodopsin structure, to V. Yee, ClevelandClinic Foundation, Cleveland for critical reading of themanuscript, and to R. Abagyan, Scripps Clinic, La Jolla,for advice with the ICM program. This work wassupported in part by grants from the National Health &Medical Research Council (9938388) and support fromAnalytica Ltd (J.N.).

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Edited by G. Von Heijne

(Received 18 October 2000; received in revised form 4 December 2000; accepted 5 December 2000)

Non-α-helical elements modulate polytopic membrane protein architecture

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