Proc. Nat. Acad. Sci. USA
`Vol. 71, No. 9, pp. 3440--3444, September 1974
`
`The Three-Dimensional Structure of the Fab' Fragment of a Human
`Myeloma lmmunoglobulin at 2.0-A Resolution
`(p-sheets/sequence aligmnents/hypervariable regions/active site)
`
`R. J. POLJAK, L. M. AMZEL, B. L. CHEN, R. P. PHIZACKERLEY, AND F. SAUL
`
`Department of Biophysics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
`
`Communicated by Max F. Perutz, June 14, 1974
`
`The structural analysis of the Fab' frag(cid:173)
`ABSTRACT
`ment of human myeloma immunoglobulin IgGl(X) New
`has been extended to a nominal resolution of 2.0 A. Each
`of the structural subunits corresponding to the variable
`and to the constant homology regions of the light and
`heavy chains contains two irregular f:l-sheets which are
`roughly parallel to each other and surround a tightly
`packed interior of hydrophobic side chains. About 50-60%
`of the amino-acid residues are included in {:j-pleated
`sheets. Sequence alignments between the homology
`regions of Fab' New obtained by comparison of their
`three-dimensional structures are given. Some of the
`sequence variations observed in light and heavy chains and
`the role of the regions of hypervariable sequence in de(cid:173)
`fining the size and shape of the active site of different
`immunoglobulin molecules are discussed on the basis of
`the three-dimensional model of Fab' New.
`
`In a previous paper (2) we described the three-Oimensional
`structure of the Fab' fragment of human myeloma immuno(cid:173)
`globulin lgG New based on the interpretation of an electron
`density map at 2.8-A resolution. The molecule was found to
`consist of four globular subunits which correspond to the
`variable (VL, VH) and constant (CL and Cal) homology regions
`of the light (L) and heavy (H) polypeptide chains, arranged
`in tetrahedral configuration. The homology subunits*, which
`closely resemble each other, share a basic pattern of poly(cid:173)
`peptide chain folding. This basic pattern (immunoglobulin
`fold) and an additional loop of polypeptide chain describe the
`more complex folding of the variable subunits. The additional
`loop in the VL subunit of lgG New is shortened by a deletion
`of seven amino acids which is unique to this L chain. The re(cid:173)
`gions of hypervariable sequence in the L and H chains were
`found to occur in close spatial proximity, at one end of the
`molecule. Two reports dealing with the three-dimensional
`structure of human immunoglobulin L chains have been
`published (3, 4). In this paper we extend-the structural analysis
`of Fab' New to a nominal resolution of 2.0 A and continue the
`description and discussion of its three-dimensional structure.
`
`METHODS
`Preliminary measurement of 13,000 x-ray reflections with
`spacings ranging from 2.8 to 2.0 A was carried out on native
`Fab' crystals. About 2000 of these reflections had intensities
`
`Abbreviations: The nomenclature of irnmunoglobulins, their
`chains and fragments is as recommended by the World Health
`Organization (l); Dnp, 2,4-dinitrophenyl.
`* A homology subunit of an irrmmnoglobulin is defined as the
`globular unit of three-dimensional structure containing the
`amino-acid sequence of a homology region.
`
`significantly above background scattering and were selected
`for intensity measurements with techniques previously out(cid:173)
`lined (5). Procedures for the preparation of heavy atom de(cid:173)
`rivatives and phase determination have been previously given
`(5, 6). Three heavy atom compounds, phenylmercuric acetate,
`diacetoxymercury dipropylene dioxide and l,4-diacetoxy(cid:173)
`mercuri-2,3-dimetoxybutane, were used to obtain isomorphous
`replacements to extend the phase information from 2.8- to
`2.0-A resolution. The average figure of merit obtained for the
`2000 reflections with spacings between 2.8 and 2.0 A that
`were phased was 0.69. A 2.0-A resolution Fourier map was
`calculated including a total of 12,000 reflections with an
`average figure of merit of 0.76. Based on this map a model
`was constructed using an optical comparator (7) and Kendrew
`skeletal models.
`Purified Fab New was digested with CNBr and the resulting
`fragments were separated by gel filtration on Sephadex G-100
`columns. The amino-acid sequences of some of the tryptic and
`chymotryptic peptides of the CNBr fragments belonging to
`the VH region were determined by procedures previously out(cid:173)
`lined (8). These peptides were aligned by homology with other
`human H chains, by sequence overlaps, and by information
`derived from the Fourier map.
