Solution structure of human and mouse immunoglobulin M by synchrotron X-ray scattering and molecular graphics modelling

J. MOE. Hiol. (1991) 221. 1345-1366

Solution Structure of Human and Mouse Immunoglobulin by Synchrotron X-ray Scattering and Molecular Graphics Modelling

M

A Possible Mechanism for Complement Activation Stephen J. Perkins?, Adam S. Nealis Department of Biochemistry and Chemistry Royal Free Hospital School of Medicine Rowland Hill Street, London NW3 2PF, U.K.

Brian J. Sutton Biomolecular Sciences Division, Biophysics Section King’s College London, 26-29 Drury Lane London WCSB 5RL, U.K.

and Arnold Feinstein Department of Immunology, The Middlesex Hospital Cleveland Street, London (Received 10 January

Windeyer Building Medical School WI P 6DB, U.K.

1991; accepted 18 June 1993)

The pentameric 71-domain structure of human and mouse immunoglobulin M (IgM) was investigated by synchrotron X-ray solution scattering and molecular graphics modelling. The radii of gyration R, of human IgM Quaife and its Fc,, IgM-S, FablZ and Fab fragments were determined as 12.2 nm, 6.1 nm, 6.1 nm, 4.9 nm and 2.9 nm in that order. The R, values were similar for mouse IgM P8 and its Fab’, and Fab fragments, despite the presence of an additional carbohydrate site. The IgM scattering curves, to a nominal resolution of 5 nm, were compared with molecular graphics models based on published crystallographic a-carbon co-ordinates for the Fab and Fc structures of IgG. Good curve fits for Fab were obtained based on the crystal structure of Fab from IgG. A good curve fit was obtained for Fab’,, if the two Fab arms were positioned close together at their contact with the ($2 domains. The addition of the Fc fragment close to the ($2 domains of this FabfZ model, to give a planar structure, accounted for the scattering curve of IgM-S. The Fc, fragment was best modelled by a ring of five Fc monomers, constrained by packing considerations and disulphide bridge formation. A position for the J chain between two ($4 domains rather t,han at the centre of Fc, was preferred. The intact IgM structure was best modelled using a planar arrangement of these Fab’, and Fc, models, with the side-to-side displacement of the Fab’2 arms in the plane of the IgM structure. All these models were consistent with hydrodynamic simulations of sedimentation data. The solution structure of IgM can therefore be reproduced quantitatively in terms of crystallographic structures for the fragments of IgG. Putative Clq binding sites have been identified on the ($3 domain. These would become accessible for interaction with Clq when the Fab’, arms move out of the plane of the Fe, disc in IgM, that is, a steric mechanism exposing pre-existing Clq sites. Comparison with a solution structure for Clq by neutron scattering shows that two or more of the six globular Clq heads in the hexameric head-and-stalk structure are readily able to

t Author

to whom

all correspondence

should

be addressed.

1345 0022-2836/91/201345-22

$03.00/O

0 1991 Academic Press Limited

S. J. Perkins

1346

et al.

_.--___---

make contacts w&h the putative Clq sites in the C,U~ domains of t’rep IgM if the (‘I(1 arm axis angle in solution is reduced from 40-45” to 28”. This would br the trigger for (‘1 activation. Keywords:

immunoglobulin M; solution scat,tering: molecaular graphics; hydrodynamic modelling; complement

1. Introduction Immunoglobulin M (Igt) plays a central role in the initial response of the immune system to foreign antigenic material (Feinstein cf al., 1986). TgM is a pentameric molecule. in which each monome consists of 14 immunoglobulin fold domains located in four polypeptide chains (Fig. 1). The pentamer also contains one further domain. the joining (J) chain, which is also thought to fold as an immunoglobulin domain (Zikan et al.. 1985; Pumphrey, 1986) and which plays a role in assembly (l)a.vis & Shulman, 1989). IgM occurs at concentrations of 0.5 to 2.0 mg/ml in human plasma. Even when its binding to single determinants is weak, it’s pentamerit structure enhances the binding to antigens with multiple epitopes. On the basis of electron microscopy and chemical evidence, IgM has been modelled as an extended structure wit,h a csentral Fc, disc and five Fab’, arms. Once IgM is bound polyvalently to an individual particle, the attachment st’abilizes a five-legged “st*apIe-like” or “tablelike” c>onformation (Feinstein 8~ Munn, 1969). This altered struct’ure is able to bind complements coniponent Clq and thereby activates the classical pathway of complement, (Lachmann R: Hughes,Jones. 1984: Feinst’ein et al.. 1986). Even though the general morphology of the Ighl structure is known from electron microscopy. no successful crystallisations of IgM or its fragments ha,ve been reported to date. The precise manner in which the 70 immunoglobulin fold domains and ,J chain of IgM are associated t,o form a disulphidelinked pentameric structure remains unclear. The IgM structure, however. shows structural homologies with immunoglobulin C: (IgC). which is formed from 12 immunoglobulin fold domains. Models for the structure of TgM have been proposed, based on electron microscopy and crystallographic data for other immunoglobulin classes (Feinstein. 1974: Beale & Feinstein, 1976). The crystal struc&tures of the Fc fragment of IgG (Deisenhofer. 1981) and the Fab fragment from IgGl Kol (Marquart rf al.. 1980) and ot,her sources are now known. Molecular graphics modelling can therefore bt~ employed to predict structures for the fragments of IgM. as well as that’ of intact TgM. These models require experimental testing. t Abbreviations used: Ig. immunoglobulin; elbow angles. angle between the pseudo Z-fold axes of VL-Vn and C&,-C,1 domain pairs: Eu/Ou. Kabat et al. (1987) residue numbering syst’em for IgG and IgM. respectively.

Solution scattering (Glatter bz Krat ky. IY82: Perkins. 1988a.h) is a powerful technique for IISV in prostructural studies of domains. in multidomnin tein structures, in particular when atomic structures are availablr for t’he dornains (Perkins 8 Sim, 1986: Perkins d al., 1986, 199Oa,b). Studies using S-ra) analysis on JgM and its monomer JgM-S have brrn described by Wilhelm rt al. (1978, 1980. 1984) and Kayushina it al. (1985. 1986). Her?. wp rekport a srf of synchrotron X-ray scattering data for lgM and its IgM-S. Fc,. Fah’2 and Fail t’ragmentls. If tht 0t the scattering curve. is interpret,ation constrained by sequence informat ion. homologous crystal structures and molecular graphicas (Smith et al.. 1990: Perkins rf a/., 199Oh). the arrangement) of immunoglobulin domains that best replicates the scattering curv(b of each fragment can be modrllrd. Since each of t’he four fragment’s is testrtl as thtl IgM model is built up. this permit,s more reliablr caor&ruction of a model for intac+ lghl. Sinct, t hc st)ructures are measured in condit,ions ~~10s~~t 0 physiological, this is an advantage over the IIN of’ electron tnic~rosc~opy. A molecular model of the domain structure within TgM will rst,ablish some of its structural and func tional properties. These are: ( 1) t,hth assembly of th iti human and mouse IgM. respectively: (3) an assessment of c+onformational flexibility of the Pab’z arm?\ relative to the F(a5 disc of IgM: (4) the loc&ion ant1 relative disposit’ion of the ($3 domains in JgM that are thought t*o contain t,he binding site for. (‘1~1 (Shulman et al.. 1986. 1987: Wright pi al.. 198X): (5) the mode of interaction of TgM with (‘I q ill t hv mechanism of classical pathway acGvat,ion of c~omplemenl.

2. Materials and Methods Two TgM inlrnunoglobulins were studied. human IgAl from a patient QLT with Waldenstriim’s &east>. HWI mouse IgM PX (MOIY 104E). Human IgM was purified from serum as described by BeaIr & Feinstrin (l!%!f). Mouse IgM was purified from the serum of mice bearing the plasmac*yt,oma MOP(’ 104E (MrTntjirr P! (I/.. 19%). using the method described by Milstein rt N/. (1975). This IgM is an antibody to z-1 ,J Ilnked dext,rans (I,eon rl rtl.. 1970). Human monomeric IgSM-S was I)repared from 1gAI t)?; selective redu&ion. using mrrcaptoet,h?-lanlirlr sllch that dissociation with an approximately SO”,, yield of IgM-S was obtained. 1gM and IBM-H werr t,hen separated by gel filtration (Be& $ Frinstein. 1969). Human t,ryptic.

Solution

&ructure

Fc, was prepared by digesting human IgM with trypsin (in the ratio of 50 : 1. w/w) for 45 min at 60°C as described by Plaut & Tomasi (1970). Note that a portion of the Cp2 polypeptide chain together with carbohydrate remains attached t.o the Fc, preparation (Fig. 1). The treatment of human IgM with pepsin yielded a mixture of the Fab’, and Fab fragments in the ratio of 1 : 2. whirh were separated by gel filtration (Beale & Buttress, 1969). The treatment of mouse IgM with pepsin yielded the Fab’, fragment but not the Fab fragment (Beale & Van Dart. 1982); treatment, with papain gave a 1 : 1 mixture of the Fab’, and Fab fragments. Sedimentation data on fresh13 gel-filtered and ultracentrifuged material in phosphate/ XaCl buffer (pH 7.8) and I=@], were obt,ained by the method of Yphantis (1964). For checks of purity and the hydrodynamic calculations see Beale & Buttress (1969) (&. E. Richardson. unpublished data). IgM and its fragments were stored frozen at -70°C OI -20°C until required. Preparations of human and mouse 1g.M were also examined using freshly gel-filtered eluates in order to avoid the presence of dimeric aggregates. Solution scattering experiments were performed in buffers caont,aining 20 rnM-sodium phosphate, @5 M-NaCI. @05% (w/v) Sax, (pH 7-O), or 12 m&I-sodium phosphate. 200 rnM-SaCl, with or without 0.5 mM-EDTA. (pH 7.0). SDS/polyacrylamide gel electrophoresis under reducing and non-reducing conditions was used to verify the int,egrity of the samples.

