solution structure of the spectrin repeat: a left-handed antiparallel triple-helical coiled-coil

J. Mol. Biol. (1997) 273, 740±751

Solution Structure of the Spectrin Repeat:a Left-handed Antiparallel Triple-helical Coiled-coil

Jaime Pascual, Mark Pfuhl, Dirk Walther, Matti Saraste*and Michael Nilges

European Molecular BiologyLaboratory, Meyerhofstr. 169012 Heidelberg, Germany

Current addresses: M. Pfuhl, DepBiochemistry and Molecular BiologLondon, WC1E 6BT, UK; D. WalthCellular and Molecular PharmacoloCalifornia, San Francisco, CA 9414

Abbreviations used: R16, 16th rea-spectrin; 2D and 3D, two and thrNOE, nuclear Overhauser effect; Cspectroscopy; TOCSY, total correlaTOWNY, TOCSY without NOESY;single quantum coherence; T1, longtime; T2, transverse relaxation timesquare deviation; ARIA, ambiguouiterative assignment.

0022±2836/97/430740±12 $25.00/0/mb

Cytoskeletal proteins belonging to the spectrin family have an elongatedstructure composed of repetitive units. The three-dimensional solutionstructure of the 16th repeat from chicken brain a-spectrin (R16) has beendetermined by NMR spectroscopy and distance geometry-simulatedannealing calculations. We used a total of 1035 distance restraints, whichincluded 719 NOE-based values obtained by applying the ambiguousrestraints for iterative assignment (ARIA) method. In addition, we per-formed a direct re®nement against 1H-chemical shifts. The ®nal ensembleof 20 structures shows an average RMSD of 1.52 AÊ from the mean for thebackbone atoms, excluding loops and N and C termini. R16 is made upof three antiparallel a-helices separated by two loops, and folds into aleft-handed coiled-coil.

The basic unit of spectrin is an antiparallel heterodimer composed of twohomologous chains, b and a. These assemble a tetramer via a mechanismthat relies on the completion of a single repeat by association of the par-tial repeats located at the C terminus of the b-chain (two helices) and atthe N terminus of the a-chain (one helix). This tetramer is the assemblageable to cross-link actin ®laments. Model building by homology of the``tetramerization'' repeat from human erythrocyte spectrin illuminates thepossible role of point mutations which cause hemolytic anemias.

# 1997 Academic Press Limited

Keywords: cell elasticity; membrane skeleton; hemolytic anemias;heteronuclear NMR
*Corresponding author
Introduction

Spectrin (also called fodrin) is a common com-ponent of cytoskeletal structures associated withthe cell membrane in metazoan organisms (Shenk& Steele, 1993). Electron microscopy studies ofspectrin samples reveal a ¯exible elongated mole-cule composed of two loosely intertwined strands

artment ofy, University Collegeer, Department ofgy, University of

3-0450, USA.peat of chicken brainee-dimensional;

OSY, correlatedtion spectroscopy;HSQC, heteronuclearitudinal relaxation; RMSD, root-mean-s restraints for

971344

that appear to be tightly associated at both ends,where the antiparallel or head-to-tail dimerizationoccurs (Shotton et al., 1979). Each strand is made oftwo homologous chains (b and a) which associateinto tetramers through a head-to-head interaction(the C terminus of the b subunit with the N termi-nus of the a subunit) at the tetramerization site.The tetramer exposes actin-binding sites at its endsand is thus able to cross-link membrane-associatedactin ®laments (Bennett & Gilligan, 1993).Although spectrin is present in most animal tis-sues, it has been most thoroughly studied in redcells.

The human erythrocyte is characterized by a dis-tinctive biconcave shape and remarkable elasticity.These properties are largely determined by themembrane skeleton, a ¯exible meshwork of pro-teins on the inner surface of the cell membranewhich is mainly formed by non-covalent inter-actions between spectrin, F-actin and integralmembrane proteins (Bennett & Gilligan, 1993).These interactions result in a relatively uniform

# 1997 Academic Press Limited

Solution Structure of the Spectrin Repeat 741

two-dimensional network which is primarily orga-nized in the shape of hexagons. Actin and theassociated proteins are located at the center and onthe six corners of the hexagon and spectrin lies onthe sides. This network is intimately coupled to themembrane at a limited number of sites via twomolecular contacts between integral membraneproteins and spectrin. These attachments are pro-vided by ankyrin which interacts with the band 3protein and spectrin, and by protein 4.1 whichinteracts with glycophorin C and spectrin.