`
`RESULTS AND DISCUSSION
`The 2.0-A map shows increased resolution with respect to the
`previous 2.8-A Fourier map even though only 2000 additional
`reflections were included. The amino-acid sequences of the
`CL, Cal, and VL regions and the tentative sequence of Va (see
`Fig. 1) correspond very closely to the features of the 2.0-A
`Fourier map. The positions of most of the carbonyl groups
`of the polypeptide chain can be easily identified in the elec(cid:173)
`tron den8ity map. Careful placing of the carbonyl and a(cid:173)
`imino groups invariably maximized features of secondary
`structure such as hydrogen bonding between adjacent
`stretches of polypeptide chain. The VL, Va, CL, and Cal
`homology subunits are predominantly folded in {:j-pleated
`sheet conformation. The CL homology subunit, for example,
`consists of a {:j-pleated sheet made up by four hydrogen-bonded
`antiparallel chains (residues 116-120, 132-140, 160-169, and
`173-182) and another {:j-pleated sheet containing three anti(cid:173)
`parallel chains (residues 147-151, 193-199, and 202-208, see
`Fig. 2). These two twisted and roughly parallel sheets sur(cid:173)
`round a tightly packed interior of hydrophobic side chains in(cid:173)
`cluding the intrachain disulfide bond which links the two
`sheets in a direction approximately perpendicular to their
`planes. About 60% of the CL residues are included in these
`
`3440
`
`1 of 5
`
`BI Exhibit 1115
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`

`

`Proc. Nat. Acad. Sci. USA 71 (1974)
`
`Structure of Human Fab' at 2.0 A-Resolution
`
`3441
`
`27 • b c
`50
`40
`30
`20
`10
`1
`- Z S V LT Q P P S V S GA P - G Q RV T I S C T G S S S II I GAG II H V It WY Q Q L P GT A P IC - LL I F H II II A -
`
`-
`
`-
`
`-
`
`-
`
`-
`
`60
`50
`40
`30
`20
`10
`1
`- Z V Q L P E S G P E L V S P - G Z T L S L T C T G S T V S T F A V - Y I V W V R Q P P G R G L E W I G Y V F Y H G T S D T D T
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`VL
`
`VH
`
`CL
`
`150
`140
`130
`120
`110
`Q P IC A A P S V T L F P PS S E E L QA II IC AT L V C L I S D F Y P GAV - T VA W IC -
`
`- A D S S -
`
`CH 1
`
`150
`140
`130
`120
`A S T IC G P S V F P L A P S S IC S T S G G T A A L G C L V IC D Y F P E P V - T V S W II -
`
`-
`
`160
`- S G -
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`VL
`
`VH
`
`-
`
`-
`
`-
`
`-
`
`60
`- R F S V S IC S G -
`
`-
`
`-
`
`70
`- P L R S R V T M L V II T - S -
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`CL
`
`- P V KA -
`
`109
`100
`- L R - V F G G G T IC L T V L R
`118
`110
`100
`90
`80
`IC II Q F S L R L S S V T A A D T A V Y Y C A R B L I A G - C I B V W G Q G S L V T V S S
`180
`190
`200
`210
`214
`170
`160
`- G V E T T T P S IC Q S II II ICY A A S S Y L S L T P E Q W IC S H IC S Y S C Q V T R -
`- E G S T - V E IC T - V A P T E C S
`
`90
`80
`70
`- S S A T L A I T G L Q A E D E A D Y Y C Q S Y D R S -
`
`CHI
`
`- A L T S -
`
`220
`210
`200
`190
`180
`170
`- G V H T F P A V L Q S S G L Y S L S S V V T V P S S S L G T - Q T Y I C 11 V 11 H 11: P S 11 T IC - V D 11: R - V E P IC S C
`
`Fm. 1. Amin<>-acid sequences of the VL, CH, VH, and CHI homology regions of Fab New, aligned by comparison of their'4;hree-di(cid:173)
`mensional structures (see text). Parts of the tentative VH sequence given here were obtained by Drs. Y. Nakashima and W. Konigsberg
`(personal communication). The CHI sequence is taken from ref. 9. Abbreviations are from ref. 10.
