Structure of an Influenza Neuraminidase–Diabody Complex by Electron Cryomicroscopy and Image...

Structure of an Influenza Neuraminidase–DiabodyComplex by Electron Cryomicroscopy andImage Analysis

Peter B. Rosenthal1*, Lynne J. Waddington2 and Peter J. Hudson2

1MRC Laboratory of MolecularBiology, Hills Road, CambridgeCB2 2QH, UK

2CSIRO Health Sciences andNutrition and CRC forDiagnostics, 343 Royal ParadeParkville, Vic., Australia 3052

The structure of a complex between a bivalent diabody and its antigen,influenza neuraminidase, has been determined by electron cryo-microscopy of single particles and image analysis. A three-dimensionalreconstruction has been interpreted in terms of high-resolution X-raymodels of the component proteins. The complex consists of two neura-minidase tetramers cross-linked by four diabodies with 422 pointsymmetry. The structure and symmetry of the complex is determineduniquely by packing constraints consistent with the maximum possiblenumber of diabody cross-links. Diabodies may provide a useful approachto the structure determination of small proteins by incorporating theproteins into large symmetric complexes followed by single-particle elec-tron microscopy.

q 2003 Elsevier Ltd. All rights reserved.

Keywords: electron cryomicroscopy; single-particle reconstruction;influenza neuraminidase; recombinant antibody; diabody*Corresponding author

Introduction

Recombinant antibodies and diabodies

Antibodies function in vivo by evolving high-affinity, antigen-binding sites on multivalentmolecules. Antibody fragments or recombinantantibodies retain the monovalent antigen-bindingactivity of antibodies, and have clinical appli-cations in cancer therapy, diagnostics, andimaging.1,2 Recombinant single-chain Fv molecules(scFv; 28 kDa) consist of VH and VL hypervariabledomains joined by a peptide linker and possess asingle binding site for antigen.3,4 To increaseavidity for applications such as tumour-imaging,scFvs can be oligomerized by several methods.Diabodies (56 kDa) are stable Fv dimers that resultfrom association of two (28 kDa) scFv molecules.Diabodies form when the polypeptide linker ineach scFv is too short to span the 35 A path fromthe VH C terminus to the VL N terminus requiredfor monomeric VH–VL association. Instead, twoscFv molecules associate together, forming twoantigen-binding sites, each consisting of a VH and

VL from different chains (Figure 1(a) and (b)).5 Tria-bodies and higher-order multimers can be formedby the same domain-swapping mechanism.6,7

Recent pharmacokinetic analysis has shown thatradiolabelled diabodies are ideal tumour-imagingreagents,8,9 and for clinical applications that requirecross-linking of different antigens, such as bindingcytotoxic T-cells to a tumour cell, bispecificdiabodies can be designed (Figure 1(c)).10

Structures of diabodies and triabodies have beendetermined by X-ray crystallography in theabsence of antigen, and confirm the connectionsand interactions between swapped domains.7,11,12

Structures of multimeric scFvs bound to antigenare needed to understand the constraints on multi-valent cross-linking presented by the distance andorientation of bound antigens. The kinetic stabilitythat arises through multivalent binding is essentialto the function of many immune and signallingcomplexes, and is the basis for clinical applicationsof diabodies.

The neuraminidase glycoprotein (NA) is areceptor-destroying enzyme on the membrane ofinfluenza virus. It is essential for virus infectionand has been an excellent target for drugdesign.13,14 In addition, the NA tetramer is a goodmodel system for the study of scFv–antigen inter-actions. X-ray structures of NA complexed withscFvs have demonstrated that scFvs bind antigen

0022-2836/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.

E-mail address of the corresponding author:[email protected]

Abbreviations used: NA, neuraminidase glycoprotein;CTF, contrast transfer function.

doi:10.1016/j.jmb.2003.09.077 J. Mol. Biol. (2003) 334, 721–731

in a manner similar to the parent antibody(Figure 1(d)).15,16 However, no three-dimensionalstructure showing a bivalent diabody unambiguouslycross-linking antigens has been determined.

In this study, the structure of such a complex ofinfluenza NA tetramers cross-linked by diabodieshas been obtained using electron cryomicroscopyand image analysis. Cryomicroscopy can be usedto determine the structure of single particles inamorphous ice without the need for crystals. TheNA–diabody complex is shown to be symmetric(422 point symmetry) with four diabodies cross-linking two NA tetramers. The structure of thecomplex has been interpreted in terms of high-resolution models of the component proteinsdetermined by X-ray crystallography. The detailedstructure of the assembly is determined uniquelyby steric packing and distance constraints posedby peptide linkers in the cross-linking diabodies.

