1\) ~ '1'1 ~
`~. "3d..ip
`v~ - (Q lt I
`C -~
`
`RESEf-?VE COPY
`
`RETlJRr~ TO
`C I R C U L 1\ T I 0 IJ D ESt(
`
`PFIZER EX. 1100
`Page 1
`
`

`

`nature
`
`26 March 1987
`Volume 326
`Issue no. 6111
`
`Crystals of a complex of antibody
`Fab fragment and influenza virus
`neuraminidase. Photogra ph by
`Julie Macklin and Stewart Butter(cid:173)
`worth in backlighting to illumin(cid:173)
`ate different crystal face ts in
`diffe rent colours. See p.358 and
`News and Views p.334.
`
`• Plastic magnets?
`... pages 335 and 370
`Organic polymers can be fer(cid:173)
`romagnetic, a property hither(cid:173)
`to .known only in inorganic
`materials.
`• Electro-detection in
`duck-billed platypus
`... page386
`
`Electroreceptors in the bill of
`the duck-billed platypus can
`pick up muscular electrical
`activity fro m the tail flick of
`freshwater shrimp on
`the
`which it feeds.
`• Snow goose probes
`... page392
`Detection of multiple mater(cid:173)
`nity and paternity in single
`broods of lesser snow geese
`pioneers the use of polymor(cid:173)
`phic DNA analysis to study
`ecology and population gene(cid:173)
`tics.
`• Nerve growth factor
`... page353
`Sensory neurons innervating
`developing skin do not synthe(cid:173)
`size nerve growth facto r until
`their fibres reach their cuta(cid:173)
`neous targets.
`• 3D protein structure
`... page347
`Techniques fo r predicting the
`tertiary structure of proteins
`may lead to the design of novel
`molecules .
`
`NEWS
`317 Towards the next trade war?
`318 US-French AIDS accord • Plutonium dispute in Ger(cid:173)
`many
`319 US fights to protect semiconductor industry • Japan
`fights to protect telecommunications
`320 US Air Force overhauls ASAT programme •
`321 Europe acts on ozone layer • Another crisis for SERC
`322 Eye institute proposed • Japanese X-ray satellite •
`AIDS institute for India?
`323 India studies herbal remedies • Japan plans to sequ(cid:173)
`ence humane genome
`324 Physicists brood at Trieste
`325 More links urged with Chinese industry • Doubts on
`Soviet dam
`CORRESPONDENCE
`326 Hazards of genetic engineering • Megafauna versus
`megafauna • Chinese universities
`NEWS AND VIEWS
`327 New ways with matter/antimatter
`328 A quiet supernova? David Lindley • Fluctuations in a
`patchy world John H Lawton
`329 Single-atom masers and the quantum nature of light
`Peter Knight
`330 Tumour necrosis factor: Polypetide mediator net(cid:173)
`work Lloyd J Old
`331 Mass extinctions: iridium anomalous no longer?
`Stephen K Donovan
`332 Eukaryotes with no mitochondria T Cavalier-Smith
`334 Antibody-antigen
`flexibility Robert Huber &
`William S Bennett
`335 Organic ferromagnets: Polymers from the Soviet
`Union Richard Friend
`336 Developmental neurobiology: Trophic factor theory
`matures J W Lichtman & PH Taghert
`SCIENTIFIC CORRESPONDENCE
`337 Nomenclature of immunological markers C Milstein
`• Soliton theory and Jupiter's great red spot P L Read
`338 Glycine-binding sites and NMDA receptors in brain
`N G Bowery • Can a negative quantity be deemed a proba(cid:173)
`bility? P T Landsberg
`BOOK REVIEWS
`339 The Shaping of Modern Psychology by L S Hearnshaw
`Alan Costall
`340 Helium Cryogenics by S W Van SciverP V E McClintock
`• The Sickled Cell by S J Edelstein D R Higgs
`341 On the heart beat -
`two books on cardiac muscle
`Andrew P Somlyo
`342 The Forms of Color by K Gerstner J D Mallon
`COMMENTARY
`343 The sombre view of AIDS M Rees
`346 Science meets Islam in the Saudi desert P Newmark
`Contents continued ..,..
`
`THIS WEEK
`• Sombre view of AIDS
`... page343
`A worst-case study shows a
`surprisingly high fraction of
`those infected wi th the AIDS
`vi rus may go on to develop the
`fu ll disease.
