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doi:10.1016/j.jmb.2006.10.016 J. Mol. Biol. (2007) 365, 226–236

Low Resolution Crystal Structure of ArenicolaErythrocruorin: Influence of Coiled Coils on theArchitecture of a Megadalton Respiratory Protein

William E. Royer Jr ⁎, Michael N. Omartian and James E. Knapp

Department of Biochemistry andMolecular Pharmacology,University of MassachusettsMedical School, Worcester,MA 01605, USA

Abbreviation used: HBL Hb, hexahemoglobin.E-mail address of the correspondi

[email protected]

0022-2836/$ - see front matter © 2006 E

Annelid erythrocruorins are extracellular respiratory complexes assembledfrom 180 subunits into hexagonal bilayers. Cryo-electron microscopicexperiments have identified two different architectural classes. In one,designated type I, the vertices of the two hexagonal layers are partiallystaggered, with one hexagonal layer rotated by about 16° relative to theother layer, whereas in the other class, termed type II, the vertices areessentially eclipsed. We report here the first crystal structure of a type IIerythrocruorin, that from Arenicola marina, at 6.2 Å resolution. The structurereveals the presence of long continuous triple-stranded coiled-coil “spokes”projecting towards the molecular center from each one-twelfth unit;interdigitation of these spokes provides the only contacts between thetwo hexagonal layers of the complex. This arrangement contrasts with thatof a type I erythrocruorin from Lumbricus terrestris in which the spokes arebroken into two triple-stranded coiled coils with a disjointed connection.The disjointed connection allows formation of a more compact structure inthe type I architecture, with the two hexagonal layers closer together andadditional extensive contacts between the layers. Comparison of sequencesof the coiled-coil regions of various linker subunits shows that the linkersubunits from type II erythrocruorins possess continuous heptad repeats,whereas a sequence gap places these repeats out of register in the type Ilinker subunits, consistent with a disjointed coiled-coil arrangement.

© 2006 Elsevier Ltd. All rights reserved.

Keywords: erythrocruorin; hemoglobin; HBL Hb; coiled coils; proteinassembly
*Corresponding author
Introduction

In many annelids, oxygen transport relies on giantextracellular respiratory proteins (∼3.6×106 Da),known as either erythrocruorins or hexagonalbilayer hemoglobins (HBL Hbs). Such large com-plexes offer a number of important advantages asoxygen transport vehicles: erythrocruorins are read-ily retained in the vascular system as freelydissolved entities, each complex possesses largeoxygen binding capacity and subunits can bearranged to permit cooperative oxygen bindingand additional regulatory features that enhanceoxygen transport.

gonal bilayer

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Electron microscopic investigations dating back tothe 1960 s established the overall shape of theerythrocruorins as consisting of two hexagonallayers.1 More recent investigations using cryo-electron microscopy with image reconstructionhave revealed two distinct forms of erythrocruorins.In one, designated as type I, the vertices of the twolayers are partially staggered with one hexagonallayer rotated relative to the other layer by about 16°.In the second form, designated as type II, the twohalves of the molecule are essentially eclipsed.2 Thetype I architecture appears to be much morewidespread, having been observed in erythrocruor-ins from oligochaetes (earthworm),3,4 achaetes(leech)5 and vestimentiferans (hydrothermal venttube worm)6 and from two polychaete chloro-cruorins.7,8 The type II erythrocruorin architecturehas been observed in three polychaete species.2,9,10

The crystal structure of a type I erythrocruorin,that from the common earthworm Lumbricus

d.

mailto:[email protected]

227Architecture of Megadalton Respiratory Proteins

terrestris, has been reported at 3.5 Å resolution.11

Lumbricus erythrocruorin assembles from144 oxygen-binding hemoglobin subunits and 36 structural“linker” subunits. The hemoglobin subunits arearranged into 12 dodecamers, which assemble ontoa central core formed from linker subunits. Theoverall D6 symmetry of the molecule, and arrange-ment of the hexagonal layers, is dictated by thecentral linker complex. Twelve interdigitated hetero-trimeric coiled-coil “spokes”, formed from the aminotermini of the linker subunits, project towards thecenter of the complex. A striking break separates along (∼40 Å) from a short (∼25 Å) coiled coil in thelinker complexes of Lumbricus erythrocruorin. Basedon electron microscopic analysis, it has been pro-posed that the alternate type I and type II archi-tectures may be coupled with differences in thecoiled-coil arrangements.10