Neuraminidase–scFv complexes

Monoclonal antibody NC10 recognizes themembrane distal side of influenza NA subtype N9originally isolated from a Noddy tern (Anousstolidus). A monomeric (26 kDa) scFv derived fromNC10 binds NA with similar binding affinity ðKa ¼5:0 £ 107 M21Þ to the (55 kDa) NC10 Fab ðKa ¼6:3 £ 107 M21Þ and with similar molecular inter-actions as shown by X-ray crystallography(Figure 1(d)).15,16 The multimeric state of the scFvis determined by the length of the linker runningfrom the VH C terminus (Ser112) to the VL Nterminus (Asp1). The NC10 scFv(15) constructwith a 15 residue linker (Gly4Ser)3 forms bothmonomers (Figure 1(a)) and dimers (diabodies;Figure 1(b)). The scFv(10) construct with a tenresidue linker (Gly4Ser)2 or the scFv(5) constructwith a five residue linker (Gly4Ser) both formdimers only. Linkers less than three residues formtriabodies (or higher multimers).17 – 19

When complexed with soluble NA tetramers iso-

lated by Pronase digestion of influenza virus, thescFv(15) monomer forms a 300 kDa complex, asdemonstrated by gel-filtration and analytical ultra-centrifugation. This is consistent with an NA tetra-mer (190 kDa) binding four monomeric scFvs(26 kDa each). The X-ray structure of the scFv(15)NA complex shows an arrangement that isconsistent with four monomer scFvs binding tothe NA tetramer (Figure 1(d)).15,16

NC10scFv(5) forms diabodies only and, whenmixed with NA tetramer in a molar excess tosaturate the NA-binding sites (molar ratio ofdiabody to NA tetramer of 4:2 or higher), forms ahomogeneous 590 kDa complex as demonstratedby gel-filtration and analytical ultracentrifugation.18

A mass of 590 kDa is consistent with four dia-bodies (52 kDa each) cross-linking two NA tetra-mers (190 kDa each). The complex is extremelystable, both to dilution and to storage at 4 8C for30 days. However, crystals of the 590 kDascFv(5)–NA complex diffract poorly and no X-raystructure has been obtained. A crystal formed bymixing diabody to NA in a molar ratio of 4:1 wassolved to 2.5 A, and the structure interpreted assingle Fv domains bound to NA tetramers withthe other half of the diabodies unbound and notvisible due to blurring from disorder. This crystalform is inconsistent with the 590 kDa complex anddoes not provide a structure of bivalent diabodiescross-linking the antigen.15

Prior to this study, therefore, the geometry anddetailed structure of a diabody cross-linking aneuraminidase tetramer in solution was unknown.Negative stain microscopy of scFv(5) bound toanti-idiotype Fabs shows a range of anglesbetween the antigen-binding sites of the diabody,suggesting that there is a flexible hinge in themolecules that can cross-link antigens in a rangeof orientations.20 Because of its size and homo-geneity, the 590 kDa scFv(5)–NA complex must,in principle, contain two NA tetramers cross-linked by between one and four diabodies,depending on steric and linker distance constraintspresented by the particular binding sites on theantigen. The symmetry of the complex would alsodepend on the number of cross-links and wasunknown. An analysis by cryomicroscopy wasexpected to elucidate the possible structures andcross-linking arrangements.

Results

Microscopy and image analysis

The purified 590 kDa NC10scFv(5)–NA complexwas first imaged by electron microscopy innegative stain on continuous carbon films. Whenimaged in uranyl acetate after glutaraldehydecross-linking (Figure 2(a)), most particles were ofthe expected size, consistent with the biochemicalcharacterization of the complex as a single molecu-lar mass species.18 The most common view was a

Figure 1. Schematic for: (a) single-chain Fv with redVH, yellow VL and cyan antigen; (b) monospecificdiabody; (c) bispecific diabody recognizing differentantigens; (d) neuraminidase (NA) tetramer with fourrecombinant scFvs (based on PDB code 1a14) colouredas in (a).

722 Cryomicroscopy of Neuraminidase–Diabody Complex

five-pointed star-shape. The very few projectionsof different size observed in negative stain can beinterpreted as an effect of the stain, since theywere not observed when the complex was imagedunstained in ice. The heterogeneity resulting fromnegative-staining was more pronounced in theabsence of glutaraldehyde cross-linking.

The complex was imaged without stain in thinfilms of amorphous ice over holes in carbon filmby cryomicroscopy. A 200 kV field emission gunmicroscope was used with large defocus values toenhance contrast (Figure 2(b)). The particles werehomogeneous even in the absence of glutaralde-hyde cross-linking and could be interpreted asdistinct views of a single complex. The mostcommon view was again a star-shape withapproximate 5-fold symmetry, although a greatervariety of views was present in ice compared to

stain. The images of 1858 particles in ice werealigned and subjected to multivariate statisticalanalysis and classification using IMAGIC-Vsoftware.21 Particles in the same class were aver-aged to produce higher signal-to-noise views ofparticle projections (Figure 3(a)). The class viewswere similar for particles from a separate datasetof 1251 particles recorded at 120 kV on a micro-scope with a thermionic gun (data not shown).

To determine a three-dimensional structure orstructures from these two-dimensional projections,it is necessary to determine the relative orientationsof the projections in three dimensions. Because theclass views did not show clear evidence of the4-fold symmetry of the neuraminidase tetramer,and the approximate 5-fold symmetry of the star-shaped views could be a false symmetry of projec-tion, no initial assumptions were made about thesymmetry of the complex. Attempts to determinethe relative orientations of the projections byangular reconstitution22 assuming C1 symmetrywere unsuccessful.

Model searches against projections

Because high-resolution X-ray structures of thecomponents of the complex were known, determi-nation of the complex structure requires findingthe relative orientations and positions of the com-ponents, assuming they are rigid bodies not under-going conformational changes. Correct models forthe complex should be identified by excellentoverall agreement between projections from thethree-dimensional model of the complex and theexperimental class views even without the neces-sity for independent determination of the relativeorientations of the class views in three dimensions.

