`
`PFIZER EX. 1115
`
`Page 1
`
`PFIZER EX. 1115
`Page 1
`
`

`

`
`
`PFIZER EX. 1115
`
`Page 2
`
`PFIZER EX. 1115
`Page 2
`
`

`

`
`
`PFIZER EX. 1115
`
`Page 3
`
`PFIZER EX. 1115
`Page 3
`
`

`

`
`
`PFIZER EX. 1115
`
`Page 4
`
`PFIZER EX. 1115
`Page 4
`
`

`

`
`
`PFIZER EX. 1115
`
`Page 5
`
`PFIZER EX. 1115
`Page 5
`
`

`

`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
`(/3-sheets/sequence alignments/hypervariable regions/active site)
`
`0
`
`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 mye)oma immunoglobulin lgGl(~) 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 /3-sheets which are
`roughly paraJleJ to each other and surround a tightly
`packed interior of hydrophobic side chains. About 50-60%
`of the amino-acid residues are included in /3-p)eated
`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
`immunog)obulin molecules are discussed on the basis of
`the three-dimensional mode) of Fab' New.
`
`In a previous paper (2) we described the three-dimensional
`structure of the Fab' fragment of human myeloma immuno(cid:173)
`globulin IgG 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 CHl) 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 IgG 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 extenlthe ~tructural 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 immunoglobulins, their
`chains and fragmen ts is as recommended by the World Healt h
`Organization ( 1) ; Dnp, 2,4-dinitrophenyl.
`* A homology subunit of an imniunoglobulin is defined as the
`globular unit of three-dimensional structure cont aining 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 1,4-diacetoxy(cid:173)
`mercuri-2,3-dimetoxybutane, were used to obtain isomorphous
`replacements to extend the pliase 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 ot?er
`human H chains, by sequence overlaps, and by information
`derived from the Fourier map.
`
`RESULTS AND DISCUSSION
`The 2.0-A m~p shows increased resolution with respect.~ th~
`previous 2.8-A Fourier map even though only 2000 add1tiona
`reflections were included. The amino-acid sequences of the
`
`CL, CHl, and VL regions and the tentative sequence of VH (A
`
`Fig. 1) correspond very closely to the features of the 2.0-
`Fourier map. The positions of most of the carbonyl gro~ps
`of the polypeptide chain can be easily identified in the e ec·
`tron density map. Careful placing of the carbonyl and a(cid:173)
`imino groups invariably maximized features of seco~da~
`b t
`n adiacen
`b d.
`structure such as hy rogen
`on mg
`e wee
`C 1
`d
`stretches of polypeptide chain. The VL, VH, CL, an~ ~
`homology subunits are predominantly folded in /3-P ea le
`sheet conformation. The CL homology subunit, for exarn~ed
`consists of a {:l-pleated sheet made up by four hydrogen-boll nd
`antiparallel chains (residues 116-120, 132-140, 160-169, :ti·
`173-182) and another {:l-pleated sheet containing three; see
`parallel chains (residues 147-151, 193- 199, and 202- 20 'sur·
`i.n(cid:173)
`Fig. 2). These two twisted and roughly parallel shee~s
`rou~d a tight~y packe? in~erior of hydroph~bic s_ide c~~;two
`their
`cludmg the mtracham disulfide bond which lmks
`sheets in a direction approximately perpendicular ~o the88
`planes. About 60% of the CL residues are included in
`
`3440
`
`PFIZER EX. 1115
`Page 6
`
`