Arenicola marina erythrocruorin is probably themost well studied of the known type II erythro-cruorins, and recently has been proposed as apotential blood substitute.12 It shows cooperativeoxygen binding, with maximum Hill coefficientsabove 4, and strong sensitivity to divalent cationsand protons.13 Mass spectrometry experimentshave identified eight distinct hemoglobin subunits,five of which have now been sequenced and twodistinct linker subunits, one of which has beensequenced.14,15 As in the case of Lumbricus ery-throcruorin, dissociation reveals both disulfidelinked hemoglobin trimers and hemoglobin sub-units not disulfide linked to other subunits.14

We have undertaken crystallographic analysis ofArenicola erythrocruorin in order to examine thestructural determinants underlying the alternatearchitectures of the two classes of annelid erythro-cruorins. The low resolution crystal structure pre-sented here reveals long uninterrupted triple-stranded coiled coils formed from linker chains intype II erythrocruorin. Coupled with alternatecoiled coil arrangements are striking differences inpacking between hexagonal layers in type I and IIarchitectures.

Results

Structure determination

The crystal structure of Arenicola erythrocruorinwas determined by a combination of molecularreplacement and non-crystallographic symmetryaveraging. Use of a search molecule comprisingone-half of Lumbricus erythrocruorin (excluding thelong coiled coils) allowed placement of both halvesof this 3.6×106 Da complex in the crystallographicasymmetric unit. This molecular replacement solu-tion permitted calculation of preliminary phases at8.5 Å resolution and determination of initial non-crystallographic symmetry operators relating the 12protomers in the whole molecule. Molecular aver-aging, with phase extension to 6.2 Å, resulted in areadily interpretable map revealing the disposition

of 144 hemoglobin subunits and clear rods ofdensity corresponding to the 12 triple-strandedcoiled-coil spokes of the linker subunits. In addition,density features for the non-helical linker LDL-Aand β-barrel domains are consistent with thestructures and disposition of these domains inLumbricus erythrocruorin.

Overall structure of Arenicola erythrocruorin

The final electron density map at 6.2 Å resolutionfor one whole molecule is shown in Figure 1.Hemoglobin subunits, whose density is coloreddark violet, occupy the surface of the molecule,with linker subunits (orange and blue) forming acentral core. The molecule exhibits overall D6symmetry. Perpendicular to the molecular 6-foldare dyad axes oriented every 30°. Two unique dyadaxes are present; similarly to our earlier descriptionof Lumbricus erythrocruorin,16 we designate theseaxes as P and Q (Figure 1). These molecularsymmetry axes relate 12 protomers, each of whichis composed of 12 hemoglobin and three linkersubunits.

Arrangement of hemoglobin subunits

The hemoglobin subunits of Arenicola erythro-cruorin are arranged into dodecamers that are verysimilar to the dodecamers found in Lumbricuserythrocruorin. A molecular 3-fold within a hemo-globin dodecamer relates three tetrameric units that,by analogy with Lumbricus erythrocruorin,11 mostlikely correspond to a heterotetramer. The arrange-ment of hemoglobin subunits is similar to that inLumbricus erthrocruorin. The 12 highest electrondensity peaks in each protomer, all above 6.8σ,correspond to the 12 heme iron atoms. Iron peaksare present in pairs, 19–20 Å apart; these pairscorrespond to the iron atoms within hemoglobin EFdimeric assemblages that are characteristic of allcooperative invertebrate hemoglobins investigatedto date.17