`
`two (3-pleated sheet.s. The homologous subunits CHI, VL, and
`VH (see Figs. 2 and 3) can be described in similar terms. In
`addition to the features of secondary structure just discussed,
`VL residues 26-27c appear in a helical conformation close to
`that of a r-helix (11). Also, VL residues 78-82, and the ho(cid:173)
`mologous VH residues 87-9I, fold with the conformation of a
`distorted r or a-helix. In general, the precise conformation of
`amino-acid residues that do not belong to the regions of secon(cid:173)
`dary structure discussed above is more difficult to define,
`especially when they correspond to regions of lower electron
`density on the surface of the molecule.
`Amino-acid sequence comparisons have been extensively
`used in several laboratories to align the primary structures of
`
`1
`2
`3
`u ~-- 4
`"66=69
`.u==5
`M ro=~ 6
`1....._ 102--
`64 = n
`21
`:;=~~=~~ =-~~
`
`10::::105
`
`74= 18
`61
`60-15
`17
`
`93 92
`94=91
`90_31
`95
`~=$ ll
`99 e=33
`~= 34 - "
`101
`=
`35
`45
`-~=~;=~
`38 =41
`
`83
`
`42
`
`r 25 26 27 27a 27b 27c 28 29 30 ~
`11_....106 _J 39 40
`L6
`
`12, 107
`...... 13 '108
`4
`109
`
`I
`I
`76 77 78 79 80 81 8z
`- - - - - - - -52 51 50 49 48 47 - - - - - - - -
`
`I
`
`I
`
`different immunoglobulin molecules and their homology re(cid:173)
`gions. The alignment of VL and VH sequences with those of
`the CL and CH homology regions is less straightforward than
`alignments between VL and VH or between CL and CH regions.
`This problem can now be approached by matching their
`similar three dimensional structures and aligning residues
`that occupy an identical or similar position in the constant
`pattern of the tertiary structure of the homology subunits.
`Alignments obtained by this procedure, which is independent
`of amino-acid· sequence homologies, are shown in Fig. 1.
`Where homologies have been detected by sequence compari(cid:173)
`son, it can be safely assumed that the three-dimensional
`structure of the homologous polypeptide chains will be very
`CL
`
`141 142 143 144 145 146 :-1
`210 J 154
`
`201 200
`202-199
`203 198=47
`204=97
`148
`205 ~ 149
`20~ 150
`207 194=51
`208=93
`152
`209
`192
`153
`
`lk~l 155
`EY
`214
`
`110
`111
`112
`171
`113
`170
`172
`114
`169-173
`115
`l~ 174-140
`167=175 139=116
`165166
`176=138
`117
`164-177
`J.llr=ll8
`163 17~ 119
`162=79 135=20
`161
`1SO::l34
`121
`160-is1
`133
`122
`182-132
`123
`
`126
`129
`128 127
`
`l g~ ~~~
`
`183 184 185 186 187 188 189 190 191
`
`- - - - - - - - 159 158 157 156
`
`CH1
`
`9
`120
`177
`lso=121
`176
`178
`149
`122
`175=79
`174 180-148
`123
`mg:~:k:~!~-g~
`170-183 ~126
`169 l~ 127
`168:::185 14=128
`167 186=42
`129
`166-is1
`141
`130
`188
`140
`131
`139
`lll
`138
`133
`lD
`134
`136 135
`
`151 152 153 154 ~
`
`201 206
`208
`205
`20=04
`~~~~g~=~~~
`212
`,l2J..=57
`213~ 158
`214 199-i59
`215-i98
`160
`
`217
`218
`m
`~
`
`162
`163
`
`216 J 161
`l.h •Mm•• m •H m •H m
`
`Fm. 2. Diagram of the hydrogen bonds (broken lines) between main chain atolilS for VL, CL, VH, and CHI. The hydrogen-bonded
`clusters correspond to the two P-sheets of each subunit (see text). Cysteine residues that participate in the intrachain disulfide bonds
`linking the two P-sheets are enclosed in rectangles; CL residue 213 and CHI residue 220 form the interchain disulfide bond and are also
`enclosed in rectangles. Numbers refer to residues as given in Fig. 1.
`
`.._ ______ 165 164 - - - - - - - -
`
`2 of 5
`
`BI Exhibit 1115
`
`

`

`3442
`
`Immunology: Poljak et al.