(b) Composition of IgM and its fragments The human IgM Ou and the mouse IgM MOPC 104E amino acid sequences were taken from Kabat et al. (1987) as representative of the human IgM QU and mouse IgM P8 sequences. Based on the Kabat Ou index for residue numbers. the proteolytic site giving human Fab Ou was taken to lie between Lys224 and Asx225 in Cpl, that for human Fab’, Ou between Arg346 and Va1347 in Cp3, and that for human Fe, between Arg326 and Gly327 in Cp2. In human IgM. the 5 Asn-linked oligosaccharide sites are located as shown in Fig. 1 at residues 170 (Cpl), 332 (C&2), 395 and 402 (Cp3), and 563 (tailpiece, at the C terminus of Cp4). Xote that the position of cleavage gives 4 oligosaccharide sites per heavy chain on PC, and not 3 as anticipated from Fig. 1. The carbohydrate compositions of human IgM and its fragments were determined from oligosaccharide analyses of IgM QU, the IgM QV t’ailpiece, TgM QU Fc, (tryptic), IgM Mann. IgM Mann Fab’, and IgM Mann Fab (S. Amatayakul. T. W. Rademacher, R. A. Dwek & A. Feinstein, unpublished results). Both high-mannose and complex-type ohgosaccharides are present (Cahour et al., 1984; Ohbayashi et aZ., 1989). To derive the composition of IgM,S from that of TgM, that of the J-chain was taken from Niedermeier et al. (1972) and Shimizu et al. (1971). The human compositions are as follows: IgM; Gal,,, Man,,,, GlcNAc,,,, NeuNAc,,, Fuc,, TgM-S; Gal,,, Man,,, GlcNAc,,, NeuNAc,, Fuc, Fc,; Gal,,, Man,,,, GlcNAc,,,, NeuNAcze, Fuc,, Fab’,; Gal,, Man,,, GlcNAc,,, NeuNAc,, FUG, Fab: Gal,, Man,, GlcNAc,, NeuNAc,. The proteolytic site resulting in mouse Fab MOPC104E is between Lys220 and Asn221 in Cpl, and that for mouse Fab’, is between Lys361 and Ser362 in ($3. The 6 mouse otigosaccharide sites are at residues 56 (V,), 170 (Cpl), 332 (Cp2). 364 and 402 (Cp3), and 563 (tailpiece). The caomposition of the V, site is taken to be the same as that on t.he Ckcl sit.e. and those for all the fragments were taken

of IgM

1347

from the human analyses. contents were taken as:

The

mousr

carbohydrate

IgM; Gal,,. Man,,,, GlcNAc,,,. NeuNAcs,,. Fuc,, Fab’,; Gals. Man,,, GlcNAc,,. SeuNAc,. Fuc, Fab; Gal,. Man,,. GlcNAc,, NeuNAc,. This determination that derived from MOPC104E which IgM:

Gal,,,.

is considered to be more accurate than Anderson et al. (1985) for mouse TgM had given for 5 sites:

Man,,,.

Glch’A~,,~.

NeuNA(a9,.

Fuc,,.

Concentrations for molecular mass c*alcula.tions were calculated from absorbance measurements at 280 nm using absorption coefficients (Table 1) calculated from the tot,al compositions by the corrected Wet.iaufer procedure (Perkins. 1986).

(c) Synchrotron X-ray data collection X-ray scat&ring curves were obtained at the Synchrotron Radiation Source (Daresbury. I1.K.). Tnitial experiments on all 8 preparations, including most of the IgM scattering curves. were recorded in 3 sessions on the low-angle solution scattering camera at Station 7.3 (Nave et al., 1985). These employed measurement times of 4 t,o 15 min, sample-detector dlgtances of 2.180 m to 2.185 m, beam currents of 88 mA to 226mA, and storage ring energies of 1.8GeV or 2.0GeV. The available Q ranges were between 0.05 and 1.1 nrn- ’ Final experiments on the preparations were carried out in 3 sessions on the camera at Station 8.2 (Towns-Andrews et al.. 1989). using measurement times of 10 min, a sample-detector distance of 2.830m. currents of 109 mA to 195 mA. and a ring energy of 2.0 GeV. The available Q range varied from @05 to l.Onm-’ to (PO9 to 1.2 nm-’ (Q=4nsino/i: scattering angle = 20; wavelength = 1). A linear detector was used in all sessions, bar 1; the number of usable detect)or channels per scattering curve ranged from 300 t>o 355. 4 quadrant detector was used in the final 3rd session on St*ation 8.2 to study the Fc, fragment. In all runs, the detector was calibrated using wet, stretched rat-tail collagen (diffraction spacing of 67.0nm). All samples were measured in alternation with their buffer background for equal counting times in the same cell. in order to minimise background subtraction errors and any effects due to the gradual decrease in beam current with time. Data collections on Station %2 were recorded as 10 separate time frames. to permit subsequent checks for the absence of beam-induced radiation damage, after which the 10 curves were averaged. Data were reduced at Daresbury using either SWANAL (Station 7.3) or OTOKO (Station (Nave et al., 1985; P. Bendall, *J. Bordas, %2) software M. H. C. Koch & G. R. Mant, EMBL Hamburg and SERC Daresbury Laboratory, unpublished software). After normalization based on the ion chamber reading (monitor positioned between the sample and the detector tube). the buffer background was subtracted from the sample. The resulting curve was normalised using the detector response measured using a uniform radioactive source. Data were transferred to London for final analyses using an interactive program SCTPL2 (Perkins & Sim, 1986; A. S. Nealis & S. *J. Perkins, unpublished software).

(d) Data analysis In a given solute-solvent contrast, the radius of gyration R, is a measure of structural elongation if the internal inhomogeneity of scattering densit,ies can be

1348

et al.

8. J. Perkins

Table 1 Composition Mouse IgM

Human IgM Total Ig folds (not including the *J chain) Amino acids: residues volume (nm3) Carbohydrate: residues volume (nm3) weight o/0 Total: M, (1O-3) Azso (l’$&, 1 cm) M, ratio (seq.) (ew) (exp.)

Human Fc,

and its fragments Human IgM-8

Human Mouse Fab’, Fab’, __-__._-.

Human Fab

Mouse Fab

70

70

20

14

10

10

4

4

7926 1112

7967

2766 384

1558 218

1096 154

1136 158

427 60

424 59

420 83 20

103 21

45 9

65 13

10 2

20 4

10

7

9

4

ri

I29

136 @726 147

49 0729

.50 0724 17.0

1115

523

623

103 10

123 12

966 0724

F Wig)

of IgM

986 0724

12.5

136

1a0

84

1.02

@39

1.02

0.30 034

190 14Cl

379 0712

190 0725

0727 13.7

12.7 @20 0.20

013

0.18

017

153

0.13

0.05

045 0.1 1

023 016

0.05

0.06

The experimental M, ratios were obtained from 2 independent beamtime sessions, and normalized relative to IgM as unity. Each value was obtained from the mean of 2 to 4 measurements in all but I case, and are compared with the values calculated from compositions.

neglected. Analyses R, and the forward

at small Q in Guinier plots give the scattering at zero scattering angle

Z(0) (Glatter & Kratky,

1982):

In Z(Q) = In Z(0) - Ri &‘/3. In X-ray scattering, the relative values of Z(O)/c (c; sample concentration) for samples measured in the same buffer during a data session gives the relative molecular masses (M,) of the proteins (Kratky, 1963). This is a control of the data collection. If the structure is elongated, the radius of gyration of the cross-sectional structure R,, and the cross-sectional intensity at zero angle [Z(Q).Q]o+O are obtained from:

ln[Z(Q).Ql= ~n[~(Q)~Qla~o - Ris Q2P. For immunoglobulins, the cross-sectional plot exhibits 2 regions, a steeper innermost one and a flatter outermost one (Pilz et al., 1973; Wilhelm et al.. 1984), and the 2 analyses are identified by Rx,-, and Rx,-,, respectively. The R, and R,, analyses lead to the triaxial dimensions of the macromolecule. If the structure can be represented by an elliptical cylinder, L = ,/12(R,Rx,‘), where I, is its length (Glatter & Kratky, 1982). Alternatively, I, is given by nZ(0)/[Z(Q).Q]o,o (Perkins et al., 1986). (e) Molecular

graph& model&g four fragments

of IgM

and its

Models represented by cc-carbon co-ordinates were built up progressively, starting from the smallest Fab fragment, through the Fab’,, IgM-8 and Fc, fragments, to whole IgM, using an Evans and Sutherland PS390 graphics system with the modelling programs FRODO (Jones, 1985) and INSIGHT (Biosym Technologies Inc.. San Diego, CA. U.S.A.). Insertions and deletions in the IgM sequence relative to the IgG crystal structures were considered only in the inter-domain regions, where they would affect the conformations accessible to the model. Amino acid residue substitutions of IgM relative to IgG

were not considered. except for the cysteine residues that form interdomain disulphide bridges in the Fab, Fab’, and Fe, fragments. (i) a-Carbon model for the Fab fragment Three Fab crystal structures with different “elbow” angles (the angle between the pseudo 2-fold axes of the V,-V, and CL-C!,1 domain pairs) were used, i.e. Fab riew. Fab Kol and Fab McPC603 (Saul et al., 1978; Marquart of al., 1980; Satow et al.. 1986). Only that of Fab Kol was used in the modelling of the other IgM fragments. To model the Cpl domain, 3 residues were deleted from the final B-strand fy3 of Cyl (Fig. 2). At the C terminus of (‘IL of Fab Kol, two residues were deleted and t(he preceding 3 residues slightly adjusted. This permitted construction of the disulphide bridge between Cysl40 (($1) and Cys214 (C,) (Ou index) at the end of the 1st /?-strand fxl of ($1. (ii) a-Carbon model for the Fab’, frayncetxt This fragment contains 2 Fab arms held together by t~he disulphide-linked ($2, domain pair. which has no counterpart

in

any

known

crystal

structure

(Fig.

1).

Attempts were made to model it upon the Cy3, pair of IgG Fc. and the Cyl-C, pair of Fab Kol. While the Cy3, pair has S-fold symmetry as expected for ($2,. the 2 a-carbon atoms that correspond to Cys337 (Ou index; Fig. 1) in Cp2 are 2.3 nm apart. Substantial remodelling of the final &strand of both domains would be required to form the Cys337Xys337 disulphide bridge. IgE has an homologous domain pair. In the IgE Fc model proposed by Padlan & Davies (1986), (~~2~was modelled upon Cy3,. However. the Cys337-Cys337 disulphide bridge rather index)

(Ou index) is not made in that IgE model, but the asymmetric bridging with Cys337--Cys246 (Ou is preferred. Since there is no c
position in IgM. the ($2 domains must be bridged between residues 337. The Cp2, pair was more satisfactorily modelled upon