Although the structure of individual hemoglobindodecamers from Lumbricus and Arenicola erythro-cruorin are very similar, arrangements of dodeca-mers in the two whole molecules are different.Our maps of Arenicola erythrocruorin confirmearlier electron microscopic results,2 indicatingthat the hemoglobin dodecamers in the twohexagonal layers are in an eclipsed orientation(Figure 2). In contrast, hemoglobin dodecamersfrom the two hexagonal layers of Lumbricuserythrocruorin are partially staggered. (Densityshown for Lumbricus erythrocruorin was calcu-lated at 6.2 Å resolution, following averagingprocedures similar to those used for the Arenicolaerythrocruorin maps.) Using the orientation shownin Figure 2(a), transition from Arenicola to Lum-bricus erythrocruorin involves rotation of the toplayer clockwise by 16° and translation of the twohexagonal layers closer together by ∼15 Å (Figure2(b) and (c)). This brings Lumbricus hemoglobin b

Figure 1. Arenicola erythro-cruorin. Electron density at 6.2 Åresolution is represented at the1.5σ level. Density correspondingto the oxygen-binding hemoglobinchains is shown in dark violet,density for linker subunits isshown in orange for the tophexagonal ring and blue for thebottom hexagonal ring. (a) Wholemolecule viewed along the mole-cular 6-fold axis. (b) Whole mole-cule viewed along the P-dyad axis,with the molecular 6-fold axisvertical and the Q-dyad axis hor-izontal. (c) Pair of one-twelfthprotomer subunits in the sameorientation as in (b). Figures 1–4were produced with PYMOL(DeLano, W. L. (2002). ThePyMOL Molecular Graphics Sys-tem: [http://www.pymol.org].

228 Architecture of Megadalton Respiratory Proteins

subunits from different hexagonal layers intoproximity at the P dyad.11 Thus, the type Iarchitecture is more compact than the type IIarchitecture. Despite the closer approach of layers,

hemoglobin subunits from one layer do notcontact hemoglobin or linker subunits from theapposing layer in either type I or type IIarchitecture.

http://www.pymol.org

Figure 2. Hemoglobin dodecamers in Arenicola and Lumbricus erythrocruorin. Density at 6.2 Å resolution is displayedat the 1.5σ level for Arenicola erythrocruorin and 1.3σ level for Lumbricus erythrocruorin. (a) Views along the molecular 6-fold axis. Note the eclipsed arrangement of dodecamers inArenicola erythrocruorin compared with the partially staggeredarrangement in Lumbricus erythrocruorin. (b) Stereo view of Arenicola hemoglobin dodecamers along the P axis. (c) Stereoview of Lumbricus hemoglobin dodecamers along the P-dyad axis.



Protomer structure

The protomer (one-twelfth of a whole molecule) ofArenicola erythrocruorin consists of a hemoglobindodecamer and a trimeric linker complex. Densitymaps for protomers of Arenicola and Lumbricuserythrocruorins are shown in Figure 3. Each proto-mer exhibits a local 3-fold axis of symmetry withinthe hemoglobin portion, with density essentiallyidentical (correlation coefficient of 0.95) for threetetramers within a hemoglobin dodecamer. A quasi3-fold axis relates the globular portion of three linkersubunits (correlation coefficient of 0.70–0.71); thisquasi 3-fold is coincident, within 0.3°, to that of themolecular 3-fold of the globin dodecamer 3-fold.

Figure 3. Stereo views of one-twelfth protomers. Surface dat 1.5σ and Lumbricus erythrocruorin at 1.3σ. Density correspviolet, that corresponding to the LDL-A and β-barrel domainsAn approximate local 3-fold (vertical) relates the hemoglobinLumbricus, the short coiled coil. The similar structures for the tw

Alignment of the 3-fold axes assures similar inter-actions between the hemoglobin subunits and eachlinker subunit. Electron density is consistent withthe LDL-A and β-barrel domains interacting withthe hemoglobin subunits in much the same way astheir counterparts do in Lumbricus erythrocruorin.Since the globular portion of the linker subunits

contains little α-helical structure, 6.2 Å resolutionmaps are not sufficient to interpret their structure.However, the known 3.5 Å Lumbricus erythrocruorinstructure11 does allow approximate placement of theLDL-A and β-barrel linker domains within theArenicola erythrocruorin maps. In addition to den-sity at the 1.5σ level that nicely outlines the main-chain β-barrel structure, three density peaks above

ensity representation is shown for Arenicola erythrocruorinonding to the hemoglobin dodecamer is depicted in darkis shown in blue and that for the coiled coils are in yellow.subunits along with the LDL-A, β-barrel domain and, foro erythrocruorins break down at the coiled coils (yellow).