`... page319
`Meanwhile, a deal between
`the French and US sides in the
`AIDS patents dispute will give
`a boost to AIDS research by
`fu nding a new resea rch centre.
`
`Population stability
`.. pages 328 and 388
`The belief that greater com(cid:173)
`plexity leads to greater ecolo(cid:173)
`gical stability is challenged by
`the observation that spatial
`heterogeneity destabilizes the
`in teractions between aphids
`and predatory ladybirds.
`
`Living fossils
`.. pages 332 and 411
`The microsporidia, a group of
`microbial parasites, may be
`living relics of the ea rliest
`phases of eukaryotic evolution.
`
`F ASEB showcase
`. page419
`
`Product Review leads with the
`increasingly popular technique
`of high-speed countercurrent
`chromatography.
`
`PFIZER EX. 1100
`Page 2
`
`

`

`~35~8------------------------------------------ARTICLES-----------------------N~A~TU~R=E_v~o~L~-~32~6~2~6~M~A~R~C~H~I~98~7
`
`13. Stoeckel, K., Schwab, M. & Thoenen, H. Brain Res. 89, 1- 14 ( 1974 ).
`14. Angeletti, R. H. & Bradshaw, R. A. Proc. natn. Acad. Sri. U.S.A. 68, 24 17-2420 ( 197 1).
`15. Scott, J. era/. Nature 302,538-540 ( 1983).
`16. Ullrich, A., Gray, A., Berman, C. & Dull, T. J. Nature 303, 82 1- 825 (1983).
`17. Darling, T. L. J. era/. Cold Spring Harb. Symp. quant. Bioi. 48, 427-434 (1983).
`18. Menescini-Chen, M.G., Chen, J. S. & Levi- Montalcini, R. Archs ita/. Bioi. 116, 53- 84 (1978).
`19. Letourn eau, P. C. Devl Bioi. 66, 183-196 (1978).
`20. Gundersen, R. w_. & Barrell, J. N . Science 206, 1079- 1080 ( 1979).
`21. Levi-Montalcini, R. Prag. Brain Res. 45, 235-258 (1976).
`22 . Jl.enehan, W. E. & Munger, B. L. J. camp. Neur~/. 246, 129- 145 (1986).
`23 . lggo, A. Br. med. Bull. 33,97- 102 ( 1977 ).
`·,
`
`24. lggo, A. & Andres, K. H. A Rev. Neurosci. S, 1-3 1 ( 1982).
`25. Davies, A. M. & Lumsden, A. G. S. J. camp. N eural. 223, 124- 137 ( 1984).
`26. Davies, A. M. & Lumsden, A. G. S. J. camp. Neural. 253, 13- 24 ( 1986).
`27. Korsching, S. & Thoenen, H. Proc. natn. Acad. Sci. U.S.A. 80,35 13- 3516 ( 1983).
`28. Bandtlow. C., Heumann, R., Schwab, M. & Thoenen, H. EMBO J. (i n the press).
`29. Rush , R. A. Nature 312, 364-367 (1984).
`30. Finn, P. J., Ferguson, I. A., Renton, F. J. & Rush, R. A. J. Neuracytol. IS, 169- 176 (1986).
`31. Lumsden, A. G. S. & Davies, A. M. Natu" 306, 786-788 ( 1983 ).
`32. Lumsden. A. G. S. & Davies, A. M. Natu" 323, 538- 539 ( 19861 .
`33. Lowry, 0. H., Rosenbrough, N.J., Farr, A. L. & Ranqall , R. J. J. bioi. Chern. 193, 265-275
`(1951 ).
`
`Three-dimensional structure of a complex of
`antibody witb influenza virus neuraminidase
`P. M. Colman, W. G. Laver·, J. N. Varghese, A. T. Baker, P. A. Tulloch,
`G. M. Airt & R~ G. Webster:t:
`CSIRO Division of Protein Chemistry, 343 Royal Parade, Parkville, 3052, Australia
`*John Curtin School of Medical Research, Australij!n National University, Canberra 2601 , Australia
`t Department of Microbiology, University of Alabama, Birmingham, Alabama 35294, USA
`* St Jude Children's Research Hqspital, Memphis, Tennessee 38101, USA
`
`The structure of a complex between influenza virus neuraminidase and an antibody displays features inconsistent with th e
`inflexible 'lock and key' model of antigen-antibody binding. The structure of the antigen changes on binding, and that of
`.