The size of the NC10scFv(5)–NA complex,590 kDa, indicates that the complex is likely to con-tain two 190 kDa NA tetramers, which implies theexistence of at least one diabody cross-link. Thesimilarity of the interaction of the Fv domain withthe neuraminidase in the NC10scFv(15)–NAcomplex15,16 compared to the NC10Fab–NAcomplex23,24 suggests that the NA tetramer–Fvinteraction will be unchanged in the diabody com-plex. Thus, one-half of the complex is likely toconsist of an NA tetramer coupled to four Fvs andat least one pair of Fvs from opposite tetramersmust form a cross-linking diabody. A systematicsearch procedure was therefore developed to findall possible models consistent with distanceconstraints of the diabody cross-links, steric con-straints (no model interpenetrations), and agree-ment with the experimental image projections.

A general six-dimensional search with coarse 108angular and 10 A translational steps was per-formed of one NA–Fv tetramer coordinate modelrelative to another. A set of parameters was con-sidered to be consistent with the complex if thegeometry allowed at least one diabody cross-link.Two Fvs on different tetramer models were con-sidered capable of forming a diabody cross-link if

Figure 2. Electron micrographs of NC10scFv(5)complex: (a) in 1% (w/v) uranyl acetate stain afterglutaraldehyde cross-linking at 100,000 £ magnification,where complexes appear white against dark back-ground; (b) frozen hydrated specimen imaged overholes in carbon at 60,000 £ magnification, 200 kV, and3.5 mm defocus, where complexes appear dark against alight background. In each case the scale bar represents500 A.

Cryomicroscopy of Neuraminidase–Diabody Complex 723

both VH(C terminus)–VL(N terminus) distanceswere less than 20 A, the approximate distance thatcould be spanned by six residues (the Gly4Ser pep-tide linker plus the N-terminal residue of VH,absent from the X-ray model). Each set of par-ameters defining a model with at least one possiblediabody cross-link was also scored for satisfyingtwo, three, or four diabody cross-links. Modelsthat satisfied the distance constraints wereevaluated for impossible steric overlaps by asimple molecular packing function: the fraction ofan artificial unit cell occupied by both models(models with less overlap cover a larger volumefraction of the unit cell).25,26 Models satisfying thedistance constraint were therefore ranked accord-ing to the packing score. The calculated three-dimensional densities for the models with the bestpacking score were then scored for agreementwith the 25 class views of the particle image data-set recorded at 120 kV, by using the programFREALIGN to carry out an orientational search ofeach model for the best phase residual againsteach projection.27 The average phase residualbetween the class views and the correspondingprojections of each model was used to provide anoverall score for each model.

It was immediately obvious from the initialcoarse search that many models satisfying a singlediabody constraint could give scores with minimalpacking violations but generally gave pooragreement with the projections. A single diabodycross-link allows very open complexes that makemaximum use of diabody flexibility, but these arevery different from the compact structuresobserved by microscopy (Figures 2 and 3). Thisinitial coarse search showed that models with thebest agreement with the experimental classes areconsistent with at least two diabody cross-links.

Two cross-links also restricted the six-dimensionalsearch space so that a finer search with 58 angularand 5 A translational steps could then be per-formed in the regions of the best packing models.The top-scoring solution of the fine search wasfound to allow four cross-links and to have nearly422 symmetry. Finally, the best 422 model wasidentified by a two-dimensional search of the rela-tive rotation (18 steps) and translation (1 A steps)

Figure 3. Image analysis of NC10scFv(5) complex in ice. (a) Average images from classification of 1858 particlesimages in ice into 25 views, showing the 15 views used to make the starting model; (b) calculated projections of thetheoretical packing model shown in (e); (c) calculated projections of the starting density map from angular reconstitu-tion shown in (f); (d) calculated projections of the final refined density map shown in (g); (e) packing model; (f) startingmap from angular reconstitution; (g) final map. CTF contrast reversals have not been applied to calculated modelprojections in (b) and (d).

Figure 4. Plot of molecular packing score for modelswith 422 symmetry as a function of the relativetranslation and rotation of two tetramer half-models.Contours show fraction of maximum volume for amodel with no overlap. Allowed models have rotationsand translations with the best packing score while simul-taneously satisfying the linker distance constraint (regionbelow and left of the broken line).


of the two tetramer half-models constrained by theallowed linker distances and scored by the packingfunction (Figure 4). Calculated projections (Figure3(b)) of the resulting model (Figure 3(e)) showexcellent agreement with the experimental classviews.

Thus a systematic and general search of thepossible geometry of the complex suggests that ithas 422 symmetry with four diabody cross-links.

This symmetry was not at all obvious from theparticle projections for which a 5-fold view wasmost common, and emphasizes the importance ofa three-dimensional interpretation of single-particleprojections recorded by electron microscopy.Though, in principle, classified projections can bealigned directly against each other, this is a difficultprocedure in practice, requiring many particles,and in the present case did not work until the point

Figure 5. (a) Fourier shell correlation versus resolution for the final reconstruction; (b) orientation angles for the 15views shown in Figure 3(a); (c) distribution of particle orientation parameters (u,f) for 3058 particles.


symmetry of the complex was established. For thecase where the structures of subunits are known orcan be guessed, the search procedure developedhere presents an alternative way of generating astarting model that can then be used for subsequentrefinement of the electron microscope images. Theapproach is complementary to other ways of gener-ating an initial approximate three-dimensionalmodel such as conical tilt28 and angularreconstitution22 procedures.