A widely held model for red cell membrane elas-ticity is based on the assumption that spectrin exhi-bits entropic-spring behavior (Svoboda et al., 1992).Several other studies indicate that the elasticdeformability of the erythrocyte depends onenthalpic events (Vertessy & Steck, 1989). Accord-ing to both models, the membrane elasticity ismainly due to the properties of spectrin. However,the rigidity of the erythrocyte membrane alsodepends on integral membrane proteins that spanthe bilayer, mainly glycophorin C and band 3 andtheir oligomerization states (Feng & MacDonald,1996), indicating that all components of the mem-brane skeleton contribute to the elasticity of thered cells.

Most of the spectrin sequence is composed of aseries of contiguous motifs called spectrin repeatsthat characterize all members of the spectrinfamily, namely spectrin, a-actinin, dystrophin, andutrophin (Hartwig, 1994). Additional structuralfeatures include an actin-binding domain made oftwo calponin homology (CH) domains (Djinovicet al., 1997), a plekstrin homology (PH) domain(Macias et al., 1994), a Src homology 3 (SH3)domain (Musacchio et al., 1992), and a calmodulin-like domain with four EF-hands (Trave et al., 1995).

The ability of the spectrin molecule to contractand expand has been attributed to the modularstructure made of repeats, initially identi®ed bypartial peptide sequences (Speicher & Marchesi,1984). The global molecular architecture was laterrevealed by complete sequencing of cDNAs for thea and b subunits from several species. The analysisof the conformational phase of the repeat usingrecombinant fragments of different lengths hasestablished the boundaries for properly foldedrepeats (Winograd et al., 1991). Despite lowsequence homology, theoretical models have pro-posed that the repeat folds into a left-handedcoiled-coil made of three antiparallel a-helices (A,B, and C) separated by two short loops (AB andBC; Parry et al., 1992). However, the crystal struc-ture of the dimeric 13th repeat of Drosophila a-spec-trin (PDB ID: 2SPC) has showed an intermoleculartriple helical bundle where helices A and B fromone molecule pack against helix C0 of the othermolecule in the dimer and the proposed BC loopappears in an a-helical conformation as part of acontinuous helix B-C(Yan et al., 1993). In the intactspectrin molecule, both termini must point inopposite directions. The termini in the crystalstructure are oriented in the same direction which

is incompatible with the global arrangement ofrepeats inside spectrin. The conformation found inthe crystal is likely to be an artifact caused by thedimerization process that this particular repeat suf-fers in solution at room temperature when it isrecombinantly produced as a GST-fusion (Ralstonet al., 1996). From the X-ray data (Yan et al., 1993),a model for the monomer was proposed thatagrees with the secondary but not with the tertiarystructure proposed by modeling (Parry et al., 1992),i.e. it proposes the existence of the BC loop butsuggests that the repeat is a helical bundle ratherthan a coiled-coil.

The importance of the spectrin repeat to the elas-tic properties of the membrane skeleton and to thestructural and functional integrity of the normalred cell is demonstrated by the fact that it is a tar-get for mutations which cause hemolytic anemias,such as some cases of hereditary elliptocytosis,pyropoikilocytosis and spherocytosis characterizedby abnormally shaped erythrocytes (Davies & Lux,1989; Winkelmann & Forget, 1993; Hassoun &Palek, 1996). Mutations associated with these ane-mias occur in helical regions of the spectrinrepeats, and frequently disrupt the spectrin tetra-mer formation (Tse et al., 1990). Impairment of thetetramer formation alters the membrane skeletonstructure and leads to reduced mechanical stabilityof the membrane. Mutations involve nucleotidesubstitutions, exon skipping, alternative splice-siteselection, short deletions and frameshifts (Hassounet al., 1996).

In earlier studies (MacDonald et al., 1994;Pascual et al., 1996), we showed that the 16threpeat from chicken brain a-spectrin (R16;Wasenius et al., 1989) in solution folds into a mono-meric helical structure composed of three helicesseparated by two loops at room temperature(298 K) and at the 1 mM concentration required forNMR. Recently, it has been demonstrated that theisolated repeat 1 from human erythrocyte a-spec-trin (DeSilva et al., 1997) and repeat 2 from humandystrophin (Calvert et al., 1996) are also mono-meric. Here, we report the complete assignment ofthe 15N, 1H and 13C chemical shifts of R16 anddetermination of its three-dimensional structure insolution, providing the ®rst experimental three-dimensional structure of a spectrin repeat in nativeconformation.

Results

Resonance assignment

The recombinantly expressed R16 domain con-sists of 110 residues spanning from Ala1763 toGlu1872 of chicken brain a-spectrin (SwissProt ID:SPCN_CHICK). Backbone and aromatic side-chainassignment has already been reported (Pascualet al., 1996). Con®rmation of the aromatic reson-ances was accomplished by analyzing a 2D 1H-13CHSQC spectrum (Stonehouse et al., 1994) where the13C carrier frequency was placed at 126 ppm. Due

Figure 1. Superposition of the Ca trace for the ®nalensemble of R16 structures (from His10 to Gln107). Thetriangles point towards the C terminus of each of thehelices.