Solution

Structure

the Cyl-C!, interaction of Fab Kol, by superimposing a second Cyl domain upon C,. This model does not have a precise 2-fold symmetry. Since the distance between the r-carbon atoms homologous to residues 337 is 1.1 nm, only slight adjustment of the preceding 2 residues on each chain was necessar.y to allow the Cys337-Cys337 bridge to be built. (1~1 and Cp2 were linked by joining the C-terminal residue of Ql to the N-terminal residue of the Cyl domain used as a model for Cp2. No further insertions or deletions were necessary. While the length of this link between ($1 and ($2 suggested that there would be interactions bet’ween the side-chain residues of these 2 domains. a range of possible orientations of the Fab fragment,s relative t.o the C&?, pair was clearly possible. (iii)

r-carbon

model for the IBM-S

monomer

The IgM-S monomer was generated by linking the Fab’, mode1 wit’h the Cp3 and Cp4 domains based on the crystal st)ructure of TgG Fc (Deisenhofer, 1981). In this crystal structure. t)he 1st residue is Pro238 (Eu/Ou index), which is structurally homologous to Ile345 (Ou index). Fc was located relative to Fab’, such that both their local 2-fold axes were coincident. Seven residues were required to link the Fc fragment to Cys337 (Ou index) at the C terminus of C@. These residues were built both in the moat ext,rnded and most compact conformations allowed. (iv)

u-Carbon

rnodd

for

the Fc,

disc,

A 5-fold symmetric Fr, model was based on the human TgG Fc structure (Deisenhofer, 1981). This was created using explicit symmetry generation within FRODO. Rot&ions about. and translations along, the local 2-fold axis of the Fc fragment were tested. In the final Fc, model. the (‘~4 domains of adjacent Fc fragments made contact with each other between the C-terminal p-strands fy3 of the domain (Fig. 2), i.e. between the edges of the S-stranded Y-faces. The closest approach of a-carbon at,oms. to within main chain to main chain hydrogenbonding distance. occurred at residues 549 to 551 (Ou index: 438 to 440 in c’y3. Eu index). So adjustment of any part, of the (!y3, domain pair was made. and yet there was a striking complementarity between the (~~4 (Cy3) domains as judged by inspection of the a-carbon coordinates when t’heg were packed in the Fc, ring. For example. the loop b3 between strands fyl and fx3 (residues 489 to 494 in Cp4; Ou index; 382 to 387 in Cy3, Eu index: Fig. 2) wraps around the adjacent Cp4 domain towards the x-t’erminal residues of strand fxl. There is also a close approach of residues of loop bl (461 to 466 in (‘~4, Ou index; 355 to 359 in (‘73, Eu index) and loop b5 (530 to 532 in Cp4: Ou index; 419 to 421 in Cy3, Eu index) at the Cterminal end of the domains. This latter interaction. however. is between domains related by a 72” rotation about the .5-fold axis, in contrast to the adjacent domains referred to above (which are related by a 72” rot,ation about the 5-fold axis of the disc plus a 180” rotation about the local 2-fold axis of the individual Fc fragment’). The final symmetric* cc-carbon model of Fc, was constrained also by t.he disulphide bridges between the ($3 domains of Fc fragments around the disc (Fig. 1). These Cys414 residues (Ou index) lie at the begining of loop b5 immediately after strand fx4, and the structurally homologous residue in Cy2 is Leu309. Although this residue lies at the begining of a loop region, which might be conformationally different between TgG and IgM, there is considerable sequence homology between Cp3 and Cy2 on either side of position 414 (309). Furthermore, the

of IgM

1349

structural constraints of strand fx4 before residue 414, and Trp418 after it (Ou index; 313, Eu index; this is an invariant residue in virtually all C domains of all classes) strongly suggests that the structure of Cp2 at Cys414 differs little if at all from that of Cy2 at Leu309. With the packing of the Cp4 (Cy3) domains as described above, and the Cp3 (Cy2) domains disposed relative to them as in the crystal structure of IgG Fc (Deisenhofer, 1981), the distance between a-carbon atoms of Cys414 (in ($3, Ou index; 309 in Cy2. Eu index) was 1.5nm. .In order to reduce this separation to a value typical for a disulphide bridge, it was necessary only to rotate the ($3, domain pair relative to the ($4, domain pair about the local 2-fold axis by approx. 5”, and translate each away from the axis by 0.1 nm. Such small alterations were readily accommodated within the Fc structure. The final distance between t)he Cys414 a-carbon atoms was @66 nm. An asymmetric Fc, model was also generat,ed with the J-chain located between 2 Fe monomers within the disc. The J chain has been predicted to have an 8-stranded antiparallel b-barrel structure (Zikan d al.. 1985). A very similar structure has been proposed independently (Pumphrey, 1986). In view of the close similarity between these predicted structures and an immunoglobulin domain. a constant immunoglobulin fold domain was taken as a mode1 for the J chain. Although this has fewer than the 137 residues of the J-chain, it is sufficient to test an asymmetric Fc, structure by X-ray scattering. It is assumed that the ,J-chain interacts with the (1~4 domains of 2 monomers within the pentamer. Since the J-chain does not have 2-fold symmetry, the interactions with the 2 Cp4 domains must be different. The .J domain was placed between two monomer units, with t.he major axis of its approximately ellipsoidal structure normal to the plane of the Fc, disc. In this orientation the J-chain extends through the full thickness of t,he disc. It is stressed that this positioning of the
a-Carbon

model for

IgM

IgM cc-carbon models were generated Fab’, and Fc, models. The polypeptide

from the final chain that links

S. J. Perkins -

1350

Cp2 and Cp3 was built in both an extended and a more compact conformation, the latter such that contact between the side-chains of the 2 domains would be possible. The disposition of the 5 Fab’, fragments relative to the PC, disc was also varied by rotating the former about their local Z-fold axes, altermg the angle between the 2-fold axes of adjacent Fab’, fragments around the disc (side-to-side movement of Fab’,), and rotating the plane of the Fab’, fragments out of the plane of the Fc, disc (up-and-down movements of Fab’,). (f) Debye sphere modelling of IgM

and its 4 fragments

Molecular modelling of the X-ray scattering curves using small spheres followed previous applications (Perkins & Weiss, 1983; Perkins. 1985; Smith et al., 1990). The co-ordinates were subdivided using a program in which cubes of side 680 nm were included in the model if they contained sufficient u-carbon co-ordinates above a cutoff value, such that the total volume of the cubes equalled that of the dry protein volume calculated from the IgM sequence (Chothia, 1975; Perkins, 1986). Spheres were added to allow for carbohydrate residues, the C-terminal tailpiece, disordered peptides and the *J-chain in order to make up the full volume (Table 1). This employed an interactive graphics program t,hat used a, cross-hair technique to identify and automate the addition of co-ordinates at the appropriate positions in the rectangular grid of IgM sphere co-ordinates, according to the expected positions in the u-carbon structure. Hydration in this model was achieved by resealing the coordinates with the cube root of the volume rat,io of the hydrated and dry models, which is 1.10 for a hydration of 63g H,O/g glycoprotein. The cube side is then 088nm. The curve simulations were based on overlapping spheres of volume @883 nm3, i.e. with diameters of 1.092 nm. This is much less than the nominal resolution of 27~/Q,,,~= of the scattering curves, which is 52 nm for Q,,,= 1.2 nm-’ in this study. Calculations of the R, of the model, the scattering curves, and the goodness-of-fit parameter R followed Perkins & Weiss (1983) and Smith et aZ. (1990). (g) Hydrodynamic

Frictional

modelling of IgM

--

3. Results and Discussion (a) Synchrotron

X-ray IgM

and

scattering measurements its fragments

on

Human pentameric IgM and four fragments of this, the IgM-S monomer? the Fc, central disc, the Fab’, arm, and the Fab fragment’ were studied by synchrotron X-ray scattering (Fig. l), together with mouse IgM and its Fab’, and Fab fragments. The scattering data were analysed in two or three distinct Q ranges in order to obtain the radius of gyration R, and one or two cross-sectional radii of’ gyration Rx,- 1 and Rx,.. 2. Represent,ative curves in Figure 3 for the 14 analyses show that the R, and R,, data were obtained from linear plots in satisfactory QRG and QR,, ranges. Time-frame analyses showed no effects from radiation damage or other exposure-dependent phenomena, in particular. at low Q. As exemplified in Figure 4, no concentration dependence of Z(O)/c was observed. Table 1 shows that, t,he relative values of Z(O)/c agree with the relative molecular masses (M,) of IgM and its fragments calculated from sequences and compositions.

and its fragments

coefficients f are calculated from f= M, where 0 is the partial specific volume, pzo+ is the density of water at 20°C. N, is Avogadro’s constant, and so2,,+ is the sedimentation coefficient. Frictional ratios f/f0 are calculated using fO= 6nqr, in which the macromolecule is assumed to be a hydrated sphere of radius r. The U values were based on sequences and the consensus volumes reported by Perkins (1986). Frictional coefficients were calculated using nonoverlapping spheres by the modified Oseen tensor procedure of Bloomfield (Garcia de la Torre & Bloomfield. 1977a,b, 1981) following Perkins (1989) and Smith et al. (1990). The available computer memory meant that, up to 190 spheres could be employed. Allowance for the void spaces between the spheres, which are not allowed to meant that a hypothetical hydration of overlap, 639 g H,O/g glycoprotein was used to calculate the total hydrated volume, in order to be equivalent to an actual hydration of 630 g H,O/g glycoprotein (Perkins, 1989a). After the direct conversion of the dry a-carbon coordinates into 10 to 188 spheres for the IgM model in question (Table 3), the sphere co-ordinates were multiplied by 1.39 to allow for both hydration and the nonoverlap of the spheres, in which the factor of 1.12 is the contribution resulting from non-overlap. (1 -~P20,w)lNaS020.w~

et al.

Figure 1. A diagram of the il immunoglobulin domains in the pentameric structure of IgM. The 10 heavy chains consist of the Vu, C&l. C&2. C,u3 and Cp4 immunoglobulin fold domains, each of which is represented by a semi-circle and 2 or 5 dots for the internal disulphide bridge. The 10 light chains consist of the V, and C, domains. Pairs of heavy chains are disulphide-linked between the Cg2 and Cp3 domains (2 dots: Cys337Cys337, marked 337). and the light chains are disulphide-linked to the Cpl domain (5 dots, CysL214-Cysn140, marked 140). Adjacent Cp3 domains are cross-linked by Cys414-Cys414 disulphide bridges (broken lines, marked 414); as are the adjacent Cp4 domains (Cys575Cys575, marked 575). The ,J chain is arbitrarily located at the centre of the pentamer between 2 Cp4 domains. and contains 8 Cys residues. The 5 oligosaeeharide sites per human heavy chain are represented by filled circles. The 6th oligosaccharide found in the V, domain of mouse IgM is depicted as an open circle. The Fab fragment, or arm, is constructed from the V,, (1,. V, and Cpl domains. The Fab’, fragment is constructed from 2 Fab arms and 2 Cp2 domains. The pentamerir Fc, fragment, or disc, is constructed from 2 ($3 and 2 ($4 domains in each of 5 Fc fragments, together with a ,J chain. The IgM-S monomer consists of a Fab’, fragment and a Fc fragment.