Angular reconstitution and refinement of thethree-dimensional structure

The results of the search procedure using high-resolution X-ray structures showed that modelswith 422 symmetry could be constructed that haveprojections similar to the experimental images.Once the symmetry had been established, angularreconstitution could be used to determine therelative orientation of the two-dimensional projec-tions, independent of the models constructed from

X-ray structures. The assumption of 422 symmetrygreatly increased the power of the angular recon-stitution procedure, and 15 of the 25 class viewsfrom the 1858 particle field emission gun datasetwere assigned relative angles and used to calculatea starting model (Figure 3(f)). Calculated projec-tions of the model (Figure 3(c)) were in excellentagreement with the experimental image classes(Figure 3(a)), and were used to iteratively refinethe relative orientation angles of the image classesby anchor set refinement.21

The dataset was then expanded to 3058 particlesfrom 24 films recorded on the 200 kV field emissiongun microscope. A wide range of defocus (3–6 mm)was used to fill in the weak regions in reciprocalspace due to the microscope contrast transferfunction (CTF). The program FREALIGN wasused for iterative model-based refinement of theinitial model obtained by angular reconstitution.For each cycle of refinement, the current modelwas used to determine orientation parameters foreach particle. In addition, the images were

Figure 6. Electron density for the complex. Surface shaded (a) side-view, (b) top-view, (c) and (d), two tetramerNA–Fv models (red and yellow) fitted into density for views as (a) and (b).


corrected for effects of the CTF. Initially, data to40 A resolution were used for refinement so thatthe first zero of the CTF for even the most highlydefocussed images remained outside this range.The resulting models possessed structural infor-mation beyond 26 A (Fourier shell correlation of0.5 between models calculated from data half-sets)and the density developed features that matchedthose known from the high-resolution structuresof the components. Additional cycles of refinementused data to 35 A, 30 A, and finally 25 A, andmodels continued to show structural informationbeyond the resolution limit used for refinement.Calculated projections (Figure 3(d)) of the finalmodel (Figure 3(g)) are similar to the originalparticle images and the experimental class views(Figure 3(a)). Figure 5(a) shows the agreementbetween models calculated from data half-sets asmeasured by the Fourier shell correlation (FSC),which crosses 0.5 at 23 A, the nominal resolutionof the final map. The slightly weak correlation at33 A (Figure 5(a)) is attributed to a zero in theCTF. The orientation parameters of the classifiedviews are shown in Figure 5(b). Star-shaped viewsare obtained from the 422 structure in directionsaway from the symmetry elements. The distri-bution of individual particle orientations for theentire dataset is shown in Figure 5(c). Surfaceviews of the density are shown in Figure 6(a)and (b).

Interpreting the map with atomic models

As described earlier, the X-ray model of an NAtetramer with four Fvs bound was likely to be agood representation of half of the complex. The

4-fold axis of the model was placed on the 4-foldaxis of the map and fit with two degrees offreedom, rotation around the 4-fold axis and trans-lation along the axis consistent with 422 symmetry.Because a map calculated from untilted projectiondata will be of arbitrary absolute hand, the X-raymodel was also fit against a map of invertedhand. The X-ray model and the map both havehanded features and a comparison clearlyindicated that the map with the inverted handwas correct. The agreement of features of the mapwith those of the X-ray models of the componentsvalidates the reconstruction. The fit of the modelto the density map is shown in Figure 6(c) and(d). The optimal agreement with the density occursfor a rotation and translation between tetramerssimilar to the best score for a packing function sub-ject to the linker distance constraints (Figure 4).

Analysis of the model

The NC10scFv(5)–NA complex has maximaldimensions of 157 A £ 125 A as shown in the viewof an a-carbon model in Figure 7(a). Four 2-foldsymmetric diabodies cross-link the NAs on theoutside of the complex, leaving a large cavity inthe centre. The antigen-binding sites on oppositeends of the diabody are 62 A apart. The surfacesof the cross-linked NA tetramers approach eachother as closely as 23 A. In the model of a cross-linking diabody shown in Figure 7(a), the C termi-nus of the VH domain model (Val111) is 20.5 Afrom the N terminus (Ala1) of the VL model. Thisdistance can be spanned by six amino acid residues(VH residue Ser112 plus the five residue Gly4Serlinker) without other intersections with the model,

Figure 7. (a) a-Carbon model for the complex with red VH, yellow VL, cyan neuraminidase, and path of diabodylinkers as a dotted white lines. (b) View of adjacent cross-linking diabodies with close approach of loops (circled) con-taining VH residues 73–76. The other two symmetry-related diabodies have been removed from the Figure for clarity.The complex is rotated by 458 about its 4-fold axis relative to the view in (a).


but the linker is near its maximum possibleextension.

In the model, an interface exists between the twoVH domains within a diabody at the end oppositethe antigen-binding sites (Figure 7(a)). At the resol-ution of the reconstruction, it is not possible todescribe the interface in detail. In addition, contactcan occur between a loop (VH 73–76) from eachVH domain and the same loop from the VH of theadjacent diabody, which binds the opposite NAtetramer (Figure 7(b)). In contrast to the VH

domain, the VL domain makes contact only withthe NA and its cognate VH, and makes no contactwith the other half of the diabody or to adjacentdiabodies. This observation suggests that sym-metric complexes with high-affinity recombinantVH domains lacking VL could be designed.