`the antibody may also change; the int£!raction therefore has some of the character of a handshake.
`
`WE report here the first analysis by X-ray diffraction of the
`three-dimensional structure 'or a viral antigen complexed with
`an antibody. Fab fragment. The antigen is the influenza virus
`enzyme neuraminidase (reviewed in ref. 1).
`Sufficiept data on the chemical 2 and spatial 3
`6 structure of
`-
`antibodies emerged in the 1970s to provide a pasis for under(cid:173)
`standing how variation in antibody structure occurs, but the
`question of how different antibodies aq:ommodate different
`macromolecular epitopes remains unresolved . The antigen bind(cid:173)
`ing fragments (Fab) of immunoglobulins t;:onsist of a light chain
`(L) and the N-terminal half of the heavy (H) chain. On each
`chain there are two globular domains of -1 OQ amino acids, the
`N-terminal domain ill each chain being variable (VL and VH)
`and the C-terminal domain conserved (CL and CH1) in their
`amino-acid sequences. VL and VH domains are associated with
`each other, forming a variable module and CL and CH 1
`for111 the constant module. Three complementarity-determin(cid:173)
`ing ' re1gions (CDR1, 2 and 3) from each of the VL and VH
`domains are clustered at the extremity of the Fab arms, and
`
`they determine the binding specificity of an immunoglobuli n
`molecule.
`11
`The three-dimensional structures of five free Fab fragments 7
`-
`and one in complex with lysozymet 2 have been reported. Four
`of the structures, New7
`, Kol 8
`, McPC6039 and 1539 10
`, are well
`refined and form the basis of the following generalizations which
`also appear valid for the other two, S10/ 1 (ref. 11 ) and 0 1.3
`(ref. 12). The pairing of VL and VH domains is determined by
`amino acids from· bpth the framework region and the CDRs.
`Most of the surface area buried in the interface derives fro m
`conserved residues and this observation explains the largely
`conserved geometry of VL-VH pairingD. Small differences in
`pairing presumably result from interactions among the CDR
`amino acids 14
`. Analysis of the VL-VH contact surface shows it
`to be unlike other interfaces between ,8-sheets 15
`. Amino aci ds
`from the outermost strands of the two sheets fold into the
`interface where they contribute the bulk of the buried surface.
`CL -CH 1 dimers are similarly conserved in their association
`pattern 16. More variable is
`the so-called elbow anglet ?.ts
`
`h
`
`Fig. I Schematic diagrams of chain fold in N2 and
`N9 neuraminidase viewed down the 4-fold axis (a)
`and perpendicular to thi s axis (b). The symmetry axis
`is bottom right in a arid stan ding vertical at the left
`rear in b. The view of the neuraminidase in b is similar
`to that shown in Fig. 5. Tagged residues and adjacent
`chain segments are referred to in the text. N2 number(cid:173)
`ing is used. In a, the side chains of amino acids 368; 370
`point towards the viewer, whilst that of Arg 371 points
`away and into the catalytic site located above and to
`the right of co· 371. Mutations at positions 367, 369,
`370, 400, and 432, as detailed in Fig. 2, abolish the
`binding of NC41 antibody to neuraminidase, whereas
`mutations at 368 and 329 reduce that binding. A muta(cid:173)
`tion at 220, which falls outside the NC41 epitope, has
`no effect on NC41 binding to neuraminidase (see Fig.
`2). In a, solid chain segments show regions in contact
`with the N C~ l antibody; contact assignments in the
`broken solid segments are ten!ative.
`
`PFIZER EX. 1100
`Page 3
`
`

`

`l "'"" 'OL " ' " MmH ""'
`
`------ARTICLES~---------------~359
`
`100~~._~~----~------~
`
`90
`
`80
`
`?{!.