`

`at. Acad. Sci. USA 71 (1974)
`
`Structure of Human Fab' at 2.0 A-Resolution
`
`3441
`
`50
`40
`30
`27 8 b C
`20
`10
`---ZSVLTQPPSVSGAP-GQRVTISCTGSSSNIGAGNHVKWYQQLPGTAPK-LLIFHNNA-
`w
`m
`10
`1
`~
`~
`~
`---ZVQLPESGPELVSP-GZTLSLTCTGSTVSTFAV-YIVWVRQPPGRGLEWIGYVFYHGTSDTDT
`
`150
`140
`130
`120
`110
`QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAV-TVAWK--ADSS
`
`160
`150
`140
`130
`120
`ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV-TVSWN---SG--
`
`60
`-RFSVSKSG---
`
`90
`80
`70
`100
`109
`-SSATLAITGLQAEDEADYYCQSYDRS--LR-VFGGGTKLTVLR
`
`70
`-PLRSRVTHLVNT-S
`
`118
`llO
`100
`90
`80
`-KNQFSLRLSSVTAADTAVYYCARBLIAG-CIBVWGQGSLVTVSS
`
`214
`210
`200
`190
`180
`170
`160
`-PVKA- -GVETTTPS KQS NNKYAAS SYLS LTPEQWKS HKSYS CQVTH- -E GS T-VE KT-VAPTECS
`
`220
`210
`200
`190
`180
`170
`-ALTS--GVHTFPAVLQSSGLYSLSSVVTVPSSSLGT-QTYICNVNHKPSNTK-VDKR-VEPKSC
`
`Amino-acid sequences of the V1, CH, VH, and CHI homology regions of Fab New, aligned by comparison of their-three-di(cid:173)
`al structures (see text). Parts of the tentative VH sequence given here were obtained by Drs. Y. Nakashima and W. Konigsberg
`I communication). The Cal sequence is taken from ref. 9. Abbreviations are from ref. 10.
`
`leated sheets. The homologous subunits CHl, VL, and
`Figs. 2 and 3) can be described in similar terms. In
`n to the features of secondary structure just discussed,
`• ues 26-27 c appear in a helical conformation close to
`a r-helix (11) . Also, VL residues 78-82, and the ho(cid:173)
`us VH residues 87-91, fold with the conformation of a
`r or a-helix. In general, the precise conformation of
`id residues that do not belong to the regions of secon(cid:173)
`cture discussed above is more difficult to define,
`y when they correspond to regions of lower electron
`on the surface of the molecule.
`o-acid sequence comparisons have been extensively
`several laboratories to align the primary structures of
`
`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 c0mpari(cid:173)
`son, it can be safely assumed that the three-dimensional
`structure of the homologous polypeptide chains will be very
`
`1
`2
`3
`-=~ M-U
`4
`68 ---24
`67
`,LJ, ::: 5
`'66 ::: 69
`~ ro=~
`1_ 102 --------------1..01
`6
`64 =n
`45
`86 ::: 35
`21
`12 ::: 20
`a __ ·103 ________________ ::::: as
`36 ::: 44
`63
`62 ::: 73
`19
`9
`:104
`84 ::: 37
`43
`2
`38 :::414
`61
`74 ::: 18
`10:· 105 ---------------------53
`60--75
`17
`
`93 92
`94 ::: 91
`95
`90 ---31
`98 ::: 89
`32
`99
`88 ::: 33
`
`12~ 26 27 27a 27b ~c 28 29 30 ~
`11 ::106 _J 39 40
`L6 -.:!: .. :g~
`
`4
`
`109
`
`r-~::=.--1
`
`76 77 78 79 80 81 82
`
`141 142 143 144 145 1461
`
`CL
`
`110
`Ill
`112
`171
`113
`170
`172
`114
`169---173
`115
`168
`174 ___ 140
`167:::175
`139:::116
`165166
`176:::138
`117
`j64 ___ 1 n m:::118
`163 178::~ 119
`162:::179
`135:::120
`161
`180:::134
`121
`160---151
`133
`122
`
`1s2l---m m
`
`126
`129
`128 127
`
`201 200
`202---199
`203
`198:::147
`~0045:::119976::=114489
`
`150
`206::~
`207
`194:::151
`205:::193
`152
`209
`192
`153
`
`fil J :J
`
`ru
`
`214
`
`183 184 185 186 187 188 189 190 191
`
`.._ __ __ ___ 159 158 157 156
`
`2627 2829 30 31 32~
`
`CH
`
`1 ~1511 s2 1s3154
`I m
`
`207 206
`208
`205
`209:::204
`
`- - - - - - - 16 5 164 - - - - - - - - -
`- - - - - - - 66 65 64 63 -
`61 60 59 - - - - - - -
`Diagram of the hydrogen bonds (broken lines) between main chain atoms for V1, C1, VH, and CHI. The hydrogen-bonded
`correspond to the two /j-sheets of each subunit (see text). Cysteine residues that participate in the intrachain disulfide bonds
`the two fj-sheets are enclosed in rectangles; C1 residue 213 and CHI residue 220 form the interchain disulfide bond and are also
`in rectangles. Numbers refer to residues as given in Fig. 1.
`
`l
`
`189 190 191 192 193 194 195 196 197
`
`102
`103
`101
`105104---100
`53 5'!. 55
`106 ··- 99 ::: 33
`52 ::_ 56
`W-~ ~=n"
`108
`97 ::: 35
`50 ::: 58
`109=1961
`36:::49
`48
`95 ::: 37
`110
`47
`111112 ::: 94
`38 ::: 46
`113
`93 ::: 39
`45
`114 ::: 92
`40
`44
`
`11 ::us J 41 42 43
`
`2
`25 --- 3
`7 5 76
`M n-H
`4
`73
`78
`23 ::: 5
`72
`79:::1221
`6
`'ff ::: 7
`71::: 80
`70
`81 ::: 20
`8
`69 ::: 82
`19
`9
`68
`83 ::: 18
`10,
`67 -84
`17
`12: 116
`85 --16
`'•15 •• 13 ··111
`18
`
`r----~
`86 ar;-;;~o ;1
`
`177
`150:::121
`176
`178
`149
`122
`175:::179
`174
`180---148
`123
`
`mg~===::~:::::~===:~1
`170---183
`.li2.===126
`169 184::~ 127
`16s:::1ss
`143:::12s
`167
`!86:::142
`129
`166---157
`141
`130
`188
`140
`131
`139
`132
`138
`133
`137
`134
`136 135
`
`216 J 161
`
`~:~:::~g~===:~~
`
`212 m===1s1
`
`158
`213::~
`214
`199---159
`215---195
`160
`
`162
`163
`
`217
`218
`219
`~
`
`PFIZER EX. 1115
`Page 7
`
`