The model of a single diabody from the complexwas compared to the X-ray structure of anunliganded diabody (L5MK16) against phospha-tidylinositol-specific phospholipase Cd1.

11 Roughlysimilar VH–VH orientations are observed and asimilar linker connectivity is inferred. However,the VH domains are at a more acute angle in theNC10scFv(5) complex, reflecting flexibility about ahinge between the VH domains. The packingbetween Fvs in the diabody is different from thatobserved in the crystal structure of the scFv(15)complex with NA, which probably representsmonomeric Fvs brought together by crystalpacking.15 The complex is similar to the simplemodel obtained initially from a molecular packingsearch constrained by the length of the linkerbetween VH and VL (Figure 4). It is similar to anearlier model (with 422 symmetry) proposed forthe NC10scFv(5)–NA complex (see Figure 1 ofMalby et al.15). Therefore, the structure of the 422complex is fully determined by the steric con-straints consistent with multivalent cross-linking.

Discussion

The NA–diabody structure described hererepresents the first three-dimensional structure ofa multimeric Fv cross-linking its antigen. Thestructure was determined to 23 A resolution byelectron cryomicroscopy and was interpreted interms of the X-ray structures of the components(NA tetramer with bound scFvs). The agreementbetween the model and all classified viewssuggests the purified complex represents a stableand homogenous particle in solution, though thepossibility of other minor species cannot beexcluded. The structure could have deviationsfrom 422 symmetry or heterogeneity at higherresolution than that achieved in this study. Inaddition, it was shown that an initial model canbe derived by systematic search of the relativeorientation of the known structures of componentsusing agreement with classified projection dataand packing as an index of success. This may findapplication in other cases where calculation of an

initial starting model from untilted data by angularreconstitution is difficult.

The ability to simultaneously cross-link twoantigens is the basis of a number of clinical appli-cations of diabodies, such as binding cells contain-ing tumour antigens to cytotoxic cells of theimmune system. The antigen-binding sites ondiabodies are much closer together (,65 A) thanthose present on bivalent immune molecules pro-duced in vivo such as IgG (,100 A), and diabodiesare likely to be less flexible.10 However, calculationsstill show a wide range of angles possible for asingle diabody,10 diabodies have been shown to beflexible by negative stain electron microscopy,20

and in this study, the flexibility is revealed in thecomparison of the scFv(5) structure with the X-raystructure of an unliganded diabody. Nevertheless,the constraints on each diabody orientation posedby multiple cross-links results in a unique struc-ture for the NA–scFv(5) complex, and furtherexplains the extreme stability of the complex ondilution through avidity.

Oligomerization reagents, adaptors,and scaffolds

Because electron microscopy of single particlesdoes not require crystalline specimens, it haspotential for the structural study of molecules thatare difficult to crystallize such as membrane pro-teins, complexes containing flexible components,and proteins undergoing dynamic transitions.Single-particle electron cryomicroscopy has beenused to determine the structure of an icosahedralprotein assembly to 7.4 A resolution,29 and a theor-etical calculation suggests that averaging less than106 images of unstained particles in ice may, inprinciple, be sufficient to determine the structureof a protein to atomic resolution, provided theposition and orientation of the particles can bedetermined from low-contrast images.30 There isenough information in the image of a larger par-ticle to determine orientation and position but notfor many smaller proteins, which are still of greatbiological interest.

A novel application of recombinant scFvs maytherefore be to form high molecular mass sym-metric complexes of an unknown antigen whosestructure could then be determined by electroncryomicroscopy. In the present study, a designeddiabody has been used as an oligomerizationreagent to incorporate NA, a 190 kDa tetramericantigen into a symmetric complex of 600 kDa. Thepossibility of selecting scFvs from a library opensthe possibility of constructing a similar large sym-metric complex from a multimeric protein ofunknown structure.

A more general strategy for structure determi-nation is to use a bispecific diabody as an adaptorto bind any small antigen to a large, symmetric,high molecular mass scaffold (Figure 8). Thoughalready less flexible than natural polyvalentimmune molecules, this will not be useful without


engineering more rigid diabodies, because dis-order will blur the reconstruction. The structure ofthe NA–scFv(5) complex suggests that the inter-face between the two VH domains (Figure 7(a)),which does not occur in vivo, could be engineeredto stabilize the diabody, creating a rigid adaptorfor attaching an antigen to a symmetric scaffold.Selection for residues that confer tight bindingbetween heavy chains without affecting antigenbinding would be generally applicable for an entireFv library. In contrast, it is likely that naturallyoccurring Fv–Fc interfaces have been selected formaximum flexibility.31

An ideal symmetric scaffold will have iso-tropically distributed diabody binding sites so thatthe antigens adopt the symmetry of the underlyingscaffold with full occupancy, and must be roughlyspherical rather than flat, so that the complexeswill not be forced into a particular orientation by athin ice layer during sample preparation. A widerange of views is essential to obtain structuresfrom single-particle images without a need for tilt-ing the specimen. Repeated use of a symmetricscaffold of known structure will make building astarting model easier and provide a standard forassessment of the quality of the refined model.The further development of such diabody adaptorsand scaffolds will provide a valuable tool for struc-tural biology as well as have other applications inprotein design and nanotechnology.