`
`1
`
`\
`.,
`
`I
`I
`I
`I
`
`1/ 100 1/ 200
`1/ .-00
`NC 41 lgG d il u ti on
`
`1/800
`
`Fig. 2 Neuraminidase inhibition ( NI ) curves of monoclon al anti(cid:173)
`body NC41 act ing on N9 neura minidase (NA) and a number of
`variants selected with different monoclonal antibodies to N9
`neuraminidase. Assays used fetuin as a substrate, as described 58
`The amino-acid sequence changes of the variants a re : •. 434 ( 432
`in N2 numbering) K-+N; 400 (400) N-+K; 371 (370) S -+ L; 370
`(369) A-+ D; 368 (367 ) S-+ N; 0 , 369 (368) I .... R; 6 , 331 (329)
`N-+ D (design ated as OX2 in text ); 0 , 222 (220) R -+ Q (this curve
`is identical to wild-type). The a mino-acid sequence changes were
`determined by sequencing e DNA of the vi ral RNA stra nd encoding
`the neuraminidase as described 59
`. The changes a re shown on the
`3-D structure of the mondmer in Fig. I.
`
`describing the angle between the local axes of symmetry in the
`V and C modules.
`The identification of antigenic regions on protein molecules 19
`has been aqempted by a variety of method~ 20- 23 but the structural
`basis of antigenicity remains unclear. Current models of antigen(cid:173)
`antibody association are based on classical ideas of lock and
`ke y complementarity. But it has been suggested24 that the anti(cid:173)
`genic regions of proteins are located in the more flexible chain
`segments, which may adopt a configuration that allows antibody
`binding. It is not known whether binding to antigen has any
`effect on the structure of an antibod/ 5
`, nor whether, if such an
`effect exists, it has biological significance. For the Fab-lysozyme
`complex 12, the parts of the lysozyme that make contact with the
`an tibody are not the more flexible regions of free lysozyme. No
`de formation of the antigen nor any structural change in the Fab
`was observed 12.
`The antigen in this study is the neuraminidase of influenza
`virus. It is a tetramer of subunits with relative molecular mass
`
`I 60,000 (M, 60 K) with circular symmetry, and is attached by a
`stalk to the viral membrane . Neuraminidase 'heads' can be
`( liberated from the virus by proteolysis26
`• There is a ntigenic
`/ variation in the heuraminidase between different strains of virus .
`l Antibodies against neuraminidase do not neutralise virus infec-
`tivity, but do modify the disease in favour of the host27
`• The
`three-dimensional structures of neuraminidase heads of sub(cid:173)
`types28 N2 (refs 29, 30) and N9 (A.T.B. , J.N .V., W.G.L., G. M.A.
`and P.M.C., manuscript in preparation ) are known and a sche(cid:173)
`matic of the chain fold is shown in Fig. I. Neuramirtidase of
`subtype N9 from a·n avian influenza virus, G70c31, has been
`used in this study. Its structure is very similar to the N2 enzyme
`as expected from the sequence homology of 50% (ref. 32).
`We previously reported electron and low resolution X-ray
`diff raction studies of Fab-neuraminidase complexes 33 and, more
`
`recently, the crystallization of another complex (with NC41 Fab)
`which diffracts X-rays to beyond 3 A resolution34. The NC41
`antibody cari suppress the yield of virus from infected cells and
`therefore
`allows
`the
`selection of antigenic variants.
`Neuraminidase-inhibition curves (Fig. 2)
`.show that some
`variants with single sequence changes .are not inhibited at all
`by NC41 antibody whereas others are partially inhibited. The
`location of these sequence changes on the three-dimensional
`structure of neuraminidase is shown in Fig. 1.
`Protomer structure
`Electron microscopy of negatively stained protomers of complex
`show tetrameric neuraminidase heads with four Fab fragments
`atta_ched (data not shown). Their image appearance is very
`stmtlar to that of protomers of N9 neuraminidase complexed
`with the Fab fragments of antibodies 32/ 3 and NC35 (ref. 33)
`and may be described as a square box (IOOx 100 x60 A) with
`four antennae ( Fab molecules) attached to one surface on the
`outer corners. The overall shape of the proto mer from the X-ray
`diffraction study (Fig. 3) is consistent with the electron micro(cid:173)
`scope images. the Fab arms subtend an angle of 45° to the
`plane of the tetrameric neuraminidase head, slightly foreshor(cid:173)
`tened in the electron microscope image33
`.