Solution Structure of IgM

X-face

I

I

1351

between approximately 2 to 30mg IgM/ml. The data were reproducibly observed in two to four sessions, and the mean values are reported in Table 2. For human IgM,, the R, of 12.17 nm is in good agreement with those of 12aOnm and 12.1 nm reported for human Waldenstrom IgM,, and IgMo,, by Wilhelm et al. (1978, 1980) after deconvolution by Glatter’s method. It is slightly larger than that of 11.5 nm for human Waldenstrom IgM,-, (Kayushina et al., 1986). For human monomeric IgM-S, the R, of 610 nm is slightly larger but is within error of that of 5.9 ( +@2) nm reported by Wilhelm et al. (1984). The IgM-S Rxs values of 254 nm and 1.70nm are also within error of the values of 2.6 ( f63) nm and 1.8 ( +02) nm given in that study. Mouse and human IgM differ in that mouse IgM possesses an additional oligosaccharide site on the Vu domain of the Fab fragment (Fig. 1). Comparison of the RG and Rx, data on the Fab’, and Fab fragments of mouse IgM with those for human IgM shows that they were very similar (Table 2). The R, for mouse IgM,, is slightly larger at 12.47 nm than that for human IgMou, but is within error (kO.4 nm). The R,IR, ratio indicates the anisotropy or the degree of elongation, where R, is the R, of the sphere with the same volume as the protein. Table 3 shows that, with the single exception of Fc,, the R,/R, ratio increases from 1.53 to 2.43 as M, increases. It is therefore concluded that the solution structure of IgM consists of an extended arrangement of its domains as expected from electron micrographs of free IgM in vacua (Feinstein et aE., 1986). The length L, of the longest macromolecular axis can be calculated by two methods, assuming that the macromolecule is an elliptical cylinder in shape.

Y-face

Figure 2. A diagram of the j-sheet topology in the immunoglobulin fold of a constant domain. The 7 a-strands are labelled using the letters A to G in the & Barclay, 1988), and the Oxford convention (Williams letters X and Y in the Cambridge convention (Beale & Feinstein, 1976). The intradomain disulphide bridge within each Ig fold (Fig. 1) connects the strands fx2 (B) and fy2 (F). The 6 bends are numbered bl to b6 in the Cambridge convention.

The scattering curves thus correspond to monodisperse preparations, which can now be analysed in molecular terms. Dilution series for the structural RG and Rx, parameters for human and mouse TgM and their fragments show no dependence on concentration

Table 2 Guinier analyses of IgM and its fragments RG

Protein Human IgM Mouse IgM Human Fr, Human IgM-S Human Fab’, Mouse Fab’, Human Fab Mouse Fab

&s.l

trim)

Q (nm-‘1 1217+0.34 607-0.14 1247f035 907-0.14 615*0.26 0.1 l-0.20 61OkO36 WlW20 493 + 0.09 OlW25 482fO.12 0.1025 2.94fo.09 0.17-O-45 290*911 @17-0.45

&.z

(nm)

(7) (11) (8) (6) (4) (5) (5) (4)

6.06+@19 0.144.23 t?o9+0.27 014-0.23 316kO.10 0.360.52 254kO.12 0.25439 2.36 f 094 0.28-054 2.41+@05 92SO.54 1.57 f 0.04 0%&@95 1.59+903 0+@@95

4

(nm)

(nm)

RG

IPI)

(13)

n.c.t

n.r.

(12)

n.c.

n.c.

(14)

17.9

(18.7)

(‘3)

192

(%1.4)

(5)

150

(161)

(4)

14.5

(154)

(5)

84

(8.5)

(4)

34

wa

Q @m-l)

Q @me’) (13) (13) (15) (6) (5) (4)

1.79+0.12 049160 1+38+@11 o-491.00 202 * 0.07 97G160 1.70+@02 0.4.5083 1.41 kO.02 0~6&190 1.41 kO.03 060-190

The RGand Rx, values are the mean + standard deviation of a total of measurements (in parentheses) in the concentration range shown in Fig. 4. The Q range used for fitting is shown beneath each R. or Rx, value. The L, values correspond to the longest dimension of the macromolecule calculated from either the RG(assuming an elliptical cylinder) or the I(0) parameters, in parentheses (assuming an elongated structure: Materials and Methods). t Not calculable (n.c.) (see the text).

S. J.

1352

Perkins

et, al.

1

100 003

cm8

0.16 o’(nm-5

0.2,

0.32

5.0 0.00

0.0

0.80 02(“m-2)

1.20

1.60

Figure 3. Representative synchrotron X-ray Guinier fl, and I?,, plots for human I~ikl and the 4 fragmwlts of human IgM; namely, Fe,, IgM-S, Fab’, and Fab. The filled symbols between the QR, and &&,, ranges as arrowed show thr data points used to determine R, and Rx, values. The & ranges are summarized in Table 2. At low & values. the Rx,_, plots show the same data points as those used in the R, plot)s to the left. In the Rx,_, plots, the data points at low Q havtx heen truncated for clarity. Each data point in the 4 Rxsm2determinations and the Rx,-, determination for Fab are shown as the mean of 3 adjacent points for clarity. Both calculations (Table 2: Materials and Methods) are in good agreement for the Fe,, IgM-S, Fab’, and Fab fragments of IgM. They are in divergence for IgM itself because of its non-cylindrical structure, and are not reported. These solut,ion data are consistent with the arrangement of domains schematically shown in Figure 1. (1) The mean L, values for human and mouse Fab’* of 153 nm is less than twice the L, of 8.4 nm for Fab. Since Fabr2 contains the ($2 domains as well as two Fab fragments, this shows that the long axes of both Fab fragments are not, colinear within Fab’,.

(2) For

IgM,

a maximum dimension c)t’ 35 IO and IgMGAL has been reporkd from the distance dist,ribution function (CVilht:lm cut d.. 1978, 1980). Since this is almost double the L, value of 19.2 to 21.4 nm for the IgM-8 monomer (Table 2) and its maximum dimension of 21 nm (Wilhelm et al., 1984), this is consistent with a circular arrangement of IgM-S monomers within IgM pentamers. In electron microscopy studies of IgM and Fc, (Feinstein & Munn, 1969; Munn et nl., 1980: Kellenberger & Villiger, 1981), the diamet’er of 1gM was reported to be 30 nm, and t,hat for Fc:, was

37 nm for IgM,,

Solution

Structure

IO.0 IgM 8.0 cI

n

u

.

6.0

5

Concentrahon

(mg/ml)

Figure 4. Concentration dependence of the Z(O)/c parameters for human and mouse IgM and their fragments. The vertical scale is arbitrary. Open symbols, human IgM; filled symbols. mouse IgM. The data correspond to those measured on Station 8.2 in the final session (Materials and Methods). The 1(0)/c data are represented for IgM-S (0) and Fab’, (A). Comparisons with the relative 1(0)/c values expected from the molecular weights are given in Table 1. The corresponding R, and Rx, parameters for human and mouse IgM and their fragments also shows no concentration dependence (data not shown). The R, and Rx, data were measured for up to 26 scattering rurves of between 1 t,o 23 mg IgM/ml in 4 sessions, while those for IgM-S. Fr,. Fab’, and Fab were measured in 2 or 3 sessions (Table 2).

1Onm. As determined by solution scattering, the maximum dimension of IgM is 35 to 37nm (Wilhelm et al., 1978, 1980) and that for Fc, is 17.9 to 18.7 nm (Table 2). Using a recently installed quadrant detector with a larger available Q range at Station 8.2, the calculation of the distance distribution function by Glatter’s Indirect Transformation method for Fc, was possible and is compatible with this diameter. The electron microscopy data appear to underestimate lengths, probably because of stain penetration. Improvements in preparation techniques have led to more precise measurements using about 100 electron micrographs of mouse IgM MOPC 104E (E.A. Munn, personal communication). About one-quarter of these micrograph images would fit a circle of diameter 37 nm, while the remaining t,hree-quarters would fit an ellipse of axes now agree with 39 nm x 35 nm. These dimensions those from scattering. The Fc, discs in these images of IgM are such that 20% would fit a circle of 14 nm diameter, while SOY/, would fit an ellipse of axes 15 nm x 13 nm. These dimensions are smaller than those from scattering; however, these do not take account of the residual peptide from the Cp2 domain (Materials and Methods, section (a)), In conclusion, present and previous solution data for IgM and its fragments are consistent with the extended, multi-armed structures suggested by electron microscopy (Feinstein & Munn, 1969; Munn et al., 1980; Davis et al., 1988). The present data can be used to develop models of the IgM structure. There is very good agreement in the Guinier Ro and R,, analyses for human and mouse IgM and their corresponding fragments (Table 2).

1353

of IgM (b) Scattering curve modelling and its fragmentn

(i) Curve $tting

oj’ IgM

for the Fab fragment

Scattering curve simulations were based initially on Fab to establish the method, since its structure is crystallographically well defined. Three crystal structures for Fab New, Fab Kol and Fab McPC603 were used (Materials and Methods, section (e)(i)). The elbow angle of Fab Kol is 170”, an almost linear arrangement, of the V and C-domain pairs, while those for Fab New and McPC60.3 are 137” and 133”. respectively. These structures represent almost the two ext.remes of elbow angles observed in Fab fragments. After addition of hydrogen atoms (Perkins. co-ordinates were 1982)> the R, of the crystal 2.55 nm (Kol), 2.46 nm (McPC603) and 2.44 nm (New). The largest RG corresponds t’o the largest elbow angle (Schiffer et al.. 1982). These were converted into 113 t’o 119 cubes of side 680 nm to define the sphere models. While the R, of the sphere models agreed with the crystallographic RG values to within 0.02 nm, all of these R, values were less than the experimental ones of 2.90 to 2.94nm (Table 2). Three further factors are involved (Smith et al., 1990). (1) The one or two oligosaccharide chains (Fig. 1) were represented by four additional spheres, but are of unknown conformation. An extended oligosaccharide conformation of length 46 nm increased the R, by 0.11 nm or 028 nm for one or two oligosaccharides. Compact conformations in contact with the protein surface increased the R, by only about) @02 nm. (2) If the protein is hydrated by 0.1, 0.2 and 0.3 g H,O/g glycoprotein (Materials and Methods), then the R, is increased by 069nm, 0.17 nm and 0.25 nm in that, order. (3) Hydrophobic amino acid residues in a central core have an electron density of 399 e nmm3, while surface hydrophilic amino acid and carbohydrate residues, and the water shell have one of 431 to 439 e nme3. These are significantly different in comparison to a solvent density of 334 e nm m3. The R, of a two-density model is increased by 0.075nm compared to a single-density model. Incorporation of t,hese factors resulted in a satisfactory curve fit for Fab with an R of 0007. This was obtained for a 117.sphere model based on the mean of the Fab Kol and Fab McPC603 crystal structure, as detailed in Figure 5(a). The R, of these models were 2.85 nm and 2.72 nm, respectively, which agree well with the experimental R, of 2.9 (kO.1) nm (Table 2). Good fibs were also obtained for the other eight available experimental mouse and human Fab curves. (Crystallographic data can thus readily account for t)he scattering curve of the Feb fragment. For reason of simplicity in the Fab’,, Fe,, IgM-S and IgM simulations. the degree of hydration was used as an empirical variable t,o allow for a range of possible oligosaccharide conformations, hydration levels and electron density distribut.ions. All these

1354

S. J. Perkins

et al.