Methods

Synthesis of neuraminidase–diabody complexes

Recombinant NC10scFv(5) was expressed in

Escherichia coli, solubilized in 6 M guanidine hydro-chloride and refolded as described.18 The diabody wasisolated by affinity chromatography on anti-FLAG M2antibody resin and then purified by gel-filtration on aSuperose 12 H10/30 column to produce pure dimer asdescribed.18 Soluble tern N9 NA was isolated by Pronasedigestion of influenza virus and purified by gel-filtration.32 The 590 kDa NA–scFv(5) complexes wereformed by adding excess of scFv(5) to NA to saturatethe NA-binding sites, and the complex peak was thenisolated by gel-filtration to remove excess scFv(5) fromthe mixture as described,18 and stored in TBS, 0.15 MNaCl, 0.02% (w/v) NaN3.

Negative stain electron microscopy

Gold grids (400-mesh) were coated with thin carbonfilm and glow-discharged in nitrogen. Samples contain-ing the complex at a concentration of 0.01 mg/ml ofprotein and 1% (w/v) glutaraldehyde were applied tothe grids for two minutes. Excess sample was drawn offwith filter-paper and the grid then washed with buffer(PBS) and stained with three or four drops of 2% (w/v)uranyl acetate. Micrographs were recorded with a JEOL100B transmission electron microscope operating at60 kV and a magnification of 100,000 £ .

Electron cryomicroscopy

Holey carbon films were prepared as described33 on400-mesh Cu/Rh grids and glow-discharged for twominutes in air. Protein (2 ml of 0.5 mg/ml in PBS) wasapplied to the carbon film in a high-humidity chamber,34

blotted for 20 seconds with filter-paper to remove excesssample, and plunged into liquid ethane at 2180 8C.Grids were stored in liquid nitrogen and transferred toa Gatan cold stage. Images of particles over holes wererecorded under low-dose conditions (10 e2/A2) on aPhilips CM12 instrument with a tungsten filamentoperating at 120 kV, a magnification of 35,000 £ , and adefocus range between 2 mm and 3 mm, or on a HitachiHF2000 instrument operating at 200 kV, a magnificationof 60,000 £ , and a defocus range between 3 mm and6 mm.

Image analysis

Images were digitized on a Zeiss-SCAI scanner with a7 mm step, selected manually using Ximdisp, and pro-cessed using programs from the MRC package.35 In all,3058 particle images, recorded on 24 films at 60,000£magnification and 200 kV, were boxed-out and adjacentpixels averaged 3 £ 3 with a resulting resolution of3.35 A/pixel in 128 £ 128 pixel squares. Particles werealigned and classified using IMAGIC V.21 Images withoutCTF correction were filtered to 35 A. Particles were trans-lationally aligned in five cycles against the rotationallyaveraged sum of all particles. Multivariate statisticalanalysis (MSA) and classification resulted in 25 classviews. Low-variance classes showing representativeviews of the dataset were then used to align all particlesby multi-reference alignment (MRA). Two more cyclesof MSA and MRA and classification followed. A similarclassification procedure was applied to 1251 particlesrecorded on three films at 120 kV. Angular reconstitutionwas used to assign orientation angles for the best 15 of 25class views of the 200 kV images assuming 422 symmetry(see Results), and a three-dimensional map was calculated

Figure 8. Schematic for a general approach ofattaching small protein antigens (cyan) to largesymmetric scaffolds using bispecific diabodies asadaptors.


by back projection. Calculated projections of the mapwere used to refine the orientations and translations ofthe 15 class views for three iterations (anchor set refine-ment).

The model was then refined against the 200 kV FEGimages with densities floated to zero average and cor-rected for the CTF using FREALIGN.27 Initial values forthe CTF were determined using CTFFIND2.35 Orien-tation and translation parameters for each particleimage were determined each cycle by minimizing thephase residual of the model calculated using parametersfrom the last cycle. Image data to 40 A resolution wereused initially, and then extended to 35 A, 30 A, and 25 Aduring a total of ten cycles. Density for a model of oppo-site hand was generated in FREALIGN by transformingthe Euler angle orientation parameters from (c,u,f) to(cþ180,u,f).

Model searches against projections

A six-dimensional search of the relative rotation andtranslation of two Na–Fv tetramer coordinates (PDB1a14) and packing analysis was performed usingXPLOR 3.8.26 For each set of parameters, VH C terminusto VL N terminus distances were calculated, and thepacking function evaluated using a cell 300 A on a side.To score models against 25 classified views from the120 kV dataset, the coordinate models were convertedto density using SPIDER.36 The optimal orientation foragreement of the model with each classified view wasdetermined using the program FREALIGN by minimiz-ing the phase residual ðPRÞ :

PR ¼

X

i

lDfiFilX

i

lFil

where Df is the phase difference and F is the amplitudeof the image structure factor. The average phase residualfor all views was taken as the score for the model.