`Surface loops of the neuraminidase in contact with the CDRs
`are 368-370, 400-403 , 430-434 and parts of 325-350. Of these,
`the conformation of the 325-350 loop is still tentative. It was
`the most difficult region of the original N2 neuraminidase struc(cid:173)
`ture29
`to
`interpret and
`remains ambiguous
`in
`the N9
`neuraminidase structure (A.T.B. et a/., manuscript in prepar(cid:173)
`ation). Such instances of ill-defined structure are typical of
`surface loops of polypeptides subject to thermal motion or
`statistical di so rder.
`Early indications on the distribution of temperature factors
`in the uncomplexed N2 neuraminidase, based on crystallo(cid:173)
`graphic refinement of that structure at 2.2 A resolution and an
`R-factor of 0.293, show above-average thermal parameters in
`chain segments 109- 111, 141-142, 221-222, 243-247, 305-309,
`316-319, 327-331, 338-343,365-369, 399-402, 429-437 and 461-
`469 (J.N.V. and P.M.C., unpublished data) . Elevated B values
`around residue 330 may correlate with inaccurate modelling as
`discussed above. All the segments listed here are surface loops.
`Note that only one of the hotter loops is underneath the
`neuraminidase head . We emphasize that the temperature factor
`analysis is preliminary, and indeed relates to N2, not N9,
`neuraminidase.
`The monoclonal variant of N9 neuraminidase known as OX2
`(As n 329-> Asp 329, N2 numbering) crystallizes iso morphously
`with wild-type N9-NC41 Fab complex34. NC41 inhibits the
`OX2 neuraminidase va riant less than it does wild-type (Fig. 2).
`Refinement of the N9- NC41 Fab structure using data collected
`from the OX2-NC41 Fab complex yields an R~ factor of 0.36
`for a model with tight geometry. This result is comparable with
`the current R-factor agai nst the native dataset and confirms our
`earlier conclusion 34 that the binding of NC41 Fab to wild-type
`and OX2 N9 neuraminidases is isosteric. Either some bonding
`'glue' is absent or repulsive forces have been introduced by the
`mutation (Fig. 2), but there is no detectable rearrangement of
`the antibody on the antigen at this stage. The precise lo.cation
`of residue 329 (N2 numbering) in the N2 and N9 structures is
`unclear. Refinement of the OX2-NC41 Fab structure should
`eventually reveal the structural basis for the reduced binding of
`NC41 to OX2 compared with wild-type N9.
`The third CDRs on both heavy and light chains are not yet
`completely modelled, and the conformation of the 325-350
`region on the antigen remains uncertain, so we cannot identify
`the amino acids that interact in the interface. All the CDRs,
`with the possible exception of light-chain CDRI appear to ma ke
`contact with the epitope. A preliminary assessment is that there
`are no more contacting residues than the 16 or 17 observed in
`the Fab-lysozyme structure12.
`
`PFIZER EX. 1100
`Page 4
`
`

`

`M603
`4.2 (0.7)
`
`12.4 (2.4)
`2.1 (0.7)
`
`KOL
`6.5 (0.4)
`5.0 (0.3 )
`
`10.8 (2. 1)
`
`NC41
`12.2 ( 1.0)
`8.8 (1.3)
`12.1 (0.7)
`
`Comparison of four different Fab quaternary structures. Upper right
`triangle: VL domain of the row Fab was mapped into the VL domain
`of the column Fab using the c. coordinates of residues 33-39, 43-47,
`84-90, 98- 104 (Kabat2 numbering). These amino acids were chosen
`because they define the interface between VL and VH domains". The
`calculated transformation was applied to the V H domain of the row Fab
`and the additional rotation in degrees (and translation in A) to optimize
`overlap of the two VH domains in question was calculated and is given
`above. c. coordinates used for overlap of VH domains were 34-40,
`44- 48,88-94 and 103- 109. Lower left triangle: CL domain of the column
`Fab was mapped into the CL domain of the row Fab using the c.
`coordinates of residues 118-124, 131- 137, 160- 162 and 174- 178 (Kabat 2
`numbering). These amino acids were chosen because they define the
`interface between CL and CH, domains16. The calculated transformation
`was applied to the CH 1 domain of the column Fab and the additional
`rotation in degrees (and translation in A) to optimize overlap of the
`two CH, domains are shown. c. coordinates used for overlap of CH ,
`domains were -121 - 125, 139- 145, 172- 179 and 186-190. Amino acid>
`from the interface were used because if this set is expanded to include
`other pans of the domain structure, the correlation between small
`rotation angles and similar chain classes that is observed for the C
`module is less good. The largest r.m.s. distance between domain structu re
`alignments is 0. 7 A. A similar comparison was done for the V modules
`of the M603 (antiphosphocholine) and uncomplexed SI0/ 1 (antine(cid:173)
`uraminidase) Fab fragments. No measurable difference in the way the
`V L and V H domains associate in those antibodies was detected. A
`comparison elsewhere of Kol and New V modules8 yields a value of
`9•. The value reponed here (6.5•) is smaller because it is uninfluenced
`by structural differences outside of the interface zone.