Fob

‘5nm

2 o-0

o-4

O-8

I-2

’

’

5nm

’

I.6

Q (nrn-‘I

(a)

8

0.0

o-4

0.0 Q (nm-‘I

(b)

8

2 o-0

o-4

0.8

1.2

1.6

Q (nrn-‘1

cc 1

Q (nrn-‘I

(d)

8

Fig. 5.

models involved physically realistic 91 to 92 g H,O/g glycoprotein. (ii) Curve jitting

hydrations

of

for the Fab’, fragment

In the modelling of the Fab’, fragment, the position of each Fab arm relative to the Cp2, domain pair has to be determined, as well as the angle between the two arms. The effect of rotation of each arm about its own long axis could not be detected. Molecular graphics a-carbon models were generated and converted into spheres in order to explore these possible conformations. Starting with a reasonable

but arbitrary symmetrical orientation of’ the E’ab fragments relative to Cp2,, the inter-arm angle was varied between 0” and 180”. For an inter-Fab angle of 50”, Figure 5(b) shows that the simulated and experimental curve (R of 0.019) deviated beyond & even though this model reproduced of 0.25 nm-‘, wide-angle the experimental R, value. Improved curve fits resulted from an inter-Fab angle of 90”, although the R, of these models were unsatisfactory. This showed that the simulations were sensitive to the relative dispositions of the three fragments.

Solution

0.4

O-0 Q (nm-’

i-2

Structure

of IgM

1355

l-6

1

(e)

8

o-0

o-4

0.8

1.2

l-6

0 (nm-‘I

o-0

o-4

0.8

(f)

l-2

I.6

Q (nm-‘I

(g)

8 6

0 G C

4

o-4

0.8

I.2

Q (nm-‘1

(h)

Fig. 5.

The final Fab’, model was derived from testing 69 possible sphere models. The inter-Fab angle was fixed at 99”. One Fab arm was fixed in position, while the C,U~, domain pair and the other Fab arm were moved in one-sphere increments. This trialand-error search gave structures that agreed well with the experimental curve and R, value. In these, the two Fab arms were as close to each other as possible and to the Cp2, domain pair in the same plane. This result was used to create the final a-carbon model and sphere model that gave the good curve fit shown in Figure 5(c) with an R of

9012 and an R, of 476 nm in good agreement with the observed R, value of 4.8 to 4.9 (kO.1) nm. This curve simulation was also consistent’ with eight other available experimental curves, although minor alterations in the position of the Fab arms could not be ruled out. (iii) Curve jltting for the IgM-S monomer The modelling of the IgM-S monomer was aimed at an assessment of the contact made between the Cp2 and ($3 domains starting from the above structure for Fab’,. Figure 5(d) showed a good curve fit

A’. J. Perkins

1356

et al.

I 0.0

0.4

I.2

0.8

Q(nm

1

Figure 5. Comparisons of the curve simulat,ions for hydrated Debyr sphere models of human alid mouse Ig1l and t heit, fragments wit,h the experiment,al synchrotron X-ray sea&ring curves. For each model. t,he z-carbon skelet,orr of’ the immunoglobulin folds used in the curvt‘ simulation are shown. viewed along the 5* axis. The unhvdratrd IJrbyt~ spheres models are shown in the same view and scale. Additional spheres allow for the carbohydrat,e moieties (and the .I-chain in the Fc, and TgM models). The number of ticks inside each sphere corresponds t)o the total of spheres superimposed on each other in projection. The direction of each tick corresponds to the z co-ordinate. with Id o’clock bring the starting 2 co-ordinate at) low z. (a) The Fab model is based on the a-caarbon co-ordinates for Fab Ii01 and Fab JlcFY’603 (elhon angles of 170” and 133”. respectively). The two hydrated Debgr models (117 and 123 spheres) from these wire rquall~weighted to give the curve fit as shown. losing an extended single-oligosaccharide structure, a hydration of O-2 g H,O/g glycoprotein, and a single density. Experimental R, 2.9 nm, simulated H, 2.X nm. K of 0007. at a sample c~ottcent~ratiorl of 194 mg Fab/ml. (b) An initial Fab’, model shows the sensitivity of the curve fitting t.o an arrangement of the (‘+ domains with a wide separation of the 2 Fab domains within the strurturr. Total of 306 spheres: experitnental tZ, 4.9 nm simulated Ro 49 nm, R WO19. at a satnplr concentration of 6.9 mp Fab;/ml. ((3) The final Fab’, tnodel sham-s the improved curve fit obtained when the 2 Fab arms are moved together. Total of 303 spheres, hydration of 0.2 g H,O/g glycoprotein, experimental RG 4.9 nm, simulated RG 4% nm. R of 0012. (d) The IgM-S model is based on I-4 Ig fold domains arranged as in the final Fab’, model and the Fc cbrystallographic model. Total of 466 spheres. hydratiort of (k I g H,O/g glycoprotein, experimental RG 6.1 nm. simulated RG 6.3 nm. R of 0.010. at, a sample concertt,ration of 14.4 “1,~ IgM-S/ml. (e) The Fc, model is based on a 5-fold symmetric arrangement of thr a-carbon atoms in 20 (‘~3 and (‘/l-l domains. to which extended polypeptide chains are attached to correspond t.o the peptide PIravage sitr in ($2. Totaal II~ 904 spheres. hydration of@I g H,O/g glycoprotein. experimental RG 61 nm. simulated R, 6.0 nm. I)ata are based on thrs use of the linear detector (upper curve: R of @029; at a sample c~oncentration of 18% tng Fc,/ml) and t hr quadrant detector (lower rurve R of 0.021 out to 0 = 0.09 nm-‘. at a sample conrentmtion of X.4 mg Fc,/ml). (f) Thr Fv, modt~l is based on an asymmetric arrangement of the a-carbon atoms in 20 (‘~3 and (‘~4 domains. Thr .I-(*hain is positicmtrl between 2 Cp4 domains as arrowed. Spheres were added as in (e). Total of 911 spheres. hydration of 0.1 g H,O/g glycoprotein. experimental R, 6.1 nm. simulat,ed R, 6‘2 ntn. R of’ 0.011 (upper curve) and OW8 (lower curtt’). (g) ‘I’htA symmetric planar TgM model is constru&ed from the planar arrangement of 5 TgM-9 models from (d). whit+ follows t,he asymmetric Fcg model of (f) with the ,J-chain positioned within the Fc 5 ring as arrowed. Total of 2360 spheres, experimental R, 12.3 nm. simulated R, 12% run, K of W30. at, a sample conc,rntration of X.5 mg [gM/ml. (h) 7’11~ asymmetric IgM model is constructed from the sphere model in (g). and atnrndetl by the 45” rotation of 2 Fab’, unit,s to one side as arrowed. The double-headed arrows signify that side-to-side tlispla~t~mt~nt (‘an o(*(‘ur for any of t ht. Fab~, units. Total of 2360 spheres, hydration of @I g H,O/g glvqoprotein. experimental /tG 12.3 nm. simulated K, 12.3 rrnl. I? of 0.019. (i) A symmetric IgM model with the .5 Fab’, units from ((a)set perpendicular to the plane of tht, Fc~, disc.. Total of 2363 spheres, experimental KG 12.3 nm. simulated I& I I.5 nm. K of 0.041.

based on the Fab’, structure of Figure S(c) and t,he Fc crystal struct.ure: provided that the Op2-($3 contac&ts were made cornpart. This was confirmed by comparisons with five other experimental curves measured for TgM-S. and tests with other Fab’, models with similar inter-Fab angles based on compact (:pLa-(:p3 contacts. The KG of this model is 626nm, in good agreement, with an experimental K, of 6.1 (+04) nm (Table 2). and K is 0.010. Models with the most rxtended Cp2-Cp3 arrangement possible led to R, values that were at least 0.3 nm higher, and worsened (WPW fits with K between 0.011 and 0.014. However. ot)her arrangrrnents based on compact CpLB-Cp3 contacts in which either the Fc was rotated by 90” about its local 2-fold axis or the two ($4 domains were separated from each other by about 2nm (to test, whether these domains might not be closely associated) also gave comparable fit.s with R between 0.009 and

O+l I, and caarinot Ir)tJ ruled out. In summary. I hc> modelling of lyl\;lGS is consistent uith the Fal)‘, modrlling. and is also sensitive to the tlistarlc~f~ bet)ween t,hcaFC and Fab’, fragments. It is of int)cr.est t,hat. while Wilhelm it CC/. (1984) also tleducA an inter-Fah an@> of 90” from their studies of TgMGS. in agreementj wit’h our results for Pah’, and IgSS. the proposed extra loose rings of spheres in’thrir Tghl-5 model were not required here in order to obtain a good curve fit,

The curve simulations for Fc, were limited by the constraints inherent in the molecular graphics packing of the five Fc fragments into a ring (Materials and Methods). The symmetric Fc, model is discoid with a diameter of approxirnatelg 15 nm. and a thickness of approximately 4 nm. The addi-

Solution

Structure

tion of the J-chain in the asymmetric Fe, model between two adjacent Cp4 domains distorts the Fe, disc into an approximately elliptical shape with major axes approximately 17 nm x approximately 15nm. While the dimensions of these FcS models were in reasonable agreement with the above recent electron microscopy estimates of 15 nm x 13 nm, the aim of the curve modelling was to distinguish between these alternative models for the location of the J-chain. To evaluate the symmetrical Fc, model, the a-carbon co-ordinates were converted into 646 spheres. A further 120 spheres were added to represent the 30 oligosaccharides on the Cp2 and Q3 domains on the ten heavy chains (Fig. I). There is a central hole of diameter approximately 3 nm (Fig. 5(e)), in which the C termini of the ten p chains, their associated carbohydrate chains and the J-chain are located. The total volume of these additions is 67 nm3, that is 131 spheres. Only 52 of these 131 spheres could be inserted into this hole. If the remaining 79 spheres were placed above and beneath t’he centre of the pentamer to form two central lobes of height 1.6nm each (Fig. 5(e); t.hird panel). This model has an R, value of 6.0 nm, which agrees with the experimental R, of 6.1 (kO.2) nm (Table 2). However, R is high at 0.029, and the experimental and simulated curves deviate beyond & of 0.6 nm-‘. This deviation is clearly seen with the use of the quadrant detector, which gave enhanced I(&) data at large & (Fig. 5(e)). In edge-on views in electron micrographs of Fc, discs (Feinstein & Beale, l977), t)here is no evidence of a central lobe, a,nd certainly not one of thickness 7.2 nm. compared with 4.Onm for the rest of the disc. A second symmetrical Fe, model was created in which the height of the two central lobes was reduced t,o 0.8 nm and the lobes were extended over the Cp4 domains. This corresponded to a possible external position for the tailpiece peptides on the surface of the (‘~4 domains and a centrally located .J-chain. Improved curve fits were not found. In the asymmetric Fc, model, the central hole became enlarged to dimensions of approximately 4 nm x approximately 3 nm. In this sphere model. X9 spheres were now required for the tailpiece glycopeptides. These were readily incorporated into the hole without any excess. The R, was increased to 6.2 nm as the result of inserting the J-chain into the Fe ring. and this agreed well wit,h the experiment.al va,lue of 6.1 (kO.2) nm. The residua,l K was much improved at 0.011, and good agreement’ was seen between the experimental and calculated curves in the & range between 0.6 and 1.0 nm-l, in particular using the data. collected with t.he quadrant detector (Fig. a(f)). Of I2 scattering curves measured for Fr,, 1 I led to better fits for the asymmetric Fc, model t.han for the symmetric one. The asymmetric model was therefore preferred. This model resembles tha,t reported by E’umphrey (1986), alt.hough we have not analpsed how the ,J-chain might interact with adjacent IgM subunits. nor how the disulphide pairing might be organised.

