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  • doi:10.1016/j.jmb.2006.10.016 J. Mol. Biol. (2007) 365, 226236

    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 resolution. The structurereveals the presence of long continuous triple-stranded coiled-coil spokesprojecting 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


    In many annelids, oxygen transport relies on giantextracellular respiratory proteins (3.6106 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

    ng author:

    lsevier Ltd. All rights reserve

    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


    mailto:[email protected]

  • 227Architecture of Megadalton Respiratory Proteins

    terrestris, has been reported at 3.5 resolution.11

    Lumbricus erythrocruorin assembles from144 oxygen-binding hemoglobin subunits and 36 structurallinker 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 ) from a short (25 ) 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.


    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 3.6106 Da complex in the crystallographicasymmetric unit. This molecular replacement solu-tion permitted calculation of preliminary phases at8.5 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 , 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 -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 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 6.8,correspond to the 12 heme iron atoms. Iron peaksare present in pairs, 1920 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 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 (Figure2(b) and (c)). This brings Lumbricus hemoglobin b

  • Figure 1. Arenicola erythro-cruorin. Electron density at 6.2 resolution is represented at the1.5 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