`
`Table I Quaternary structure comparisons of Fabs
`
`NEW
`M603
`KOL
`NC41
`
`NEW
`
`12.7 (2.3)
`2.5 (0.0)
`11.5 (0.4)
`
`~~~---------------------------------------ARfiCL£$,--------------------~N~A~TU~R~E ~v~o=L.~3~26~2~6~M~AR~C~H~t~98~7
`Quaternary structure of the Fab
`The amino-acid sequence of the Fab is known (G.M.A., unpub(cid:173)
`lished data). The assignment of heavy and light chains in the
`Fab structure
`is consistent with side-chain densities at
`homologous positions on the two chains, such as heavy-chain
`( H) Trp 47 and light-chain (L) Leu 46, H Val 37 and L Tyr 36,
`and, H Trp 103 and L Phe 98. Heavy-atom binding sites also
`show an asymmetry which is consistent with these observations.
`The quaternary structures of known Fab fragments were com(cid:173)
`pared as described in Table 1. The lower left triangle of entries
`indicates that CL -CH, pairing is a function of immunoglobulin
`chain subtype since -y1: A chains (as in Kol and New) show a
`consistent mode of association as do a or -y2. : K chains (as in
`M603 and NC41). Small but significant alterations in Y-domain
`pairing are observed between Kol, New and M603 (Table I,
`upper right). This variation in VL-VH association has been
`attributed to the fact that_ the CDRs contribute partially to the
`dimer interface between the two domains in the Y-module14,
`although in the case of Kol we suggest that intimate crystal
`contacts involving the CDRs may contribute.
`Comparison of these three Fabs with NC41 complexed with
`neuraminidase shows, in each case, a significantly larger
`difference in V L-V H association. Compared with Kol, the dis(cid:173)
`tance between H CDR2 and L CDR3 is larger and the relative
`positions of the CDRs around the antigen-binding surface are
`altered by up to 4 A. A movement of this magnitude is of the
`order of the distance between adjacent a-carbon atoms on a
`polypeptide and may be considered as dramatic as a sequence
`insertion or deletion in a structurally critical part of the molecule.
`As well as the bulk movi:ment of domains described here, the
`CDR loops may be flexible and able to adapt further to an
`epitope.
`Building an atomic model into tlie penultimate electron
`density map (that in which no phase information from the Fab
`structure had been included) required the rotation of H Trp 47
`180° around C 0 -C13 compared with its position in Kol. M603
`and New are the same as Kol at this point. The positions of the
`loops at residue 40 on both heavy and light chains also required
`remodelling. Therefore the reorganization of the interface
`through sliding of domains has consequences for the domain
`structure of the VL and VH.
`The sequence of the NC41 VL and VH domains shows that
`those framework amino acids on both the light chain (Tyr 36,
`Gin 38, Pro 44, Tyr 87, Phe 98) and the heavy chain (Val 37,
`Gin 39, Leu 45, Trp 47, Phe 91, Ala 93, Trp 103) that are buried
`in the interface15 are conserved as expected. There is nothing
`in the primary sequences of the CDRs of NC41 to suggest a
`cause of the unusual domain association. An independent analy(cid:173)
`sis of the N C41 sequence confirms this view (C. Chothia, per(cid:173)
`sonal communication).
`Two other Fab structures are known to fit the normal pattern
`of VL-VH association, the free antineuraminidase monoclonal
`antibody SI0/ 1 (ref. II ) and the complexed anti-lysozyme
`monoclonal antibody D1.3 (ref. 12). Among light-chain dimers
`the picture is less clear. Although several examples ofYL- Ywlike
`association have been observed3·35"37
`, there are two clear excep(cid:173)
`tions38-39, one of which is said to be caused by the CDR residue
`L 91, or, in our view, perhaps by a His at residue 38.