of IgM

1357

In all these models, the conformation of the C,U~ polypeptide extensions left after tryptic Fc, preparation (Materials & Methods, section (a)) was considered. In Figures 5(e) and 5(f), these are shown in an arbitrary, extended conformation which gave the best agreement with the observed R, of the Fc, preparation. This is not) intended to represent a probable solution conformation. However, less extended arrangements of these Cp2 polypeptides led to smaller R, values a.nd worsened agreements. (v)

Curve

fitting

for lg M

Models of the full IgM pentamer were constructed. The arrangement of the two Fab and the c~L2~ moieties in Fab’, and IgM-S (Fig. 5(c) and (d)) was retained in IgM. IgM models based on bot’h the symmetric and asymmetric Fc, models have an overall diameter of approximately 36 nm, which is in excellent agreement with the estimates of 37 nm from elect’ron microscopy and solution scattering. IgM models in which the plane of the Fab’, unit is coincident with the plane of the Fc, disc gave much improved fit.s t.o t,he experimental data (K of 0.030: Fig. 5(g)) compared to models in which the two planes were perpendicular to one another (R of 0041; Fig. 5(i)). All IgM models with symmetric or asymmetric Fc, discs gave rise t’o a curve simulation with a poor curve fit caused by an inflexion close to Q=0.4nmm* ( Pi g. 5(g)), although slightly improved curve fits were obtained with the asymmetric Fc, structure. This inflexion was also visible in earlier IgM curve modelling (Wilhelm ef a,/., l!#O). Attempts t.o remove this inflexion in models in which one, two or three Fab’2 fragments; initially disposed as in Figure 5(g), were rotated up or down out of the plane by 45” at, the conne&on between the Cp2 and Cp3 domains, left the R of t,he simulations unchanged at 0030. No information is therefore available on this extent moderate of conformational change by solution scattering. However: similar angular movements by 90” of all the Fab’, arms caused large departures from the observed scat.tering curves (Wilhelm et al., 1980). \Vhile this conformational change could be ruled out for free ZgM in solution, this TgM staple conformation is seen in elect)ron micrographs of IgM molecules when bound t,o flagellae (Feinstein & Munn, 1969), and is clearly a result of combination wit,h antigen. The inflexion could be removed in planar IgM models in which one, two or three Fab’, units were rotated sideways by 45” in the plane of the Fc, disc. Similar sideways rot,ations of the Fah’, units in the model of IgM in which the plane of each Fab’, is perpendicular to the plane of the disc resulted in less successful curve fits. The best agreements (R=0.019) were obtained with the asymmetric Fc, struct,ure and the sideways rotation of two Fab’, units (Fig. 5(h)). This gave an KG of lP3 nm, in agreement wit’h the experimental R, of 12.3 ( + 0.3) nm. This int’erpretation is sqjported by

S. J. Perkins

1358

noting that the peak of the inflexion at &= 0.44 nrn-’ in Figure 5(i) corresponds to a distance of 14 nm, which is close to the mean distance between two neighbouring Fab’, units in the IgM models in Figure 5(g) and (i), and to the maximum at 15 to 16 nm found in the distance distribution function calculated for these theoretical curves. The intensity of this maximum is increased on going from the model of Figure 5(h) to that of Figure 5(g), and is much increased in the model of Figure 5(i). In the model of Figure 5(h), the sideways rotations of the Fab’, units cause these distances to be distributed more evenly. In conclusion, solution scattering is sensitive t.o the side-to-side displacement of the Fab’, units relative to Fc, in IgM, and shows that these IgM conformations exist in solution. The IgM solution structure is best viewed as a family of structures in which any one Fab’, arm is rotated to one side or the other by up to 45” at any given instant. Explicit calculations of all possible conformations for t’he five Fab’, arms, and the averaging of their scattering curves, were not performed for reason of the approximate 5-fold symmetry of this model. This means that the models tested above correspond closely to this average. Solution scattering was able to rule out IgM structures in which the plane of t’hr Fab’, fragment is perpendicular to that of the Fc, disc (Fig. 5(i)). However. it was insensitive to limited rotations of the Fab’, units above and below the plane of the PC, disc, so no conclusions can be drawn about the existence of such IgM conformations in solution. (vi) Confidence

in the scattering

models

The extent to which the constrained modelling procedure based on molecular graphics represents a safe interpretation of the solution structure can be judged from the number of parameters that can be determined. This is given by (D,,, &,,,/n)+2 Gn the Sampling Theorem (Luzzati & Tardieu, 1980). in the macrowhere D,,, is the largest dimension molecule and can be set as L, (Table 2). The nominal resolution of the scattering curves extends generally to 5 nm at &,,,= I.2 nm- ‘. Error-free data are assumed. Here the repeated measurement of the scattering curves increases the confidence level of the final result. (1) For the Fe, and Fab fragments, eight and five parameters. respect,ively. are available. Since these fragments are well defined, the structural analyses are over-determined. The good curve fits for Fc, and Fab therefore substantiate the modelling procedure used for all the systems. The asymmetric Fc, model could be distinguished from the sytnmet,ric model, and the former favoured. (2) For the Fab’, and IgM-8 fragments, seven and nine parameters, respectively, are available. If the Fab’, fragment is constrained to be planar with three subunits of defined structure, four parameters will define its structure (i.e. the distances and angles of the two Fab arms relative to the Cp2, domains).

et al.

Models in which the two Fab arms were positioned close to each other by the Cp2, domains satisfied the scattering data. In the IgM-S analysis, one parameter defines the distance of t’he F(+ fragment relat.ive to the Fab’, fragment (which was fixed in its structure). A compact location of t,he Fe fragment relative to the Fab’, fragment agreed with the scattering curve. Even though only a family of possible structures could be assessed. thr principal features of these t)wo IgLM fragments in br determined. (3) For ZgM, I4 xtruct.ura,l paratneters are acailable. If each Fab’, fragtnent is considered to bet a rigid entity, at it fixed distance from the Fe, disc,. 15 parameters will be required to define the sidts-to side. up-and-down. and axial robation of all tivtb Fab’, fragments relative to Fc,. While this system is apparently under-determined. ea,ch of the t,hrpc’ rotations was considered separately. Only the large side-to-side rotations involving t’wo or t hrre Fxb’? arms in the plane of the Fv, disc rrsulted in good IgM curve tits for IpM (Fig. 5(h)). and this is a wt>ll defined result, (c) Hydrodynamic

rnodelling ,fmgvwnts

of lgN

arm! its

SedimentaGon data for human and mouse IpSl and it,s fragments are reported in Table X. (Wculation of the frictional ratios .f’/i(, (Materials and Methods) show t,hat these increase in lint, with the macromolecular M,. A linear retationshif) ratios id not stsrn between thr f/f0 and l&/K, (Perkins. 1989~~). This shows that t,hr sedimentation dat’a act as an independent monitor ot’the structures. Two strategies fhr hydrodynamic rnodelling wt’rth followed. In the first. all the dry I)ebyr models for IgM and its fragments were directly converted in1 o hydrodynamic models. which were then correetetl

y---

-IgM-S

Fab

Fab @? CL

Figure 6. Hydrodynamic models for 1gM ad its i’ragments. These were individually calculated from the Debye models shown in Fig. 5(a). (c). (d), (f’) and (h). and employed for the calculations of Table 3. All are drawn to the same scale. The sphere diameter is 2.302 nm.

1359

Solution Structure of IgM

Table 3 Sedimentation data for IgM and its fragments

Protein Human Mouse Human Mouse Human Mouse Human Mouse

IgM IgM Fc, Q,U IgM-S P8 104E Fah’, (pepsin) Fab’, (pepsin) (papain) Fab (pepsin) Fab (papain)

ROIR, ratio

flfo

ratio

Hydrated volume (nm3)

239 243 1.64 n.d.$ n.d. 1.90 1.82 n.d. 1.56 1.53

1.78 1.77 1.60 1.47 1.47 1.36 1.41 1.39 1.14 1.17

1609 1641 621 315 315 215 227 227 82 83

t The simulations (Fig. 6) are based on hydrodynamic molecules are listed in order of decreasing M,. $ Not determined.

for hydration (Materials and Methods; Fig. 6). In the second, the resulting hydrated IgM model was subdivided to form the fragments. The results were insensitive to the method employed, and in the case of IgM also to the side-to-side displacement of the Fab’, fragments. The calculated sozo,w values are almost all within (+0*2 5) of the experimental values in Table 3, which is the expected accuracy of the calculations (Perkins, 1989a). Errors on the experimental data are estimated as (kO.2 S). While the calculated soz,,w for IgM of 17.7 S is at the limit of error, the use of a slightly reduced hydration of 0.3 g H,O/g glycoprotein (Materials and Methods) gave a good sozo,+,calculation of 18.1 S. These agreements show that the sedimentation data are fully consistent with the modelling of the X-ray scattering curves. (d) (Slycosylation

of the

IgM model

The locations of carbohydrate attachment sites (Fig. 1) in the final molecular graphics a-carbon model for IgM were examined. In the C,U~, pair, Asn332 (Ou index) is fully solvent-exposed as required. In Fc,, Asn402 (Ou index) is homologous to Asn297 in IgG Fc, and carbohydrate is expected t’o cover much of the four-stranded X-face of Cp3 (Fig. 2). This suggests that the same disposition of ($3 domains will be found in Fe, as for the Cy2 domains in IgG. In Fc,, the glycosylation site at Asn395 (Ou index) lies on strand fx3 of Cp3 and is totally accessible at the edge of the four-stranded X-face in the pentamer (Fig. 8(a)). In mouse IgM, glycosylation occurs at the nearby Asn364 (Ou index), and this site is also accessible at the beginning of strand fx2. These observations, together with the ease of docking the Fe subunits into a ring and connecting the Cys414-Cys414 disulphide bridges in Fcg (Materials and Methods), and the good agreement with dimensions calculated from solution scattering data and electron microscopy all support the molecular graphics modelling of the Fc, central disc of IgM.