`

`3442
`
`Immunology: Poljak et al.
`
`Proc. Nat. A cad. Sci. USA 71 (19'1.f)
`
`FIG. 3. Stereo pair drawing of the a-carbon backbone of the VH structure. Thin lines connect the a-carbon atoms of residues that are
`hydrogen-bonded; see Fig. 2. PCA is pyrrolidone carboxylic acid.
`
`similar. Since the amino-acid sequence of /3rmicroglobulin is
`highly homologous to that of CL and CHI (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 'A. 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 Inv 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 'A. 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 VL sequences
`can be discussed in terms of the three-dimensional structure
`of Vx New. Hairpin bends in the polypeptide chain of Vx
`New occur around positions 14-15, 27-30, 39-40, 67-68, 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 'A. 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 'A. 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 VH, 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 Vx (or Gly 109 in VH) is tightly
`packed between the intrachain disulfide bond and Leu 4 (a
`constant residue in VL and VH); (c) Gly 101 is relatively close
`to the constant Gin 6, although here there is room for a side
`chain as observed in V. (Gin 101) or in VH (position 110);
`(d) Gly 102 (111 in VH) is very close to a constant aromatic
`residue (Tyr/ Phe, 86 in Vx 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 Vx 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 :fl
`and Tyr 85, Glu 82 and Tyr 142 (CL) make internal hydrogen
`bonds. In addition to the residues just discussed, most of t_he
`nonpolar, hydrophobic amino acids that occur in the interior
`of the structure between the two /3-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-
`r con·
`d
`t
`stant or nearly constant residues that appear at ben s O
`tribute to intra- and inter-subunit bonds seem to be importan f
`for the preservation of structure. Mutations that alter anY ; 1
`these residues, which constitute more than 50% of th~t ed
`number of residues of the V x sequence, cannot be cons! er of
`to be "neutral or modulating" (14). Different combinationslJI·
`these invariant and semi-invariant residues that a~ coen·
`patible with the requirements of the constant three ~ ..,
`.
`.
`ur vie .. ,
`gene
`sional structure of the homology subumts are, iil O•
`better explained by a process of evolutionary germ hnet •4
`.
`·
`By con r ..... '
`ari·
`divergence than by random somatic mutations.
`the nature of the residues that occur in the regions of hype:a to
`able sequence, at one end of the molecule and fully expo jnts·
`the solvent, is not limited by any visible structural constra ence
`It has recently been pointed out (15) that the seQ~talli
`Arg/ Lys-Phe-Ser-Gly-Ser-Lys (positions 60-65) is con .-(cid:173)
`in 'A. chains from several animal species that have been
`
`PFIZER EX. 1115
`Page 8
`
`