`If changes in the amino-acid sequence of the CDRs (which
`are only a small part of the interface) can modulate the pairing
`pattern, binding of the six CDRs to a macromolecula r surface
`might similarly perturb the V L-V H interface. Without knowing
`the three-dimensional structure of the free NC41 Fab we cannot
`definitely conclude that the antibody structure has changed on
`binding antigen, but we believe that this explanation is more
`plausible than the alternative, which requires that special
`sequences in the C DRs alone have determined the pairing
`pattern.
`Other data support conformational changes in antibodies
`when antigen or hapten bihds40"44. Changes in circular dichro-
`
`ism40 or circular polarization of Huorescence42 indicate an
`altered environment for aromatic residues after antigen bi nds
`to antibody, and kinetic data support a bi-molecular process
`with distinct conformational states for bound and free Fab41·43•44
`•
`Both of these aspects are embodied in the sliding V L-VH model
`presented here.
`Direct structural evidence has been presented for hapten(cid:173)
`•46. In that
`induced structural changes in a Bence- Jones protein45
`case hydrophobic ligands can apparently penetrate the V L-V L
`interface and signal their presence to• the C-module. Strain
`introduced in the structure of the C-module can be relaxed by
`reducing and re-oxidizing the disulphide bond between the
`C-terminal regions of the two light chains46. Table I indicates
`that in the neuraminidase- NC41 complex no significant changes
`in the quaternary structure of the C-module have occurred .
`Analysis of the pseudo-symmetry axes of theY and C modules
`shows that the angle between these a xes, the elbow angle, is
`- 150°, which is intermediate in the observed range of 130- 180°
`for other Fab structures. The sense of the bending is the same
`as in all other Fab fragments, with VH and CH, domains closer
`together than the VL and CL domains.
`Conformational changes in the antigen
`The neuraminidase upper surface loop involving residues 367-
`371 was remodelled to better fit the density as observed in the
`complex. Adjacent parts of the loop which were also omitted
`from the phasing process (residues 364- 366 and 372- 373) fit the
`density as if they are rigidly attached to the N9 core. The C~
`atoms of residues 368-371 have been moved by I A or more
`from their position in N9 neuraminidase (Fig. 4). Least-squares
`refinement of the structure to R = 0.35, gave a mean shift of
`0.4 A in all C., atoms with a standard deviation of 0.2 A. The
`current position of the 370 loop (Fig. 4) shows displacements
`of more than 1.2 A for c. atoms 369- 371 compared wit h their
`
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`'·~~T~U~R~~~·~vo~L~-~3~26~26~M~A~R~C~H~t~98~7------------------------ARTICL£S--------------------------------------------~~~t
`fig. 3 Stereo images of the c. skeleton of the
`protomer, showing four Fab molecules attached
`tetrameric neuraminidase
`'head'.
`to one
`Neuraminidase, purple; heavy chain, green;
`light chain, blue. View with the protomer 4-fold
`axis vertical, but tipped towards the viewer. The
`crystals of N9- NC41 Fab belong to the space
`group P42 12 with a= 167 A, c = 124 A (ref. 34).
`There is one quarter of a protomer per asym(cid:173)
`metric unit and 70% of the cell volume is
`occupied by solvent. Data were collected photo(cid:173)
`graphically to 3 A resolution. In all, 67,976
`measurements of 25,241 independent reflections, representing 76% of the data in the 3 A sphere, were merged with an R-factor on intensities
`of 0.12. Phasing was initiated through two heavy atom derivatives (potassium tetra-chloro-platinate and diamino-dinitro-platinum). An envelope
`was computed60 on the basis of a 5 A resolution electron-density map allowing the location of the neuraminidase tetramer on the crystallographic
`4-fold axis to be determined first by inspection and subsequently by correlation methods in real space and rigid body least-squares refinement61
`,
`resulting in a correlation coefficient of 0.204 between Fo bs and Fcak for data in the range 5-3.5 A. It was now clear which surface regions of
`the neuraminidase were in contact with the remaining electron density in the image, and, in subsequent use of neuraminidase as partial
`structure in the phasing process, those regions were excised. A second electron density map, now at 3.5 A resolution, was phased on the two
`derivatives (one to 5 A and one to 3.5 A), the neuraminidase partial structure, and solvent flattening from a redetermined envelope. The four
`domains of the Fab fragment were recognised in this map and fitted independently by real-space correlation methods with the known Fab
`structure Kol8. Rigid-body least-squares refinement with five groups, neuraminidase, VH, CH., VL and CL produced shifts of up to IS in
`group orientations and led to an R-factor of 0.425 and a correlation coefficient of 0.308 for data between 5 and 4 A resolution. The structure
`is based on a 3 A electron density map, which is phased in addition to the information listed above with the structure of the Fab fragment
`excluding the complementarity-determining loops. Thus there is no structural prejudice in the image of those parts of the antigen or antibody
`that are near, or in, the bonding interrace. Further refinement62 has since reduced the residual to 0.35 for data between 6 and 3 A resolution.