Total of spheres?

Experimental s020,w (S)

188 (188) (ii) (39) (2,

17.7 (17.7) 11.4 (7.4) (7.4) (ii)

(25)

w4

(&

37 (3.7)

spheres of diameter

Simulated s”~~,~ (6) 182 18.6 11.35 7.35 7.35 6.13 6.16 6.28 3.80 3.83

P302 nm. The macro-

(e) Zdenti$cation of a possible Clq binding site in IgM IgM and IgG are able to activate complement by the classical pathway by binding with Clq of complement. Whereas IgG aggregates complexed to antigen are required for activation, a single molecule of IgM complexed to antigen is all that is required (Borsos & Rapp, 1965a,b; Ishizaka et aE., 1968). The interaction between Clq and both IgG and IgM is electrostatic, and binding is strongly dependent upon ionic strength (Hughes-Jones, 1977; Poon et al., 1985). When Clq binds to IgG aggregates, 9 to 12 salt ions are released per Clq molecule bound (Burton et al., 1980; Emanuel et al., 1982). Mutagenesis studies (Duncan & Winter, 1988) have shown that the Clq binding site in IgG involves the charged residues Glu318, Lys320 and Lys322 (Eu index) on Cy2, so 12 is the expected total of salt ions to be released upon binding of two Clq heads. When Clq binds to free IgM, six salt ions are released (Poon et al., 1985), and this becomes eight to nine upon binding to cell-bound IgM (Wright et aZ., 1988, 1990). IgM may therefore have a similarly charged binding site for Clq. There is evidence that the Clq site lies in Cp3 of

420

Human (0~) Mouse (104E) Rabbit Dog Chicken

xenopus Catfish Ladyfish Shark

430 440 I I I WDSGERFTCTVTHTDLPSPLKQTISRPK WNNRKEFVCTVTHRDLPSPQKKFISKPN WESGEQFTCTVTHADLPFPLKHTISKSR WESGEQFTCTVTHTDLPSVLKQTISRPK WDGGDGYVCKVNHPDLLFPMEEKMRKTK WN'NLIK.FVCKVEHTELASMKEVFLFKEK WINGTEFICEVEHEAFTQQYEKVTFKRE WKNRTEYTCKVEHSDLPSPLRTSYRREC WLSGAEFYCWSHQDLPTPLRASIHKEE * *

Figure 7. Comparison of the C-terminal regions of 9 Cp3 sequences. The positions of His430 and Asp/Glu432 are marked underneath with an asterisk (*) (see the text). Sequences are those of human, mouse, chicken, dog, rabbit, Xenopus, horned shark, channel catfish and lady-

fish p chains (Kabat

et al., 1987; Schwager et al., 1988;

Kokubu et al.. 1988; Ghaffari & Litman, 1990).

& Lobb.

1989a,b; Amemiya

S. J. l’erki,ns et al.

1360

the Fe, disc, in particular the region around Pro436 (Wright. rt nl.. 1988). Fab’, fragments do not hind to (‘lq or activate complement (Feinstein & Richardson. 1978; Feinst’ein it (~1.. 1983). and a monoclonal antibody to C/.0 blocks complemrntto the other dependent Iysis, while antibodies domains do not, (Leptin $ Melchers. 1983). Tn <@Us,if’

Asp/Glu432

Srr4OB (Ou index) is replaced b>, Asn406. or I’ro43fi is replaced tty Ser436. a&vat ion of’ ~~mplemert t i,q impaired (Shulman rt ccl.. 1986. 19%; M’riyht uf rrl.. 1988, 1990). \t’hilcb the (‘lq binding motif’ of’ 1x(; (Glu318-I,~s~3~O-l,~~3~2: F:u index) in C’;:! is 1101 miservrd in C’p.3, l’ro43A in (‘$1 of IgM (On index) lies precisely in t,his homologous region of ( ‘~3. Sittc.c,

H1s430

\

I

(b)

(cl Figure 8. Location of residues implicated in the possible binding of Cl q to 1gM. (a) Thr posItions of His430. Aspi(:l~~ 432 and Pro436 (0) (see the t,ext) lie on the periphery of the Fc, disc. The relationship between 2 adjac-rnt Fc urlits of the pentamer shows how Cys414 ( n ) is able to form disulphide bridges between them. Pairs ofcarbohydrat.~ sitt, rrsidur+ (A) at .4sn395 and Asn402 are shown for each Cp3 chain. (b) Face-on and (c) side-on views of the structure seen in (a). t)ogethcr with a d-arm representation of Clq in contact with the region of His430. Asp/(:lu432 and I’ro436. In (b). (‘I q is shown below the plane of t,he Fc Aructure. A Clq arm--axis angle of 28” is required for c@irnal contacT hrtw-wn r’ (‘lq heads in the region of the shaded spheres and 2 adjacent Cl q sites on Fc indicated by 3 (a). It may hr seen that the (‘1 q head must, in part at least, lie within the plane of the Fc, disc. The Debye model for (‘I q is taken from Prrkins (1985). Figs 8 and 9 were created using INSIGHT TI (Biosym Inca., San Diego. (‘4. I:.S.A.) c,n a Silicon (;raphics -CD%‘lY~ \Vorkstation.

Solution

Structure

Pro436 is very highly conserved across all immunoglobulin classes, mutation of this residue might be expected to disrupt the local conformation (Shulman et aE., 1987). ?uTine available C,U~ sequences (Fig. 7) were readily aligned without gaps or insertions to show that only four charged residues and one histidine residue were conserved across all or almost all the sequences. One is Arg/Lys443 (Ou index; Fig. 7), which forms a salt bridge to the Cp4 domain (see Deisenhofer, 1981, IgG Fc structure), and is conserved across all immunoglobulin classes. TWO more are Lys361 and Asp/Glu417 (Ou index), which lie far apart (C”361-C”417 approximately 1.5 nm) in

qf IgM

1361

Cp3 on exposed loops on opposite faces of the Fe, disc. Interestingly, the C”361-C”417 distance of approximately 0.85 nm between residues in two adjacent Fe monomers is ideal for salt bridge formation. Since these residues are close to the Cys414-Cys414 disulphide-bridge, this would further stabilize the contact between Cp3 domains of adjacent subunits in Fe,, and might account for t’he conservation of these residues in seven or eight of the nine sequences. The two remaining conserved charged residues are Asp/Glu432 (if Glu43I in the catfish sequence is included) and His430 (Ou index; Fig. 7). Neither is conserved in other immunoglobulin classes.

Fab;

(a> F

I

1

0

(b)

10

I

20 nm

(cl

Figure 9. Proposed conformational changes implicated in the interactions between immunoglobulin IgM and Clq of complement. (a) The 10 Clq binding sites (Fig. 8(a)) are indicated on the Debye model for IgM (Fig. 5(g), (Ih)). These are located close to the central plane of the Fc, disc (Fig. 8(c)). Only 5 of these are acessible to the approach of a Clq molecule towards one face of the IgM molecule. The scale corresponds to t,his model. (b) A representation of a single Clq arm and head with a Fc and Fab’, fragment to illustrate the postulated conformational change required for a Clq head to bind. When the Fc and Fab’, fragments are coplanar (broken line), the ($2, domain pair prevents the Clq head from binding to the (‘~3 domain in the mode shown in Fig. 8(b) and (c). (c) Rotational movement of the Fab’, fragment out of the plane of Fc (shown here as a 60” rotation) removes all steric conflict between the ($2 domain pair and t’he Clq head when bound to C,U~. as shown here in 2 orthogonal views. (d) and (e) IgM together with Clq molecules drawn with armaxis angles of 46” and 28”, respectively. When this angle is 40” to 45, as found in free Clq in solution (Perkins, 1985), the Clq heads are too far apart for adjacent, Clq heads to attach to Cu3 domains on adjacent subunits in IgM. When this angle is reduced to 28”. adjacent Clq heads are sufficiently close to allow binding to 2 Cp3 domains on adjacent subunits in IgM. in the binding mode shown in Fig. 8(b) and (c).

8. J. Perkins et al.

1362

The molecular graphics model of Fc, shows t’hat Asp/Glu432 lies exposed on the loop between strands fy2 and fy3, close to the polypeptide chain that connects Cp3 to ($2 (Fig. 8(a)). Together with the adjacent His430, these residues could constitute part of a Clq site. Both are also immediately adjacent to Pro436 (Fig. 8(a)), which has been implicated (see above). As they lie close to the polypeptide connection to ($2 in Fab’,, accessibility to Asp/Glu432 and His430 would depend upon the disposition of the Fab’, arms relative to the Fc, disc. In fact, since the globular head of Clq is approximately 6.0 nm x approximately 3.6 nm (Perkins. 1985), any conformation of IgM in which Cp2, is coplanar with PC, is expected to preclude access to this loop region of Cp2 (Figs 8(b) and (c). and 9(b)).

4. Conclusions (a) Structure of IgM IgM and its Fc, fragment have been studied extensively by electron microscopy in order to propose low-resolution models of the structure (Feinstein, 1974; Beale & Feinstein, 1976). Here, molecular graphics were employed to construct improved models of IgM and its fragments from a more extensive database of immunoglobulin crystal structures. While the number of models tested was restricted by the model building, other scattering equivalent models may not be excluded on this basis. These models have been tested in detail using X-ray solution scattering curves and hydrodynamic sedimentation data for Fab, Fab’,, IgM-S, Fc, and IgM. This has led to an improved understanding of the solution structure of IgM. The IgM structure is essentially planar. It has a highly extended multidomain structure, which is similar to that measured for many complement proteins (Perkins et al., 199Oc). The overall dimension of the IgM molecule (tip-to-tip distance between diametrically opposed Fab arms) is approximately 36 nm, and that of the Fc, disc is approximately 17 nm x 15 nm (and thickness analyses 4 nm). Comparative approximately suggest that the J-chain is incorporated within the ring of five Fc fragments, rather than at the centre of the Fc, disc. X-ray crystallographic analysis will be required to establish this result unequivocally. Within the Fab’, fragment,, the ($2, domain pair appears to hold the two Fab units close together at an angle of about 90” between them. With extensive contact possible between both Fab fragments and the ($2, pair, the Fab’, unit may behave as a crystallographic rigid unit. Again, relatively analyses will be required to establish this definitively. Since the IgM scattering curves were sensitive to this movement, there is good evidence that, the side-to-side displacement of the Fab’, fragments relative to Fc, (in the plane of the disc) is an important feature of the solution structure of IgM. The IgM solution structure is best viewed as a family of structures in which any one Fab’, arm is