Table 1. Experimental restraints and structural statisticsfor the best 20 structures

Experimental restraintsTotal number of restraints 1035

NOE restraints 719unambiguous 602

intraresidue 157sequential 75medium-range 324long-range 46

ambiguous 117dihedral angle restraints 170hydrogen bond restraints 146

Deviations from experimental restraints:RMSD:

of unambiguous NOE restraints (AÊ ) 0.025 �0.003of ambiguous NOE restraints AÊ ) 0.015 �0.005of dihedral angle restraints (�) 0.287 �0.019of hydrogen bond restraints (AÊ ) 0.003 �0.001of 1H chemical shifts (ppm) 0.241 �0.012

Structural statistics for the ensemble:Quality indices:

overall G value (PROCHECK) 0.15 �0.03what-check score (WHATIF) 0.47 �0.18combined z-score (PROSA) ÿ5.44 �0.52

RMSD from the average structure:all residues (10-107)

backbone (N, Ca, C) (AÊ ) 1.83 �0.12heavy atoms (AÊ ) 2.71 �0.19

residues in a-helical conformationbackbone (N, Ca, C) (AÊ ) 1.52 �0.15heavy atoms (AÊ ) 2.44 �0.20

742 Solution Structure of the Spectrin Repeat

to the high a-helical content, the spectral dispersionof the Ca and Ha chemical shifts was poor, pre-cluding the extension of the assignment to theside-chains. Taking advantage of the 1H-13C-13C-1Hcorrelation, we could obtain side-chain assign-ments, primarily from an analysis of the 3DHCCH-COSY and TOCSY spectra. We used the13Ca and 1Ha chemical shifts as a starting point forthe validation of the previous sequential backboneassignment. In order to help the assignment, infor-mation from the expected 13C chemical shift rangesand 1H spin system topology was used as an initialcriterium. Further con®rmation was obtained bychecking the resonances in the 3D 13C HSQC-TOWNY spectrum. A list containing the 15N, 1H,and 13C chemical shifts assigned and referencedaccording to Wishart et al. (1995) is available assupplementary material.

Experimental restraints

Table 1 shows the experimental restraints usedto calculate the structure. Due to severe overlap,we could manually identify only 355 unambiguousdistance restraints between protons. Application ofthe ``ambiguous restraints for iterative assignment''(ARIA) method (Nilges et al., 1997) increased thenumber of NOE-based distance restraints. Asdescribed in Materials and Methods, after eightiterations a total of 719 distance restraints could beobtained, where 602 were unambiguous and 117ambiguous. The former contained 157 intraresidue,75 sequential, 324 medium-range and 46 long-

range restraints. The elongated shape of the mol-ecule results in a lower number of long-range NOEcontacts in comparison to a globular protein ofsimilar size where the closer proximity of differentsegments allows more interactions. The ambiguousrestraints contained 70 with two, 36 with three,seven with four, three with ®ve and one withseven assignment possibilities. Inside this group ofambiguous restraints, two long-range restraintswere found, the ®rst with two assignment possibi-lities and the second with three.

Measurement of the 3JHN-Ha coupling constantfor R16 (Pascual et al., 1996) allowed the constraintof 85 f angles to ÿ60(�20)�. Since the pattern ofsequential and medium-range NOEs and the con-sensus chemical shift index for these 85 residuesindicate a helical conformation, their c angles were

Figure 2. View of the backbone average structure of R16(from His10 to Gln107). From the N to the C terminus,helix A is colored green, the AB loop red, helix B yel-low, the BC turn red, and helix C cyan. Some of theside-chains of residues implicated in interhelical contactsare shown in white and labeled according to the oneletter code for amino acid residues and in parenthesisaccording to the helices nomenclature.


also constrained to ÿ40(�20)�. In order to de®nethe helices better, we added 73 hydrogen bondscanonical of a-helical conformation along the mainchain as two distance restraints (2.50 < dOi-NHi � 4 < 2.70 AÊ and 2.5 < dOi-Ni � 4 < 3.70 AÊ ).These were assigned to the residues in the centralpart of the helices. Proline residue 61 (the sequenceis shown in Figure 6) and residues around it wereleft unrestrained.