`
`Proc. Nat. Acad. Sci. USA 71 (197~)
`
`Fm. 3. Stereo pair drawing of the a-carbon backbone of the Vu structure. Thin lines connect the a-carbon a.toms of residues that are
`hydrogen-bonded; see Fig. 2. PCA is pyrrolidone carboxylic acid.
`
`similar. Since the amino-acid sequence of Prmicroglobulin is
`highly homologous to that of CL and CHl (12), it is reasonable
`to conclude that it shares the basic three dimensional structure
`or immunoglobulin fold of CL and CHI.
`As discussed before (2), the Kern and Oz serological markers
`of human X chains, which correspond to positions 154 and 191
`in the L chain sequence, occur on the surface of the molecule,
`in close spatial proximity. The Iw allotypic markers of human
`K chains have recently been shown (13) to involve Ala/Val
`and Val/Leu substitutions at positions 153 and 191, respec(cid:173)
`tively, which closely correspond to the positions of the Kern
`and Oz markers in human X chains. Replacements at positions
`153 and 191 in K chains will also affect antigenic determinants
`of the molecule that are recognized by anti-allotypic antisera.
`Since the distance between the a-carbon atoms of the ho(cid:173)
`mologous residues is about 8-10 A, replacements involving
`both positions can be simultaneously recognized by a single
`anti-allotypic antibody molecule.
`Some of the variable and constant features of V L sequences
`can be discussed in terms of the three-Oimensional structure
`of V>. New. Hairpin bends in the polypeptide chain of V>.
`New occur around positions 14-15, 27-30, 39-40, 67~, and
`92-93 and an approximate 90° bend around residues 75-76.
`Except for the bend at positions 92-93 (a hypervariable re(cid:173)
`gion) all others involve a Gly residue that is constant in
`human X chain sequences. Most of these bends also involve a
`constant or nearly constant Pro-Gly or Ser- or Thr-Gly se(cid:173)
`quence. A similar conclusion has been obtained from the study
`of a crystalline V« fragment (4). Glycine residues also con(cid:173)
`tribute to a constant sequence (Phe-Gly-Gly-Gly, positions 99-
`102) that is not part of a bend. The constant character of
`this sequence in all X chains can be explained by the following
`observations: (a) Phe 99 is located in an internal, interchain
`hydrophobic pocket that includes the homologous constant
`Trp 108 in VB, related to Phe 99 by a local pseudo 2-fold axis
`of symmetry; it can be assumed that Phe 99 (and Trp 108)
`make an interchain contact that is important for VL-VH
`
`assembly; (b) Gly 100 in V>. (or Gly 109 in VB) is tightly
`packed between the intra.chain disulfide bond and Leu 4 (a
`constant residue in VL and VB); (c) Gly 101 is relatively close
`to the constant Gln 6, although here there is room for a side
`chain as observed in v« (Gin 101) or in VB (position 110);
`(d) Gly 102 (111 in VH) is very close to a constant aromatic
`residue (Tyr/Phe, 86 in V>. or 95 in VH) so that the limited
`space available requires the presence of a Gly residue at this
`position. Some other residues that are constant in V>. or that
`show only a limited degree of variability, such a Tyr 35, Gin
`37, Ala 42, Pro 43, and Asp 84 are involved in close contacts
`with the VH subunit. Other constant residues such as Gin 37
`and Tyr 85, Glu 82 and Tyr 142 (CL) make internal hydrogen
`bonds. In addition to the residues just discussed, most of the
`nonpolar, hydrophobic amino acids that occur in the interior
`of the structure between the two (j-sheets are invariant or are
`replaced by other hydrophobic residues. They are Leu 4, Gin
`6, Val 10, Val 18, Ile 20, Cys 22, Val 32, Trp 34, Leu 46, Phe
`61, Val 63, Ala 70, Leu 72, Ile 74, Leu 77, Ala 83, Tyr 85, Cys
`87, Ser 89, Val 98, Thr 103, Leu 105, and Val 107. All the con(cid:173)
`stant or nearly constant residues that appear at bends or con(cid:173)
`tribute to intra- and inter-subunit bonds seem to be important
`for the preservation of structure. Mutations that alter any of
`these residues, which constitute more than 50% of the total
`number of residues of the v >. sequence; cannot be considered
`to be "neutral or modulating" (14). Different combinations of
`these invariant and semi-invariant residues that are com(cid:173)
`patible with the requirements of the constant three-dimen(cid:173)
`sional structure of the homology subunits are, in our view,
`better explained by a process of evolutionary germ line gene
`divergence than by random somatic mutations. By contrast,
`the nature of the residues that occur in the regions of hypervari(cid:173)
`able sequence, at one end of the molecule and fully exposed to
`the solvent, is not limited by any visible structural constraints.