5.5σ clearly pinpoint the calcium ions bound to theLDL-A domains. These peaks suggest slightlydifferent orientations of the LDL-A modules inArenicola and Lumbricus erythrocruorin that pre-sumably result from their close proximity to thecoiled-coil helices (see below).The major difference between protomers of

Arenicola and Lumbricus erythrocruorins occurs inthe triple-stranded coiled coils (shown in yellow inFigure 3). In both molecules, each coiled coil projectstowards the molecular center from the globularportion of the protomer. Density in Arenicolaerythrocruorin indicates a continuous ∼70 Å longcoiled coil. In contrast, the coiled-coil density inLumbricus erythrocruorin is broken up into a long(∼45 Å) coiled coil and a shorter (∼20 Å) coiled coilwithin the globular portion of the linker headregion.The density for the coiled-coil helices also pos-

sesses a quasi 3-fold axis; however, the coiled coil 3-fold is inclined in Arenicola erythrocruorin relative tothose of the dodecamer and linker globular portionby 15°. This inclination angle is larger than, butoriented in a similar direction to, the 9° angleobserved between the long coiled coil and hemoglo-bin dodecamer 3-fold in Lumbricus erythrocruorin.(The short coiled-coil in Lumbricus erythrocruorinpossesses a quasi 3-fold that is essentially coincidentwith that of the hemoglobin dodecamer.) Asdiscussed below, the different arrangements ofcoiled coils in Arenicola and Lumbricus erythrocruor-ins have profound implications for the overallarchitecture of these complexes.

Central linker complex

The differences between type I and II architecturesin arrangement of the hemoglobin dodecamers(Figure 2) are evident in, and indeed dictated by,the arrangement of the 36 linker subunits at the coreof these complexes. Views along the 6-fold axis(Figure 4(a)) show the linker globular region ofArenicola erythrocruorin to be slightly staggeredbetween layers, but with the local 3-fold orientedsuch that bound hemoglobin dodecamers attain aneclipsed arrangement. (The local 3-fold axes of thelinker head region and dodecamers do not projectradially from the center of the complex).The effect of the linker coiled coils on the overall

type I and type II architectures can be appreciatedfrom views of the complete linker complex alongthe P-dyad (Figure 4(b) and (c)). The continuouscoiled coils of Arenicola erythrocruorin limit theextent of contacts between the two hexagonallayers. Introduction of a break in the coiled coils,as observed in Lumbricus erythrocruorin, iscoupled with rotation of one layer about the 6-fold axis and translation of layers closer together(∼15 Å). This results in a more compact linkercomplex and greater contacts between layers inLumbicus erythrocruorin. Contributing greatest tothe difference is Lumbricus linker chain L1, whoseconnection between long and short coiled-coil

helices exhibits the longest inter-helical segmentand whose β-barrel domains participate in exten-sive contacts at the Q-dyad of the bilayer inter-face.11 These bilayer interactions likely providegreater stability for the central linker complex inLumbricus erythrocruorin. It appears, at this reso-lution, that the bilayer interactions are limited tothe coiled coils themselves in Arenicola erythro-cruorin, with no contacts between bilayers invol-ving the β-barrel linker domain. The less extensivebilayer interactions in Arenicola erythrocruorin maycontribute to its lower stability at alkaline pHcompared with Lumbricus erythrocruorin.18,19

Discussion

Design of extracellular oxygen carriers requires abalance between large size, permitting a largenumber of oxygen binding sites and vascularretention, and adequate solution properties. It wasshown many years ago that the blood viscosity inArenicola marina is less than 25% of that predicted fora linear polymer of comparable molecular mass.20