Structure analysis

The termini are highly disordered, as indicatedby their 15N-T1, T2 and 15N-1H heteronuclear NOE

values (Pascual et al., 1996), and therefore a sensi-ble superposition cannot be made with the fullsequence. Consequently, calculation of an averagestructure and analysis of the ensemble was carriedout with structures spanning from His10 toGln107. Table 1 shows the structural statistics ofthe ensemble. The analysis shows that the struc-tures barely violate the empirical restraints andonly slightly deviate from ideal geometry having asmall force ®eld total energy. None of the struc-tures showed NOE violations bigger than 0.5 AÊ

nor dihedral angle violations bigger than 5�. TheRMSD value for the 1H chemical shifts is indicativeof a generally correct assignment. The averageRMSD from the mean structure for the backboneatoms (N, Ca, and C) of residues in helical confor-mation is 1.52 AÊ , and 2.44 AÊ for all heavy atoms.Regarding the quality indices, the overall G value(Laskowski et al., 1996) should be above ÿ0.5, the``what-check'' score value (Vriend & Sander, 1993)for a good structure is expected to be above ÿ0.5and the combined z-score (Sippl, 1993) varies withthe sequence length and for a 100-residue proteinis expected to be ÿ8.0 � 2.0. As shown in Table 1,these criteria are met by the R16 structure.

The ensemble is shown in Figure 1, and the aver-age structure with key residues implicated in inter-helical contacts in Figure 2. The Ramachandranmap (Figure 3) for all 20 structures shows thatmore than 90% of the residues appear in theallowed regions. Just a few loop residues in someof the structures have f/c values in disallowedregions. A plot showing the distribution of thenumber of NOE-restraints per residue and back-bone atoms RMSD from the average per residue isshown in Figure 4a and b. Figure 4c shows therelative solvent accessibility. This plot reveals thatthe most buried residues match with the a and dpositions of the heptad pattern (McLachlan &Stewart, 1975), shown in Figure 6, characteristic ofcoiled-coils (Crick, 1953; Lupas, 1996, 1997). Animprovement in the ®t between the observed andcalculated 1H chemical shifts when comparingensembles calculated without and with 1H chemi-cal shift re®nement can be observed in Figure. 5aand b. The analysis of the ®nal ensemble by PRO-CHECK-NMR indicates that the overall structuralcharacteristics are equivalent to a typical 2.5 AÊ res-olution crystal structure.

Description of the R16 fold

Analysis of the secondary structure of the aver-age structure using the program DSSP (Kabsch &Sander, 1983) shows that R16 consists of threea-helices separated by two loops. The ®rst helix, A,spans from Gln11 (A6, according to the nomencla-ture of Yan et al. (1993); see Figure 6) to Ser32(A27); the second, B, begins with Val42 (B1) andends with Asp76 (B35) accommodating the trans-Pro61 (B20); the last one, C, extends from Lys81(C1) to Gly106 (C26). Helices A and C have seventurns and are smoothly bent throughout their

Figure 3. Ramachandran plot of the f (Phi) and (Psi) torsion angles for all 20 structures in the ensemble generatedusing PROCHECK-NMR. Each square represents the f/c values for a residue from the structure of the ensembleindicated by the number inside the square. Small triangles represent Gly residues. A, B, and L de®ne the mostfavored regions; a, b, l, and p de®ne the allowed regions; �a, �b, �l, and �p de®ne the generously allowed regions.Squares that appear on the latter region are topped by the residue name and number. Notice that all of them belongto loop regions and none appears on the disallowed region systematically.


length. Helix B is the longest with ten turns. It hasa kink (y � 21.8�; Barlow & Thornton, 1988) whichis caused by Pro61. A loop, from Glu33 to Thr40connects the ®rst two helices, whereas a tight turn(Ca

i to Cai � 3 distance� 7.0 AÊ ) from Asn77 to Gly80

makes the connection between helices B and C.Most of the contacts at the interface between the

helices are made by hydrophobic side-chains,which are highly conserved in the spectrin repeatsand occupy the positions a and d of the heptad pat-tern of coiled-coils. However, ionic interactionsbetween charged side-chains of variable residuesmainly at g and e positions also appear to stabilize

the structure. A total of 46 interhelical NOEs de®neseveral contact regions and allow the mapping ofthe interior of the structure (Figure 2). At the top,there are interactions between the side-chains ofPhe12 (A7), the variable Gly70 (B29) and Ile84(C4); below, contacts appear between Met16 (A11),Val66 (B25) and Phe91 (C11). In the middle, thealmost invariant Trp22 (A17) in the g positioninteracts with the variable His59 (B18) and withTrp95 (C15). Near the bottom, contacts involvingVal30 (A25), Leu52 (B11) and Ala102 (C22) can beobserved. Finally, an electrostatic rather than ahydrophobic interaction occurs at the bottom

Figure 4. Plot showing the distribution of the number ofNOE restraints (a), backbone atoms RMSD from theaverage (b) and the relative solvent accessible area (c)per R16 residue (from His10 to Gln107).