`The r.m.s. error in the atomic positions is of the order of 0.5 A at this stage.
`
`Fig. 4 Stereo image of the electron density
`map (2Fo- Fc, R = 0.35) of the neuraminidase
`loop 368-372 in the complex with NC41 Fab.
`Residues here are labelled 868-872. The yellow
`model shows the c11rrent fit of the loop in the
`complex structure. The purple model is the
`position of this loop in uncomplexed N9
`neuraminidase.
`
`Fig. 5 Stereo image of one quarter
`of the protomer, showing the Fab
`bound to the side of the active site
`cavity on the enzyme. The enzyme
`active centre is facing the viewer,
`below and to the left of the antibody
`binding site, and is coloured yellow.
`The neuraminidase perspective is as
`in Fig. 1 b. Neuraminidase, green;
`heavy chain, purple; light chain,
`blue.
`
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`
`position in free N9 neuraminidase. Thus the antigen is distorted
`by interaction with the antibody.
`inhibit
`Most monoclonal anti-neuraminidase antibodies
`neuraminidase activity against fetuin, but only some of these
`also inhibit activity against the trisaccharide sialyl-lactose47.
`Although NC41 Fab cannot sterically block entry of a trisac(cid:173)
`charide into the active site (Fig. 5), NC41 does inhibit this
`activity (R.G.W., manuscript in preparation). We suggest that
`one possible mechanism for enzyme inactivation is the small
`displacement of Arg 371, which points directly into the catalytic
`site. We cannot yet rule out other interpretations, such as the
`effect of the antibody on steering substrates and products into
`or out of the active site. It has been shown that changing Arg 371
`for Lys by site-specific mutagenesis results in the loss of90-95%
`of enzyme activity, although the mutated protein is correctly
`folded (M. R. Lentz, R.G.W. and G.M.A., manuscript in prepar(cid:173)
`ation).
`Deformation of antigens by antibodies has been proposed to
`explain a variety of experimental data, including single-hit
`kinetics for neutralization of polio virus48 and altered binding
`affinity for second antibodies binding noncompetitively to the
`antigen49
`• The capacity of anti-apomyoglobin antibodies to,
`effectively, expel the haem from myoglobin has long been
`known50. There are also many reports of antibodies inhibiting
`enzymes51 . Our results directly demonstrate that conformational
`changes can be induced in antigens by antibodies. Furthermore,
`the change observed here, although small, correlates with the
`inactivation of the neuraminidase towards sialyl-lactose. Other
`anti-neuraminidase antibodies
`that
`inhibit neuraminidase
`activity only towards large substrates and, like NC41, can select
`antigenic variants, probably inhibit enzyme activity by blocking
`access of large substrates to the active site. We are testing the
`hypothesis that such antibodies do not distort the active site of
`the enzyme by an X-ray diffraction study of the N9-NC10 Fab
`complex34
`• Unlike NC41, this antibody does not inhibit enzyme
`activity on sialyl-lactose. Antigen distortion is likely to be but
`one of a number of possible mechanisms for virus neutraliz(cid:173)
`ation52.
`
`Conclusions
`The structure of a complex between an antibody and influenza
`virus neuraminidase shows features inconsistent with a rigid
`'lock and key' model for antibody-antigen interactions. In con(cid:173)
`trast to the findings from a lysozyme-antibody complex12 we
`observe ( 1) an unusual V L-V H pairing in the V module of the
`Fab, and (2) local perturbation of the antigen at the centre of
`the epitope. The interaction therefore has some of the character
`of a handshake. NC41 Fab has not yet been crystallized, and
`we can only suppose on the basis of sequence data that its
`structur