Fitting X-ray structures to the map

Tetrameric models for the NA with four scFvs (PDBcode 1A14) were placed in map density using the pro-gram O,37 and the hand of the map determined asdescribed in the text. The map density was placed in anartificial unit cell and Fourier-transformed using pro-grams from the CCP4 suite38 to create crystallographicstructure factors. A two-dimensional search of the rela-tive rotation and translation of the two tetramer modelswith 422 symmetry was scored by the Fourier shell corre-lation between structure factors from model and mapusing XPLOR 3.8.26 Figures 1(d) and 3(e) were madewith Rasmol.39 Figures 3(f) and (g), and 6(a) and (b)were made using Surf.40 Figures 6(c) and (d), and 7were made using Setor.41

Acknowledgements

P.B.R. thanks Richard Henderson for advice andsupport, and gratefully acknowledges supportfrom a Human Frontier Science Program LongTerm Fellowship, an Agouron Institute Fellowship

(Research Grant AI-SA1-99.3), and the MedicalResearch Council of the UK. We thank membersof the Laboratory of Molecular Biology, AlexKortt, and Airlie McCoy for discussions, JohnBerriman for advice on microscopy, and JohnBurns for assistance in the preparation of proteincomplexes.

References

1. Carter, P. (2001). Improving the efficacy of antibody-based cancer therapies. Nature Rev. Cancer, 1,118–129.

2. Hudson, P. J. & Souriau, C. (2003). Engineered anti-bodies. Nature Med. 9, 129–134.

3. Huston, J. S., Levinson, D., Mudgett-Hunter, M., Tai,M. S., Novotny, J., Margolies, M. N. et al. (1988). Pro-tein engineering of antibody binding sites: recoveryof specific activity in an anti-digoxin single-chain Fvanalogue produced in Escherichia coli. Proc. NatlAcad. Sci. USA, 85, 5879–5883.

4. Bird, R. E., Hardman, K. D., Jacobson, J. W., Johnson,S., Kaufman, B. M., Lee, S. M. et al. (1988). Single-chain antigen-binding proteins. Science, 242, 423–426.

5. Holliger, P., Prospero, T. & Winter, G. (1993). Diabo-dies: small bivalent and bispecific antibody frag-ments. Proc. Natl Acad. Sci. USA, 90, 6444–6448.

6. Iliades, P., Kortt, A. A. & Hudson, P. J. (1997).Triabodies: single chain Fv fragments without alinker form trivalent trimers. FEBS Letters, 409,437–441.

7. Pei, X. Y., Holliger, P., Murzin, A. G. & Williams, R. L.(1997). The 2.0-A resolution crystal structure of a tri-meric antibody fragment with noncognate VH–VL

domain pairs shows a rearrangement of VH CDR3.Proc. Natl Acad. Sci. USA, 94, 9637–9642.

8. Tahtis, K., Lee, F. T., Smyth, F. E., Power, B. E.,Renner, C., Brechbiel, M. W. et al. (2001). Bio-distribution properties of (111)indium-labeledC-functionalized trans-cyclohexyl diethylene-triaminepentaacetic acid humanized 3S193 diabodyand F(ab0)(2) constructs in a breast carcinoma xeno-graft model. Clin. Cancer Res. 7, 1061–1072.

9. Yazaki, P. J., Wu, A. M., Tsai, S. W., Williams, L. E.,Ikler, D. N., Wong, J. Y. et al. (2001). Tumor targetingof radiometal labeled anti-CEA recombinant T84.66diabody and t84.66 minibody: comparison to radio-iodinated fragments. Bioconjug. Chem. 12, 220–228.

10. Holliger, P., Brissinck, J., Williams, R. L., Thielemans,K. & Winter, G. (1996). Specific killing of lymphomacells by cytotoxic T-cells mediated by a bispecificdiabody. Protein Eng. 9, 299–305.

11. Perisic, O., Webb, P. A., Holliger, P., Winter, G. &Williams, R. L. (1994). Crystal structure of a diabody,a bivalent antibody fragment. Structure, 2,1217–1226.

12. Carmichael, J. A., Power, B. E., Garrett, T. P., Yazaki,P. J., Shively, J. E., Raubischek, A. A. et al. (2003).The crystal structure of an anti-CEA scFv diabodyassembled from T84.66 scFvs in VL-to-VH orientation:implications for diabody flexibility. J. Mol. Biol. 326,341–351.

13. Gubareva, L. V., Kaiser, L. & Hayden, F. G. (2000).Influenza virus neuraminidase inhibitors. Lancet,355, 827–835.

14. Varghese, J. N., Epa, V. C. & Colman, P. M. (1995).Three-dimensional structure of the complex of


4-guanidino-Neu5Ac2en and influenza virusneuraminidase. Protein Sci. 4, 1081–1087.

15. Malby, R. L., McCoy, A. J., Kortt, A. A., Hudson, P. J.& Colman, P. M. (1998). Three-dimensional struc-tures of single-chain Fv-neuraminidase complexes.J. Mol. Biol. 279, 901–910.

16. Kortt, A. A., Malby, R. L., Caldwell, J. B., Gruen, L. C.,Ivancic, N., Lawrence, M. C. et al. (1994). Recombi-nant anti-sialidase single-chain variable fragmentantibody. Characterization, formation of dimer andhigher-molecular-mass multimers and the solutionof the crystal structure of the single-chain variablefragment/sialidase complex. Eur. J. Biochem. 221,151–157.

17. Atwell, J. L., Breheney, K. A., Lawrence, L. J., McCoy,A. J., Kortt, A. A. & Hudson, P. J. (1999). scFv multi-mers of the anti-neuraminidase antibody NC10:length of the linker between VH and VL domainsdictates precisely the transition between diabodiesand triabodies. Protein Eng. 12, 597–604.