Figure 5. Plots of the difference between the averageobserved and calculated 1H chemical shifts versus thesequence number (from His10 to Gln107). In a, anensemble of ten structures coming from the last ARIAiterative calculation without 1H chemical shift re®ne-ment was used. In b, the ®nal 20 structures re®ned invacuum and against 1H chemical shifts were the inputfor the calculation.


between the variable Glu33 (A28), the conservedAsp34 (A29) and two highly conserved positivelycharged residues, namely Lys48 (B7) and Arg105(C25) both in g positions.

Analysis of the tertiary structure of the averagestructure of R16 was performed using the programHELIX (Walther et al., 1996). Table 2 shows thegeometrical and packing parameters measured foreach pair of helices. Notice that the helices A/Band B/C are antiparallel while helices A/C areparallel. The column gives the crossing anglevalue for each pair of helices. Depending on the

orientation of the helices in the pair (parallel orantiparallel), the crossing angle varies by 180�.Regarding the helical packing, the ratio columnpresents the percentage of residues in close contact(all column) that pack in ``knobs into holes'' man-ner (153 column). Measurement of these par-ameters shows that R16 folds into a left-handedantiparallel triple helical coiled-coil with crossingangles for the parallel (A/C) and antiparallel (A/Band B/C) helices of 30.8�, ÿ144.7� and ÿ162.3�,respectively, and an average pitch of 178.6 AÊ . Onaverage, 52% of the residues implied in R16 inter-helical contacts show the classical ``knobs intoholes'' type of packing characteristic of coiled-coils.

A comparison of the same parameters with thecrystal structure of the Drosophila repeat (2SPC)and with coil-Ser (Lovejoy et al., 1993; PDB ID:1COS), a synthetic trimer composed of threea-helices folded into an antiparallel coiled-coil, isalso shown in Table 2. Analysis of the R16 foldagainst the PDB using the program DALI (Holm &

Figure 6. Alignment of spectrin repeats. The numbering corresponds to the fragment used in the solution structure.The sequences aligned are the 16th repeat of chicken brain a-spectrin (a16) solved by NMR, the 13th repeat of Droso-phila a-spectrin (a13) solved by X-ray, and the ``tetramerization'' repeat made of the partial repeats of human erythro-cyte b/a-spectrin (b17/a0). The consensus line was obtained from a complete alignment of all repeats from humanerythrocyte spectrin. The heptad pattern as well as the helices nomenclature are shown. The multiple sequence align-ment was made using the program CLUSTALW (Thompson et al., 1994).


Sander, 1993) reported as the highest score(z � 6.1) the match with the avian farnesyl dipho-sphate synthase (PDB ID: 1FPS). Both structuressuperimpose with a RMSD of 2.8 AÊ for 70% of theCa atoms of the repeat including fragments fromall three helices.

Discussion

The spectrin repeat folds into atriple-helical coiled-coil

As previously reported (Pascual et al., 1996), R16folds into a monomeric triple-helical structure. Thefold of R16 is composed of three long helices (A, B,C) separated by a loop (AB) and a turn (BC). Asdepicted in Figures 1 and 4b, both the AB loop andthe BC turn correspond to the more disorderedareas. However, the close correspondence betweenthe positional disorder and their mobile behaviorobserved via the relaxation measurements suggeststhat the disorder is not due to the scarcity ofrestraints for those residues but re¯ects real mobi-lity in solution. Moreover, the AB loop shows ahigher degree of disorder than the BC turn. This isin agreement with the relaxation parameters of therepeat where a shorter value for 15N-T1, longer forT2 and a smaller 15N-1H heteronuclear NOE areobserved for the AB loop residues compared tothose of the BC turn (Pascual et al., 1996). Regard-ing the helices, a high proportion of the residuesimplicated in interhelical contacts pack their side-chains following the well-de®ned ``knobs intoholes'' arrangement characteristic of a coiled-coil.The crossing angle and the pitch values corrobo-

Table 2. Helical geometry and packing parameters of R16, 2

Helices GeomeMolecule Helix 1 Helix 2 (�)

R16 A B ÿ144.7A C 30.8B C ÿ162.3

2SPC A B ÿ157.0A C0a 12.5B C0a ÿ160.8

1COS A B ÿ157.7A C 32.7B C ÿ159.3

a This helix comes from the second molecule in the crystal dimer.

rate the coiling of the helices (Table 2). The trans-Pro61 (f ÿ66.4, c ÿ29.6) is accommodated in themiddle of helix B by a kink. Other repeats alsoshow a proline residue at the same helical position(B20; Pascual et al., 1997), indicating that a singleproline residue can be present inside a helix with-out preventing its folding (Barlow & Thornton,1988).