`It has recently been pointed out (15) that the sequence
`Arg/Lys-Phe-Ser-Gly-Ser-Lys (positions 60-65) is constant
`in X chains from several animal species that have been ana-
`
`3 of 5
`
`BI Exhibit 1115
`
`

`

`Proc. Nat. Acad. Sci. USA 71 (1974)
`
`Structure of Human Fab' at 2.0 A-Resolution
`
`3443
`
`lyzed and that this sequence could, therefore, fulfill a special
`function. The side chains of Phe 6I and Gly 63 (Val 63 in>.
`Newm) are part of the internal hydrophobic core surrounded
`by the P-pleated sheets; consequently, they could be replaced
`only by other nonpolar residues. Arg 60 appears to be involved
`in an internal hydrogen bond to Glu 80 and/or Gin 78. How(cid:173)
`ever, the side chains of the other residues of that constant
`sequence are external with no apparent structural constraint.
`Residues that occur in the constant or nearly constant N(cid:173)
`terminal sequences of V>., V., and VH can be analyzed in simi(cid:173)
`lar terms: some are internal (residues 4, 6, 10, I2 in V>.) and
`would not be expected to show much change, but others have
`external side chains with no visible constraints (positions 5, 9,
`11, I3, I6in V>.).
`As discussed before (2), ttie VH subunit exposes a larger area
`in the region of the active site. Comparison of the three-dimen(cid:173)
`sional model of Fab' New with that of a >. chain dimer (3)
`and a V. dimer (4) suggests that the VH subunit plays an
`essential role in defining the conformation and the specificity
`of the antigen binding site. The VH hypervariable sequences
`extending from positions'50-60 and I00-110 are longer than
`the homologous regions of L chains. In particular, the third
`hypervariable region of VH has been found to consist of a
`variable number of amino-acid residues, ranging from I3 to 20
`when counted from Cys 96 to Trp 108, whereas the homol(cid:173)
`ogous loop of V>. and V. (Cys 87 to Phe 99) has been found to
`contain only 11 to I3 residues. This hypervariable loop of VH
`does not conform to the approximate local 2-fold axis of sym(cid:173)
`metry relating VL to VH. Instead, this loop bends towards the
`L chain (see Fig. 4), making the structural pocket at the
`active site (2, I6) narrower than it is in L chain dimers, where
`it appears as a large cavity (3, 4). The width and depth of the
`active site pocket of different immunoglobulins can, therefore,
`be altered by variations in the length of this hypervariable
`VH loop. Thus different H chains that pair with the same L
`chain in induced antibodies (I 7) could modulate affinity not
`only by changes in the amino acids present in this sequence
`but also by alterations in the length of the polypeptide chain
`in this region.
`Although the length of the L chain hypervariable regions is
`relatively constant, some human and mouse (I8, 19) K chains
`have been found to include additional residues in the first
`hypervar~able region (positions 25-32). This additional length
`of polypeptide chain will also have an important effect in
`determining the dimensions of the active site pocket and the
`antigen specificity of the immunoglobulin molecules to which
`they belong.
`With the model of Fab' New as a basic structural frame(cid:173)
`work, a striking correlation between the structure and func(cid:173)
`tion of the well-studied MOPC 3I5 anti-Dnp mouse myeloma
`protein (20, 2I) can be obtained. The L(X) chains of IgG
`New and MOPC 3I5 are highly homologous and contain an
`equal number of residues in the third hypervariable region
`between the constant amino acid residues Cys 89 and Phe 99.
`Also, a comparison of the tentative sequence of VH New (Fig.