18. Kortt, A. A., Lah, M., Oddie, G. W., Gruen, C. L.,Burns, J. E., Pearce, L. A. et al. (1997). Single-chainFv fragments of anti-neuraminidase antibody NC10containing five- and ten-residue linkers form dimersand with zero-residue linker a trimer. Protein Eng.10, 423–433.

19. Kortt, A. A., Dolezal, O., Power, B. E. & Hudson, P. J.(2001). Dimeric and trimeric antibodies: high avidityscFvs for cancer targeting. Biomol. Eng. 18, 95–108.

20. Lawrence, L. J., Kortt, A. A., Iliades, P., Tulloch, P. A.& Hudson, P. J. (1998). Orientation of antigen bind-ing sites in dimeric and trimeric single chain Fvantibody fragments. FEBS Letters, 425, 479–484.

21. van Heel, M., Harauz, G., Orlova, E. V., Schmidt, R.& Schatz, M. (1996). A new generation of theIMAGIC image processing system. J. Struct. Biol.116, 17–24.

22. van Heel, M. (1987). Angular reconstitution: a poster-iori assignment of projection directions for 3D recon-struction. Ultramicroscopy, 21, 111–124.

23. Colman, P. M., Tulip, W. R., Varghese, J. N., Tulloch,P. A., Baker, A. T., Laver, W. G., Air, G. M. & Webster,R. G. (1989). Three-dimensional structures ofinfluenza virus neuraminidase–antibody complexes.Phil. Trans Roy. Soc. ser. B, 323, 511–518.

24. Malby, R. L., Tulip, W. R., Harley, V. R., McKimm-Breschkin, J. L., Laver, W. G., Webster, R. G. &Colman, P. M. (1994). The structure of a complexbetween the NC10 antibody and influenza virusneuraminidase and comparison with the over-lapping binding site of the NC41 antibody. Structure,2, 733–746.

25. Hendrickson, W. A. & Ward, K. B. (1976). A packingfunction for delimiting the allowable locations ofcrystallized macromolecules. Acta Crystallog. sect. A,32, 778.

26. Brunger, A. T. (1992). X-PLOR Version 3.1 manual,Yale University, New Haven, CT.

27. Grigorieff, N. (1998). Three-dimensional structure ofbovine NADH:ubiquinone oxidoreductase (complexI) at 22 A in ice. J. Mol. Biol. 277, 1033–1046.

28. Radermacher, M., Wagenknecht, T., Verschoor, A. &Frank, J. (1986). A new 3-D reconstruction schemeapplied to the 50 S ribosomal subunit of E. coli.J. Microsc. 141, RP1–RP2.

29. Bottcher, B., Wynne, S. A. & Crowther, R. A. (1997).Determination of the fold of the core protein of hepa-titis B virus by electron cryomicroscopy. Nature, 386,88–91.

30. Henderson, R. (1995). The potential and limitationsof neutrons, electrons and X-rays for atomic resol-ution microscopy of unstained biological molecules.Quart. Rev. Biophys. 28, 171–193.

31. Lesk, A. M. & Chothia, C. (1988). Elbow motion inthe immunoglobulins involves a molecular ball-and-socket joint. Nature, 335, 188–190.

32. McKimm-Breschkin, J. L., Caldwell, J. B., Guthrie,R. E. & Kortt, A. A. (1991). A new method for thepurification of the influenza A virus neuraminidase.J. Virol. Methods, 32, 121–124.

33. Harris, J. W. (1962). Holey films for electronmicroscopy. Nature, 196, 499–500.

34. Jeng, T. W., Talmon, Y. & Chiu, W. (1988). Contain-ment system for the preparation of vitrified-hydratedvirus specimens. J. Electron Microsc. Tech. 8, 343–348.

35. Crowther, R. A., Henderson, R. & Smith, J. M. (1996).MRC image processing programs. J. Struct. Biol. 116,9–16.

36. Frank, J., Radermacher, M., Penczek, P., Zhu, J., Li, Y.,Ladjadj, M. & Leith, A. (1996). SPIDER and WEB:processing and visualization of images in 3D electronmicroscopy and related fields. J. Struct. Biol. 116,190–199.

37. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard,M. (1991). Improved methods for building proteinmodels in electron density maps and the location oferrors in these models. Acta Crystallog. sect. A, 47,110–119.

38. Collaborative Computational Project Number 4(1994). The CCP4 suite: programs for protein crystal-lography. Acta Crystallog. sect. D, 50, 760–763.

39. Sayle, R. A. & Milner-White, E. J. (1995). RASMOL:biomolecular graphics for all. Trends Biochem. Sci. 20,374.

40. Vigers, G. P. A. (1986). Clathrin Assemblies in VitreousIce: A Structural Analysis by Image Reconstruction,Cambridge University, Cambridge, UK.

41. Evans, S. V. (1993). SETOR: hardware-lighted three-dimensional solid model representations of macro-molecules. J. Mol. Graph. 11, 134–138. See alsopp. 127–128.

Edited by W. Baumeister

(Received 7 July 2003; received in revised form 24 September 2003; accepted 29 September 2003)


Structure of an Influenza Neuraminidase–Diabody Complex by Electron Cryomicroscopy and Image...

Documents

Transcript of Structure of an Influenza Neuraminidase–Diabody Complex by Electron Cryomicroscopy and Image...