This emphasizes the critical role of the Arenicolaerythrocruorin shape, elucidated here, which per-mits the binding of 144 oxygen molecules to a single3.6 MDa protein complex.The crystal structures of Lumbricus and Arenicola

erythrocruorin demonstrate the central role of linkersubunits in dictating the relative orientation of thetwo hexagonal rings. Most important in this regardare the arrangements of the triple-stranded coiledcoils. Linker coiled-coil sequences from differentannelids (Figure 5) are readily aligned based on thecharacteristic pattern of seven residue (heptad)repeats in coiled coils in which hydrophobic resi-dues generally occupy the first and fourth positions.These positions, designated a and d,21 line one sideof a helix such that hydrophobic contacts formbetween coiled-coil helices. Two different classes ofsequences are evident from the linker sequencealignment; erythrocruorins from polychaetes withtype II architecture show continuous heptad repeats,whereas those sequences from type I erythrocruor-ins generally show a two or three residue gaprelative the type II sequences. (The Riftia LYexception is discussed below.) The effect of thetype I sequence gap is to place sequences on eitherside of the gap out of register. As a result, suchsequences are incompatible with a continuous coiledcoil. Rather, in Lumbricus erythrocruorin, two dis-tinct triple-stranded coiled coils are formed on eitherside of the gap.11 In contrast, the coiled-coilsequences of type II linker subunits are fullycompatible with the continuous coiled coilsobserved in the Arenicola erythrocruorin structurepresented here.One exception to the above sequence pattern is

evident in Figure 5. The sequence of the Riftia LYsubunit does not display the break found in othertype I sequences, even though cryo-electronmicroscopic studies of this vestimentiferan

Figure 4 (legend on opposite page)


Figure 5. Alignment of the coil-coiled domains from available annelid erythrocruorin linker sequences. Two clearlydemarcated groups are evident. Linker sequences from the oligochaete Lumbricus terrestris,26,27 the achaete Macrobdelladecora,28 the polychaete Sabella spallanzanii,29 and the vestimentiferan Lamellibrachia30 form one group. Alignment of thisgroup with the remaining linker sequences requires introduction of a sequence gap, which has been placed between thetwo coiled coils observed in the 3.5 Å crystal structure of Lumbricus erythrocurorin (Lumbricus linker helical segments arehighlighted in light blue). The second group is formed from linkers of polychaetes Arenicola marina (CAJ00866), Alvinellapompejana (CAJ00867 and CAJ00868), Tylorrhynchus heterochaetus,31 and Neanthes (Nereis) diversicolor32; and thevestimentiferan Riftia pachyptila (CAJ00871). Lack of a gap in these sequences allows the “a” and “d” positions of theheptad repeat to remain in register for the entire sequence shown, which is necessary for a continuous coiled coil to beformed, as found in the Arenicola erythrocruorin crystal structure. Hydrophobic residues are shown in red and basicresidues are shown in blue at the “a” and “d” positions.


erythrocruorin6 show it to have type I architecture.While this is the onlyRiftia pachyptila linker sequenceavailable that encompasses the coiled-coil region, apartial sequence of chain LX is also available. TheRiftia LX sequence is very similar to the AV-1sequence of the closely related vestimentiferanLamellibrachia, with a sequence identity of 87% for123 residues in the C-terminal β-barrel domain. Suchhigh homology in the β-barrel domain suggests thatthe two chains are likely to be similar in remainingportions. Since Lamellibrachia AV-1 exhibits asequence gap that is similar to that found in othertype I hemoglobins, it is appears likely that the RiftiaLX subunit will also have a gapwithin the coiled-coilregion. This suggests that the type I architecturemight not require that all linker subunits exhibit sucha sequence gap within the coiled-coil region. Incontrast, formation of uninterrupted coiled coils, as

Figure 4. Central linker complex of Arenicola and Lumbricuthe 1.5σ level for Arenicola erythrocruorin and 1.3σ level forhexagonal layer shown in orange and other layer in blue. (a) Vlayers between Arenicola and Lumbricus complexes, which is ccoiled coils. (b) Stereo view of Arenicola linker complex vicontinuous coiled coils limit inter-layer contacts involving theLumbricus linker complex viewed along the P-dyad axis. In Lumwith rotation and translation of layers, allows tight packing oflayers.

found in type II architecture, probably does requirecontinuous heptad repeats.The structure and sequences of the type I and type