Superposition of the backbone atoms of the heli-cal residues between the average solution structure(R16) and the monomeric model from the dimericX-ray structure (2SPC) shows a RMSD of 2.1 AÊ .These repeats have diverged early in evolution(Pascual et al., 1997) and have only 22% identicalresidues at equivalent positions (Figure 6). Of these23 identical residues, 13 are highly conservedamong all spectrin repeats. Most of them arehydrophobic residues that occupy the a and d hep-tad positions (A4, B1, B4, B8, B22, B32, C15, C18,and C29) and show low dispersion of their w1

angle in the ensemble of NMR structures. How-ever, these side-chains do not superimpose wellwith those from the crystal model, showing someof them to have distinct gauche/trans w1 rotamers.Moreover, the aromatic cluster made by A17/B18/C15 shows a different disposition of the rings(Figure 7).

Contrasting their tertiary structure (Table 2),different values are obtained for the crossing angle,pitch and ratio of residues packed according to the``knobs into holes'' arrangement. For comparison,Table 2 includes the corresponding values for asynthetic antiparallel triple-helical coiled-coil(1COS) with almost identical helical length. Noticethat 1COS should provide the ideal values for an

SPC, and 1COS

try PackingPitch (AÊ ) 153 All Ratio (%)

198.4 5 10 50157.2 7 12 58180.2 7 15 47251.2 9 23 39206.1 12 26 46308.8 8 26 31197.2 14 25 56167.6 17 24 71192.8 15 19 79

Figure 7. Superposition between the X-ray monomericmodel (depicted in red) and the average solution struc-ture (depicted in green) of the conserved hydrophobicside-chains A17, B18, and C15 in the core of the repeat.These side-chains show a low dispersion of their w1

angles in the ensemble of solution structures.


antiparallel triple-helical coiled-coil since it onlycontains leucine residues at the a and d positions.Since R16 shows a higher crossing angle, lowerpitch and higher ratio of residues packed in``knobs into holes'' as compared to 2SPC, we con-clude that the repeat folds into a coiled-coil in sol-ution whereas the crystal structure of the repeat isbetter described as a bundle. These discrepanciescould re¯ect the possibility that the crystal struc-ture resembles more the ``tetramerization'' repeat(a very special repeat constituted by intermolecularinteractions of the C-terminal helices A and B ofb-spectrin and the N-terminal helix C of a-spectrin)rather than a typical internal repeat exempli®ed bythe NMR structure.

Antiparallel interactions between repeatswithin the dimer

Since the functional unit of spectrin is a dimerand that of dystrophin is a monomer, differencesin the pattern of conservation of residues betweenrepeats in both proteins could explain that differen-tial property (Winder et al., 1995). There are onlytwo highly conserved and exposed (either in b, c orf positions) residues within the helices of the spec-trin repeats that are not conserved in the dystro-phin repeats. These are Asp18 (A13) and Glu25(A20) which are both in consecutive c heptad pos-itions of the helix A (Figure 6). Conservation of aresidue or a physical property at a speci®c positionor area could indicate an important structuraland/or functional role. In this case, however, apossible involvement of these charges in functionslike repeat-repeat interactions in the antiparalleldimer is unclear since conserved and exposed resi-

dues with opposite charge are not observed in thesequence alignment of the repeats.

Molecular explanation for hemolytic anemias

Several point mutations in the repeats fromhuman erythrocyte spectrin have been related to asubset of hemolytic anemias such as hereditaryelliptocytosis, pyropoikilocytosis and spherocyto-sis. Most of those mutations have been mappedinside the ``tetramerization'' repeat. We have builta model by homology of the tetramerization repeatusing the R16 structure as a template and employ-ing the WHATIF program (Vriend, 1990) to inter-pret the puzzling fact that a single point mutationleads to the disease.