rotated to one side given instant. (b) Functional

or

the other by up to 45” at any

implications

oj the 1gM

structure

Out-of-plane displacements of the Fab’, units in free IgM in solution are unlikely to be important,. Electron microscopic images of TgM complexes with bacterial flagellae, when the IgM is in excess, reveal the TgM bound in the staple conformation to a single flagellum. When the antigen is in excess, however, the IgM is always seen in a planar conformation cross-linking two or more flagellae (Feinstein & Munn, 1969; Feinstein et ab., 1971). It might’ be expectfed that once one Fab’, arm was bound to a flagellum, t)he local concentration of ant,igenic determinants would be such that a second Fab’, arm would bind to the same flagellum and generate a staple conformation. The fact that this is not, observed, and that the formation of a planar complex with anot,her flagellum is preferred, indicates that there is an energy barrier to the out-ofplane movement of the Fab’, arm and the format,ion of the staple (Feinstein et al., 1971). Such an essentially planar structure for free IgM is consistent with electron microscopy and solution scattering Free IgM cannot activate complement, alt,hough it can bind weakly to Clq. The affinity between Cl q and free IgM lies in the region 2.5 x lo4 M ’ (I’oon ef al., 1985; Painter et al., 1982) to 5 x IO5 >I ’ (Feinstein et aZ., 1983). Since this is approximateI\ the same as the affinity of approximately 5 x lo5 M--’ between Clq and monomeric IgM-S (Feinstein et al.. 1983), the attachment’ of Clq to free IgM appears to involve a single C’lq head. lgM has to undergo conformational change upon antigen binding in order t.o enable (llq attachment and complement activation (Feinstein rt al.. 1986). At physiological ionic strength, the affinit,y between Clq and an ant,i-dextran TgM, complexed wx-it.h was found be approximatcl~ dextran. to 3 x 10’ K ’ (Feinstein et al.. 1983). In comparison the corresponding afhnities of C’lq for monomeric* dimeric and t,rimeric IgG were found to k~r 5 x IO” 2.5 x IO’ and 2.5 x 10” M- ‘, respect,ively (Hughes Jones. 1977). This suggests that the l)roductivc binding of Clq to the IgM-dext.ran complex involves two heads of Cl y. Tf one Clq head binds to free IgM. the six salt iotrs released (Poon et nl., 1985) suggest that binding involves three charged residues as for Tg(:. If’ t~wo Clq heads bind to antigen-bound 1g1M.the eight to nine salt ions released (Wright it &l.. 1988. 1990) would appear t,o implicate the interaction of two charged groups. The latter interaction is consistent with the observation of two adjacent conserved charged residues on ($3 which may constitute the Clq binding site in 1gM (Results a,nd Discussion. section (e)). (c) A poasihbr

rrrrcha~nisn~

ff)T fwr~plervrrr~l

c&rntio?~

The present result,s permit the proposition 01’ ;L mechanism for complement activation by IpM. In

Solution Structure of IgM the solution structure of free IgM, the Fab’, arms are able to move in the plane of the Fc, disc. If the Clq binding site includes Asp/Glu432 and His430 residues, the c~L2~ domains would prevent access to at least part of the site (Fig. 9(b)), since both residues are positioned close to the central plane of the Fc, disc (Fig. 8(c)) immediately adjacent to the Cp2, domains. Side-to-side displacements of Fab’, in free TgM would not expose the ten Clq sites on IgM (Fig. 9(a)), but the displacement of one Fab’, out of the Fc, plane would allow access of a Clq head to one of the two Clq sites associated with that subunit (Fig. 9(b),(c)). This will occur only upon interaction with antigen under conditions of excess antibody when the staple conformation, as seen in electron microscopy, is stabilized. These staple conformations activate complement, unlike the planar IgM conformations formed when IgM interacts with an excess of antigen (Ishizaka et al., 1968; Feinstein & Richardson, 1981; Feinstein et al., 1983). We thus propose a purely steric accessibility model. for exposure of Clq binding sites in IgM, in contrast to earlier propositions that the sites might be created by means of the conjunction of particular residues caused by a distortion of IgM following antigen binding (Feinstein et al., 1983). In apparent contradiction of this accessibility model. the Fc, fragment itself is known not to bind Clq (Feinstein et al., 1983). The preparation of Fc, using trypsin leaves a glycosylated, 19 residue peptide fragment of Cp2 attached to each ($3 (Fig. 5(e),(f)). Further treatment with papain still leaves at least eight residues including the inter heavy-chain disulphide bridge Cys337-Cys337 attached to each ($3 (A. Feinstein, unpublished results). These Cp2 peptide extensions lie immediately adjacent to the proposed Clq sites, and may partially block them. Inspection of Figures 8(b) and 9(a) show that there are five proposed Clq sites accessible from each side of the Fc, disc. Two Clq sites on the same side of the disc in adjacent subunits are separated by 9 nm between adjacent subunits; 15 nm separates sites in non-adjacent subunits. In the solution structure of free Clq (Perkins, 1985, 1989a), the distance between the tips of adjacent globular heads is 14 nm for an average Clq arm-axis angle of 40” to 45” and an arm length of 145 nm. Figure 9(d) compases these structures for free Clq and free IgM (Fig. 5(g)). It also shows that if two adjacent Fab’, arms are displaced out of the Fc, plane to expose two Clq sites, the arm-axis angle of Clq would have to be reduced to 28” in order for Cl q to bind to IgM. It’ is of course possible for two Clq heads separated by 14 nm in free Clq to bind to two exposed nonadjacent Clq sites separated by 15 nm in IgM. However, the binding of a third Clq head must involve an adjacent Clq site in IgM, and require the reduction of the arm-axis angle of Clq to 28”. We therefore propose that the binding of two Clq heads may be sufficient to cause a substantial conformational change in Clq, but that the binding of three heads must necessarily cause such a change.

1363

This reduction of the Clq arm-axis angle upon binding to IgM may be facilitated by the distribution of charged groups over the molecule. From composition data, the net charge on Fab’, is calculated to be +6 (of which +4 charges are located on Cp2,), while that on each of Cp3 and ($4 is 0, and those of the ten tailpiece peptides and the J-chain at the centre of IgM are -30 and -8, respectively. This suggests that the centre of IgM is negatively charged while its outermost periphery is positively charged. Each globular head of Clq has a net positive charge of + 10. When Clq binds to IgM, the Clq head may be attracted towards the centre of the IgM molecule, thus promoting the reduction of the arm-axis angle of Clq in the complex. The pro-enzymic tetramer Clr,Cls, binds to the collagenous stalks of Clq, and its autoactivation initiates the classical pathway of complement. Several models have been proposed for this interaction, in which Clr,Cls2 is either interwoven in and out of the six Clq stalks, or is placed fully on the outside of the Clq stalks (Schumaker et al.. 1986; Perkins, 1989b). A reduction in the Clq armaxis angle of two adjacent Clq arms when bound to IgM could bring two serine protease domains within Clr,Cls, closer together. This reduction could therefore be the trigger for the autoactivation of the protease domain in Clr within Clr,Cls,, each of which would then activate one neighbouring Cls protease domain by a similar mechanism. In the absence of models for IgM, Schumaker et al. (1986) invoked either an increase or a decrease in the Clq arm-axis angle as a possible Clq activation mechanism. These conclusions are in fact independent of the precise location of the Clq site. That proposed in Figure 8(a) lies at the edge of the Fc, disc. Any other location closer to the centre of the Fc, disc would demand an even greater change in the Clq arm-axis angle upon binding to IgM. In summary, the proposed model for the solution structure of IgM offers a simple, steric explanation for why free IgM does not activate complement, how out-of-plane displacement of Fab’, leads to the exposure of Clq binding sites on IgM, and how this necessarily involves a conformational change in Cl q upon binding to IgM that may be the trigger for complement activation. This mechanism for Cl activation may apply equally well to antigen-bound IgG. It may be that the sufficient aggregation of IgG binding sites for Clq heads in immune complexes must be achieved in order that the IgG immune complex can bind Clq sufficiently strongly, and cause a reduction in the arm-axis angle of the Clq molecule so that Cl activation results. S.J.P. thanks the Wellcome Trust, the Medical Research Council, the Science and Engineering Research Council and the Lister Institute for Preventive Medicine for support and access to the X-ray synchrotron facilities at SRS Daresbury, and for the purchase of a Silicon Graphics Workstation. S.J.P. also thanks Dr W. Bras, Dr H. Gerritsen, Dr C. Nave and Dr E. Towns-Andrews for instrumental support at SRS Daresbury. B.J.S. thanks

1364

S. J. Perkins

the Medical Research Council and The Royal Society for support in the initial stages of this work. A.F. thanks the Medical Research Council for support. We thank Dr S. Amatayakul, Dr T. W. Rademacher and Professor R. A. Dwek for allowing us access to their unpublished oligoand acknowledge generous saccharide determinations, contributions from Dr Neil E. Richardson (deceased 24.1.1988) in the early stages of this project. References Anderson. D. R., Atkinson, P. H. & Grimes. W. J. (1985). Major carbohydrate structures at five glycosylation sites on murine IgM determined by high resolution ‘H-P;MR spectroscopy. Arch. Biochem. Biophys. 243. 605-618. Amemiya. C. T. & Litman. G. W’. (1990). (lompletr nucleotide sequence of an immunoglobulin heavychain gene and analysis of immunoglobulin gent’ organisation in a primitive teleost species. Proc. ,Vaf. Acad. Sci., I:.S.A. 87, 811-815. Beale, I). & Buttress, X. (1969). Studies on a human 19-S Immunoglobulin M: The arrangement of inter-chain disulphide bridges and carbohydrate sites. Biochim. Biophys. Acta!, 181, 25Ct267. Beale, I>. & Feinstein. A. (1969). Studies on the reduction of a human 19s immunoglobulin M. Biochrm. .J. 112, 1877194. Beale. I). & Feinstein, A. (1976). Structure and function of the constant regions of immunoglobulins. Quart. Rev. Biophys. 9, 135-180. Beale. D. & Van Ejort. T. (1982). A c~omparison of t,hr proteolytic fragmentation of immunoglobulin M from several different mammalian species. c’owtp. Biochum. Physiol. 71B. 475482. Borsos, T. & Rapp, H. J. (196%). Complement fixation on cell surfaces by 19s and 7S antibodies. R&~LCP 150. 505-506. Borsos. T. & Rapp, H. ,J. (19656). Hemolysin titration based on fixation of the activated first component of complement: Evidence t,hat one molecule of hemolysin suffices to sensitize an erythrocyt,r. ,J. Immunol. 95, 559-566. Burton. D. R.. Boyd. .J.. Brampton, A. I).. Easterbrook-Smith, S. B.. Emanuel. E. J.. Xovotny. -1.. R,ademacher. T. \V., van Schravendijk. M. R.. Sternberg, M. ,J. E. & Dwek. R. A. (1980). The (‘ly receptor site on immunoglobulin (:. Z’at,clrr (London),

288. 338-344.

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