II linker subunits raise the possibility that the type IIarchitecture may represent a more primitive assem-bly. The disjointed connection between coiled coilsin Lumbricus erythrocruorin suggests a greaterdegree of specialization among linker subunits,since continuous coiled coils could be formed fromthree identical subunits. Moreover, the β-barreldomains of Lumbricus L1 chains have acquired thecapacity to form extensive contacts between hex-agonal layers, a property not shared with the otherLumbricus linker subunits. However, the Arenicolalinker subunits do have distinctions, even if lessstriking than those of the Lumbricus linkers. Lateralcontacts between protomers likely involve uniqueinteractions between different linker subunits, as

s erythrocruorin. Density at 6.2 Å resolution is displayed atLumbricus erythrocruorin, with density for linkers in oneiews along the molecular 6-fold axis. Note the rotation ofoupled with the introduction of a break in the Lumbricusewed along the P axis. In Arenicola erythrocruorin, theglobular portion of the linker subunits. (c) Stereo view ofbricus erythrocruorin, the break in the coiled coils, coupledβ-barrel domains of linker subunit L1 between hexagonal

Table 1. Statistics for the crystallographic data

Sample Arenicola erythrocruorin-CO

Space group P212121Cell constants

a (Å) 339.5b (Å) 362.6c (Å) 454.6

Resolution range (Å) 40.0–6.2(High resolution bin) (6.42–6.2)Reflections 114,201 (9152)Completeness (%) 88.3 (71.7)Multiplicity 3.6 (2.3)I/error (I) 10.2 (1.8)Rsym 0.128 (0.442)


they do in Lumbricus erythrocruorin.11 Additionaldistinctions are present in the breakdown of quasi 3-fold symmetry in the linker region of the protomerstructure. Whereas the density for the LDL-A and β-barrel domains possess a quasi 3-fold axis that isaligned with the dodecamer 3-fold axis, the quasi 3-fold axis of the coiled coil is offset. This offsetpresumably results from distinct connectionsbetween the coil helices and LDL-A modules.Interestingly, modeling experiments (data notshown) suggest that it would be possible forcoiled-coil spokes to project from each protomerwith their 3-fold aligned along the dodecamer 3-foldaxis without causing interpenetration betweenspokes. Such an arrangement might be compatiblewith assembly from just a single type of linkersubunit, if subunit flexibility allowed quasi equiva-lence as is observed in many spherical viruses.22

One can speculate that evolution of at least twounique linker paralogs, as present in Arenicolaerythrocruorin,14 has enhanced the efficiency ofsubunit assembly. Evolution of additional differ-ences between linker subunits, then, would allowformation of the more compact type I architecturewith larger inter-layer contacts, perhaps leading togreater stability of the linker core.

Materials and Methods

Protein isolation and purification

Arenicola marina worms were purchased from theMarine Biological Laboratory (Woods Hole, MA). Wormswere cut with a longitudinal slit on the dorsal side andextruded viscera were rinsed with distilled water. Bloodwas drawn from large dorsal blood vessels using a 1cctuberculin syringe (26 gauge needle). Erythrocruorin waspurified from the blood by repeated ultracentrifugationtwice (120,000g) to pellet the 3.6×106 Da complex. Thiswas followed by resuspension in 10 mM Hepes (pH 7.5),100 mM NaCl, 10 mM CaCl2, to obtain a concentration of∼100 mg/ml for crystallization.

Crystallization and data collection

Crystals of carbon monoxide (CO) liganded Arenicolaerythrocruorin were grown at 4 °C by vapor diffusionagainst a solution of 30% (w/v) PEG 400, 13 mM CaCl2,150mMNaCl, 0.1MHepes (pH 7.5), andwere successfullyflash frozen from this solution on a cryo-loop in liquidnitrogen. Diffraction data were collected from two crystalsat BioCARS beamline 14-BMC at the Advanced PhotonSource (APS), Argonne National Laboratory, Argonne, IL.Observable diffraction from these crystals was limited to6 Å at best, despite screening many crystals. Data from 195frames, each with an oscillation angle of 0.5°, wereprocessed with HKL2000 and scaled with SCALEPACK.23

Statistics are provided in Table 1.