This repeat is built by interactions between twomolecules, and it is therefore likely to be very sen-sitive to mutations that destabilize the interhelicalpacking. One of the mutations appears in positionA17 where the highly conserved Trp is mutated toArg (Parquet et al., 1994). This conserved Trp hasbeen described as one of the key residues for thefolding of the repeat where a conservativemutation to Phe already causes a signi®cantdecrease in its global stability (Pantazatos &MacDonald, 1997). Apart from this, the model indi-cates that an Arg residue in this position wouldcause a charge repulsion with the Arg in positionB21. This could prevent the proper folding of therepeat. An interesting mutation is caused by thereplacement of Ala with Pro in position B10 (Tseet al., 1990). The presence of a single Pro residue isnot rare in helix B of the spectrin repeats, especiallyin position B20. In fact, the tetramerization repeatcontains a Pro residue in position B28. The pre-sence of a second Pro in the same helix most likelycauses another kink that disrupts the proper pack-ing. An additional peculiar feature of this repeat isthe presence of ®ve Arg residues in the helix C forwhich mutations have been found (C7, C8, C14,C21, and C25; Palek & Sahr, 1992; Winkelmann &Forget, 1993). For instance, the substitution of Argwith Leu in position C8 causes the loss of an inter-helical salt bridge with the Glu at B26 according toour model. The same is applicable to the Arg toSer mutation at C25 which may lead to the break-age of another highly conserved interhelical ionicinteraction with the Asp at A29 (Figure 8).

Elastic properties of spectrin

It has been proposed (Bloch & Pumplin, 1992)that the residues connecting two sequential repeatscould act as a hinge region being largely respon-sible for the elastic properties of spectrin. However,this region is rather short, in general comprisingonly two residues which are rarely Gly or Pro.Thus, it is possible that there is a continuous C-Ahelix between the repeats, although the heptad pat-tern is lost between helix C of one repeat and helixA of the next one. The likely residues implicated ininter-repeat contacts are conserved, hydrophobic

Figure 8. Model of the arrangementof the conserved side-chains A29,B7, and C25 of the ``tetrameriza-tion'' repeat colored in green inter-acting via an intermolecular saltbridge as well as a model for theeffect of the Arg to Ser (colored inred) mutation in C25 that preventsthe tetramerization. The inset thebackbone of the model for the tet-ramerization repeat; helices A andB from the b-chain are in whiteand helix C from the a-chain is incyan. Notice the absence of the BCloop. Each triangle points towardsthe C terminus of its respectivehelix.


and exposed like A2 (His7 in R16) that occupiesthe heptad postition f, the ®rst (Tyr35) and the ®fth(Leu39) residue in the AB loop and the ®fth(Thr78) residue in the BC turn (Figure 6). Unfortu-nately, these residues show the highest disorderboth in the crystal and solution structures of singlerepeats. Consequently, further insights into themolecular mechanism of spectrin elasticity willbecome available when a re®ned structure of adouble repeat has been determined, and the struc-ture of the proposed hinge region and the inter-repeat contacts emerge.

Materials and methods

NMR spectroscopy

The expression, puri®cation and 15N/13C labeling ofR16 was carried out according to Pascual et al. (1996).The NMR sample contained 1 mM of uniformly15N/13C-labeled R16 dissolved in 90% H2O/10% 2H2O,10 mM potassium phosphate (pH 6.0). The NMR spectrawere recorded at 308 K using Bruker DMX 500/600MHz spectrometers. Water suppression was accom-plished using WATERGATE (Piotto et al., 1992) or coher-ence selection by pulsed ®eld gradients combined withsensitivity enhancement (Kay et al., 1992). The exper-iments acquired for the backbone and aromatic sidechains assignment have already been described (Pascualet al., 1996). In order to assign the side-chains 3D 13C-edi-ted HCCH-COSY (Ikura et al., 1991) and HCCH-TOCSY(Bax et al., 1990) spectra were obtained, using 20 ms asTOCSY mixing time, as well as a 3D 13C HSQC-TOWNYmodi®ed from a 13C HSQC-NOESY according toMajumdar & Zuiderweg (1993) by introducing theTOWNY scheme (Kadkhodaei et al., 1993) with a mixingtime of 60 ms. For structure calculation purpose, two 3DNOESY-type spectra, namely 15N NOESY-HSQC(Sklenar et al., 1993) and 13C HSQC-NOESY (Majumdar& Zuiderweg, 1993), were acquired with mixing times of70 ms. All 3D experiments were recorded in the phase-sensitive mode using the States-TPPI method (Marionet al., 1989). The NMR spectra were processed and ana-lyzed on Silicon Graphics workstations using the pro-grams AZARA (Boucher, 1994) and AURELIA (Neidig

et al., 1995). Linear prediction was applied to theindirectly detected dimensions. The residual water signalin some of the 3D spectra was suppressed by means ofthe Karhunen-Loeve transformation (Mitschang et al.,1991). The ®nal size of the 3D data matrices was1024 � 512 � 256 real points.