`I) and that of VH MOPC 3I5 indicates that the third hyper(cid:173)
`variable regions of VH in both chains between the constant
`Cys 96 and Trp I08 include the same number of amino acid
`residues. It is, therefore, feasible to fit the MOPC 3I5 sequence
`(I8) to the basic VL and VH structures obtained in the 2.0-A
`Fourier map of Fab' New. The model of the MOPC 3I5
`binding site that emerges from this comparison includes a
`
`Fm. 4. View of some of the amino-acid residues at the active
`site of IgG New. Residue numbers for VL (27 to 31and89 to 95)
`and V H correspond to those of Fig. 1.
`
`crevice similar to that shown in Fig. 4, in which L chain Tyr 34
`forms the "upper" limit, H chain Trp 47 and Phe 50 form the
`"lower" limit, L chain Phe 99, H chain Tyr 104 and Phe 34
`contribute to the "sides" and L chain Trp 98 and Phe 103 and
`H chain Phe 105 form the 6- to IO-A deep "floor." The high
`density of adjacent aromatic side chains that line this crevice,
`at the center of the active region, is striking and correlates
`with the observed specificity of MOPC IgA immunoglobulin
`for Dnp and other haptens that include benzene and naph(cid:173)
`thalene aromatic rings. The relatively shallow depth of the
`active center is in agreement with the electron microscopy
`study of a complex between MOPC 3I5 IgA and the bifunc(cid:173)
`tional hapten bis(Dnp-j3-alanyl)-diaminosuccinate (20)
`in
`which the Dnp groups that join the Fab arms of different IgA
`molecules end to end are only I5 A apart.
`The possibility of a conformational change as a biological
`signal triggered by antigen binding is an important question
`to be considered in discussing immunoglobulin structure.
`In this context, the Fab structure can be described as a tetra(cid:173)
`hedral arrangement of homologous subunits, covalently linked
`in pairs (VL to CL and VH to CHI) by linear stretches of poly(cid:173)
`peptide chain ("switch" regions) bent to a larger extent in the
`Fd' chain than in the L chain (2), suggesting flexibility.
`Furthermore, two identical L chains of a dimer assume differ(cid:173)
`ent conformations (3), such that one of them appears similar
`to the L chain of Fab', making an angle greater than 90°
`between the major axes of the CL and VL subunits, whereas the
`other L chain of the dimer makes an angle smaller than 90°,
`as observed between the VH and CHI subunits of Fab' New.
`These observations suggest that a conformational change
`could take place by a hinge-like movement at one or both
`switch regions. Since a disulfide bond linking V L to CL has been
`found in some rabbit IgG molecules (22, 23), the flexibility of
`the more open L chain may be more limited.than that of the H
`chain. An "opening" of the Fd' chain, as illustrated in Fig. 5,
`
`4 of 5
`
`BI Exhibit 1115
`
`

`

`3444
`
`Immunology: Poljak et al.
`
`Proc. Nat. Acad. Sci. USA 71 (1974)
`
`and vitamin K10H (16) which can only interact with some of
`the side chains of the active site.
`
`This research was supported by Grants AI 08202 from the
`Natio11al Institutes of Health, NP-141A from the American
`Cancer Society, and N.I.H. Research Career Development
`Award AI 70091 to R.J.P.
`
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`
`Fm. 5. A view of the a-carbon backbone of Fab' New. The
`V and C, domains, the L chain (open line), the Fd' chain (solid
`line) and the local, approximate 2-fold axes (broken lines)
`relating the VL to the VH subunit and the CL to the CHhubunit
`are shown. The two short arrows ·indicate the switch region of
`both chains. The longer arrow indicates a possible relative motion
`of the V and C, domains (eee text).
`
`would lead to a relative movement of the structural subunits
`and to the exposure of some of the VH and CHI side chains that
`were not previously exposed. A relative movement of struc~
`tural subunits leading to changes in quaternary structure has
`been demonstrated by crystallographic analyses of reduced
`and oxygenated hemoglobin molecules (24). Although no
`major conformational changes hHNe been observed after bind(cid:173)
`ing of ligands to Fab' fragments (16, 25)·the occurrence of
`such chimges cannot be ruled out on the basis of these experi(cid:173)
`ments carried out in the crystalline state. Furthermore, some
`of the interactions necessary to trigger the postulated con(cid:173)
`formational change may not be present in the binding of rela(cid:173)
`tively small haptenic groups such as phosphoryl choline (25)
`
`5 of 5
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`BI Exhibit 1115
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