Phasing

Molecular replacement calculations were carried outusing a search molecule created from the atomic model for

one hexagonal ring of Lumbricus erythrocruorin,11 butleaving out coordinates for the long coiled coils. Rotationand translation calculations against the Arenicola erythro-cruorin diffraction data to 8.5 Å using CNS24 were readilysuccessful at locating the two halves of the molecule in theasymmetric unit.Initial calculated phases at 8.5 Å resolution were

improved by six cycles of 12-fold averaging with theRAVE package of programs25 using a protein envelopebased on the molecular replacement model. This averagedmap permitted improved mask definition including por-tions of the coiled-coil region. The process was iteratedseveral times until a satisfactory envelope was obtainedencompassing an entire one-twelfth protomer includingthe full triple-stranded coiled coil. Phasing was extendedfrom 8.5 Å to 6.2 Å by small incremental increases inresolution using six cycles of 12-fold averaging at each of 22resolution steps. Based on this 6.2 Å electron density map,the mask envelope and non-crystallographic symmetrymatrices were improved and the phase extension proce-durewas carried out a second time. Final averaging at 6.2Åled to an averagedmap exhibiting anR-factor of 21.6% anda correlation coefficient of 0.946 between the observedstructure factors and those calculated from this map.For comparison, a 6.2 Å averaged electron density map

was also obtained for Lumbricus erythrocruorin. Phases, amolecular envelope and non-crystallographic symmetryoperators were obtained from the refined atomic coordi-nates of Lumbricus erythrocruorin.11 The electron densitymap obtained was subjected to six cycles of 24-foldmolecular averaging (two whole molecules per asym-metric unit). The map obtained in this way exhibited an R-factor of 20.9% and a correlation coefficient of 0.909between the observed structure factors and those calcu-lated from this map.

Alignment of linker sequences

Linker sequences were obtained from the NationalCenter for Biotechnology Institute (NCBI). The followingsequences (with the NCBI protein codes in parentheses)were used: L1 (AAF99389), L2 (ABB71122), L3(ABB71123), and L4 (ABB71124) from Lumbricus terres-tris; L1 (BAC82449) from Macrobdella decora; L1(CAB38536) and L3 (CAC37413) from Sabella spallanzanii;AV1 (P16222) from Lamellibrachia; L2 (CAJ00866) fromArenicola marina; L1 (CAJ00867) and L2 (CAJ00868) fromAlvinella pompejana; L1 (P18207) and L2 (P18208) fromTylorrhynchus heterochaetus; L2 (BAA09580) from Neanthes(Nereis) diversicolor; LX (CAJ00870) and LY (CAJ00871)fragments from Riftia pachyptila. Alignments were madeby hand, using the 3.5 Å crystal structure of Lumbricus


erythrocurorin as a guide. The coiled-coil domains werealigned using the absolutely conserved first cysteineresidue of the LDL domain. It was found that the linkerchain from Riftia LY and all the linker chains from type IIcomplexes could be aligned with their type I counter-parts if a two to three amino acid gap was inserted inthe type I sequences. This gap was placed into theextended peptide region between the positions of thelong and short helices observed in the structure ofLumbricus linker chains.11 The resulting alignment(Figure 5) showed that both type I and type II complexesshare homology at the “a” and “d” positions such thatone face of the helix is enriched for hydrophobicresidues, as commonly found with coiled coils.

Acknowledgements

We thank Sagar Kathuria for his early crystal-lization experiments onArenicola erythrocruorin andthe BioCARS staff for assistance with data collection.This work was supported by NIH grant DK43323 (toW.E.R.). Use of the Advanced Photon Source wassupported by the US Department of Energy, BasicEnergy Sciences, Office of Science, under contractno. W-31-109-Eng-38. Use of the BioCARS Sector 14was supported by the National Institutes of Health,National Center for Research Resources, undergrant number RR07707.

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Edited by R. Huber

(Received 13 July 2006; received in revised form 27 September 2006; accepted 5 October 2006)Available online 11 October 2006