Structure calculation

Structure re®nement was achieved employing theambiguous restraints for iterative assignment (ARIA)methodology (Nilges et al., 1997). ARIA performs anautomatic assignment of NOESY spectra peaks based onthe chemical shifts list and its compatibility with theinterproton distances measured in the initial structures.Merging of the manually assigned NOE restraints withthe automatically assigned NOE restraints is done inorder to calculate better de®ned structures that will beused in the next iteration. The structure re®nement isobtained by molecular dynamics using version 3.851 ofX-PLOR (BruÈ nger, 1992) adapted to interpret ambiguousdistance restraints (Nilges, 1995) and applying speci®csimulated annealing protocols as described by Nilgeset al. (1997). Assignment of distance restraints implyingmethylene and isopropyl group resonances is done usingthe ¯oating chirality approach (Weber et al., 1988;Folmer et al., 1997).

In our case, three data sets were used. The ®rst oneconsisted of 355 NOE-based unambiguous distancerestraints derived from manual assignment of differentNOESY spectra. The second and the third data sets wereobtained from the 3D 15N NOESY-HSQC and 13CHSQC-NOESY spectra using the automatic peak-pickingroutine of AURELIA. A manual cleaning for obviousartifacts and solvent residual signal of the peak-pickedlists was performed. Automatic assignment of the pickedpeaks was obtained using a frequency window tolerancearound their chemical shift values of �0.02 ppm in the1H acquisition dimension, �0.1 ppm in the 1H indirectdimension and �0.2 ppm in the heteronuclear one. Con-version of peak volumes into distance restraints wasaccomplished through the calibration proceduredescribed by Nilges et al. (1997). The initial structuresused for the ARIA re®nement were the seven lower inenergy from 500 structures calculated employing the dis-tance geometry program DIANA (Guntert et al., 1991)


applying the REDAC strategy (Guntert & Wuthrich,1991). They were obtained using 355 NOE-based unam-biguous distance restraints manually assigned including32 long-range NOEs; cross-peak intensities were classi-®ed as strong, medium or weak using contour levels forcalibration, and the upper limits for the distancerestraints were set to 3.0 AÊ , 4.0 AÊ and 5.0 AÊ , respectively.Eight ARIA iterations were run modifying the assign-ment parameter (p) from 0.999 to 0.8, the violationthreshold (Tv) from 2.0 AÊ to 0.0 AÊ and the violation ratio(Nv) from 0.5 to 0.75. In each iteration ten structureswere re®ned although just the seven structures withlower energy were used for assignment purpose. At the®nal iteration, a total of 719 distance restraints based onNOEs were obtained, where 602 were unambiguous and117 ambiguous.

Further re®nement to detect ionic interactions on thesurface of the molecule was accomplished by the simu-lation of non-bonded interactions in vacuum using all719 NOE-derived distance restraints and an X-PLORenergy function that included an electrostatic potentialterm and a 1H-chemical shift pseudopotential(Kuszewski et al., 1995). Starting with the best 40 struc-tures from the initial distance geometry calculation, 40new structures were calculated in vacuum. The 20 struc-tures with lower energy were selected for structure anal-ysis. All 20 structures were superimposed to well-de®ned regions (from His10 to Gln107) and an averagestructure spanning those residues was calculated. Aquality analysis of the ®nal superimposed ensemble wasassessed using the programs PROCHECK-NMR(Laskowski et al., 1996), WHATIF (Vriend & Sander,1993) and PROSA (Sippl, 1993). The coordinates of theensemble of 20 structures (PDB ID: 1AJ3) plus a list withthe experimental NMR restraints (PDB ID: R1AJ3MR)are available at the Brookhaven Protein Data Bank.

Helical geometry and packing analysis

The de®nitions and sign conventions of helical geome-try and packing parameters were adapted from those ofWalther et al. (1996). The measurements were obtainedusing the program HELIX (Walther et al., 1996) and per-formed for each pair of helices. Parameters studiedincluded the dihedral packing (or helix-crossing) angle() associated with the line of closest approach betweenthe pair of helices; the pitch or helical supercoiling, esti-mated by assigning local lines of closest approach toboth ends of a given helix-helix interface and relating thedihedral angle associated with the vector connecting themiddle points of the local lines to the length of the vec-tor; and the automated identi®cation of the so-called``knobs into holes'' (cell 153, according to Walther et al.(1996)) type of helical side-chain packing distinctive ofcoiled-coils (Crick, 1953).

Acknowledgments

We thank Y. Prigeant and D. Davoust for providingmeasuring time at the NMR laboratory of the Universitede Haute Normandie, Rouen (France). We also thankFrancisco Blanco for his constant help and supportduring the project and constructive criticism of themanuscript.

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Edited by P. E. Wright

(Received 12 June 1997; received in revised form4 August 1997; accepted 5 August 1997)

http://www.hbuk.co.uk/jmb

Supplementary material comprising one Table isavailable from JMB Online.

solution structure of the spectrin repeat: a left-handed antiparallel triple-helical coiled-coil

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