`

`Nat. Acad. Sci. USA 71 (1974)
`
`Structure of Human Fab' at 2.0 A-Resolution
`
`3443
`
`and that this sequence could, therefore, fulfill a special
`on. The side chains of Phe 61 and Gly 63 (Val 63 in >,.
`) are part of the internal hydrophobic core surrounded
`p-pleated sheets; consequently, they could be replaced
`y other non polar residues. Arg 60 appears to pe involved
`internal hydrogen bond to Glu 80 and/ or Gln 78. How(cid:173)
`the side chains of the other residues of that constant
`ce are external with no apparent structural constraint.
`es that occur in the constant or nearly constant N(cid:173)
`I sequences of Vx, V., and VH can be analyzed in simi-
`ms: some are internal (residues 4, 6, 10, 12 in Vx) and
`not be expected to show much change, but others have
`I side chains with no visible constraints (positions 5, 9,
`16in Vx).
`discussed before (2), tl'ie VH subunit exposes a larger area
`region of the active site. Comparison of the three-dimen-
`model of Fab' New with that of a >.. chain dimer (3)
`V. dimer (4) suggests that the VH subunit plays an
`"al role in defining the conformation and the specificity
`antigen binding site. The VH hypervariable sequences
`· g from positions'50-60 and 100-110 are longer than
`mologous regions of L chains. In particular, the third
`ariable region of V H has been found to consist of a
`e number of amino-acid residues, ranging from 13 to 20
`counted from Cys 96 to Trp 108, whereas the homol(cid:173)
`loop of V x and V. ( Cys 87 to Phe 99) has been found to
`• only 11 to 13 residues. This hypervariable loop of VH
`ot conform to the approximate local 2-fold axis of sym(cid:173)
`relating VL to VH. Instead, this loop bends towards the
`·
`(see Fig. 4), making the structural pocket at the
`site (2, 16) narrower than it is in L chain dimers, where
`rs as a large cavity (3, 4). The width and depth of the
`site pocket of different immunoglobulins can, therefore,
`red by variations in the length of this hypervariable
`p. Thus different H chains that pair with the same L
`in induced antibodies (17) could modulate affinity not
`Y changes in the amino acids present in this sequence
`by alterations in the length of the polypeptide chain
`region.
`ough the length of the L chain hypervariable regions is
`ely constant, some human and mouse (18, 19) K chains
`been found to include additional residues in the first
`ariable region (positions 25-32). This additional length
`ypeptide chain will also have an important effect in
`· ing the dimensions of the active site pocket and the
`specificity of the immunoglobulin molecules to which
`belong.
`h the model of Fab' New as a basic structural frame(cid:173)
`a striking correlation between the structure and func-
`f the well-studied MOPC 315 anti-Dnp mouse myeloma
`(20, 21) can be obtained. The L(>..) chains of IgG
`·
`and MOPC 315 are highly homologous and contain an
`number of residues in the third hypervariable region
`een the constant amino acid residues Cys 89 and Phe 99.
`a comparison of the tentative sequence of VH New (Fig.
`d that of VH MOPC 315 indicates that the third hyper(cid:173)
`ble regions of VH in both chains between the constant
`96 and Trp 108 include the same number of amino acid
`Ues. It is, therefore, feasible to fit the MOPC 315 sequence
`~ the basic VL and VH structures obtained in the 2.0-A
`. er map of Fab' New. The model of the MOPC 315
`g site that emerges from this comparison includes a
`
`Fra. 4. View of some of the amino-acid residues at the active
`site of IgG New. Residue numbers for VL (27 to 31 and 89 to 95)
`and Va correspond to t hose 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 10-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 irnmunoglobulin
`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 315 IgA and the bifunc(cid:173)
`tional hapten bis(Dnp-{:1-alanyl)-diaminosuccinate (20)
`in
`which the Dnp groups that join the Fab arms of different IgA
`molecules end to end are only 15 A apart.
`The possibility of a conformational change as a biological
`signal triggered py 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)
`peptid~ 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 µ 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 VL 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,
`
`PFIZER EX. 1115
`Page 9
`
`

`

`3444
`
`Immunology: Poljak et al.
`
`FIG. 5. A view of the a-carbon backbone of Fab' New. The
`V and C1 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 Vn subunit and the CL to the Cnl subunit
`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 C1 domains (see 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(cid:173)
`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 ha¥e been observed after.bind(cid:173)
`ing of ligapds to Fab' fragments (16, 25) the occurrence of
`such changes 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)
`
`)
`Proc. Nat. Acad. Sci. USA 71 (lB74
`f
`and vitamin K10H (16) which can only interact with som
`e 0
`the side chains of the active site.
`This research was supported by Grants AI 08202 from the
`National Institutes of Health, NP-141A from t he American
`Cancer Society, and N .I.H. Research Career Development
`Awatd AI 70091 to R.J.P.
`
`1. World Health Organization (1964) Bull. W.H.O. 30, 447--450
`2. Poljak, R. J ., Amzel, L. M., Avey, H. P., Chen B L .
`Phizackerley, R. P . & Saul, F. (1973 ) Proc. Nat. A~d." &/,
`USA 70, 3305- 3310.
`3. Schiffer, M., Girling, R. L., Ely, K. R. & Edmundson A B
`'
`·
`·
`(1973) Biochemistry 12, 4620--4631.
`4. Epp, 0 ., Colman, P., Fehlhammer, H., Bode, W., Schiffer
`M . & Huber, R. (1974) Eur. J. Biochem., 45, 513-524.
`'
`5. Poljak, R. J., Amzel, L. M., Avey, H. P ., Becka L. N
`Goldstein, D. J. & Humphrey, R. L. (1971 ) Cozd Sp,;,;;
`Harbor Symp. Quant. Biol. 36, 421-425.
`6. Poljak, R. J ., Amzel, L. M ., Avey, H. P., Becka, L. N. &
`Nisonoff, A. (1972) Nature N ew Biol. 235, 137- 140.
`7. Richards, F . M. (1968) J. Mol. Biol. 37, 225-230.
`8. Chen, B. L. & Poljak, R. J. (1974) Biochemistry 13, 1296-
`1302.
`9. Edelman, G. M., Cunningham, B. A., Gall, W. E., Gottlieb
`P. D., Rutishauser, U. & Waxdal, M . J. (1969) Proc. Nat'.
`Acad. Sci. USA 63, 78- 85.
`10. Dayhoff, M . 0., ed. (1972) Atlas of Amino Acid Sequence
`and Struture, Vol. 5 (National BioMedical Research Foun(cid:173)
`dation, Silver Sprµig, Md.).
`11. Low, B. W. & Grenville-Wells, H.J. (1953) Proc. Nat. Acad.
`Sci. USA 39, 785-801.
`12. Peterson, P . A., Cunningham, B. A., Berggard, I. & Edel(cid:173)
`man, G. M. (1972) Proc. Nat. Acad. Sci. USA 69, 1697-1701.
`13. Milstein, C. P., Steinberg, A. G., McLaughlin, C. L. &
`Solomon, A. (1974) Nature 248, 160--161.
`14. Weigert, M. G., Cesari, I. M ., Yonkovich, S. J. & Cohn, M.
`(1970) Nature 228, 1045-1047.
`15. Stanton, T., Sledge, C., Capra, J. D ., Woods, R., Clem, W.
`& Hood, L. (1974) J. Immunol. 112, 633- 640.
`16. Amzel, L. M., Poljak, R. J., Saul, F., Varga, J. M. &
`Richards, F. F. (1974) Proc. Nat. Acad. Sci. USA 71, 1427-
`1430.
`17. Friedenson, B., Appella, E., Takeda, Y., Roholt, 0. A. &
`Pressman, D . (1973) J. Biol. Chem. 248, 7073-7079.
`18. McKean, D ., Potter, M . & Hood, L. (1973 ) 8iochemistry 12,
`760--771.
`19. Barstad, P., Rudikoff, S., Potter, M ., Cohn, M., Konigsberg,
`x E.
`W. & Hood, L. (1974) Science 183, 962-964.
`20. Green, N. M ., Dourmashkin, R. R. & Parkshouse, R. l\,.
`. H N
`(1971) J. Mol. Biol. 56, 203-206.
`21. Francis, S. H ., Leslie, R. G. Q., Hood, L. & EIBen,
`·
`·
`Nol
`(1974) Proc. N at. Acad. Sci. USA 71, 1123-1127.
`22. Poulsen, K., Fraser, K . J. & Haber, E. (1972) Proc.
`·
`S3
`Acad. Sci. USA 69, 2495- 2499.
`23. Appella, E. (1973) Biochem. Biophys. Res. Commun.
`'
`L C
`1122- 1129.
`24. Perutz, M. F., Muirhead, H., Cox, J. M. & Goaman,
`·
`·
`. D R.
`G. (1968) Nature 219, 131- 139.
`1 245;
`25. Padlan, E. A., Segal, D. M., Spande, T. F ., Davies?
`Rudikoff, S. & Potter, M . (1973 ) Nature New Bio·
`165-167.
`
`PFIZER EX. 1115
`Page 10
`
`