J. J.lfol. Bfol. (1986) 190, 593-604
`
`Phosphocholine Binding Immunoglobulin Fab McPC603
`An X-ray Diffraction Study at 2·7 A
`
`Y oshinori Satowt, Gerson H. Cohen, Eduardo A. Padlan and David R. Davies
`
`Laboratory of 111olecular Biology
`National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases
`National Institutes of Health, Bethesda, MD 20892, U.S.A.
`
`(Received 27 December 1985)
`
`The crystal structure of the Fab of McP0603, a phosphocholine-binding mouse myeloma
`protein, has been refined at 2·7 A resolution by a combination of restrained least-squares
`refinemen t and molecular modeling. The overall structure remains as previously reported,
`with an elbow bend angle between the variable and constant moduJes of 133°. Some
`adjustments have been made in t he structure of t he loops as a result of t he refinement. The
`hypervariable loops are all visible in the electron density map with t he exception of three
`residues in the first hypervariable loop of the light chain. A sulfate ion occupies the site of
`binding of the phosphate moiety of phosphocholine.
`
`1. Intro duction
`Direct information about the three-dimensional
`strncture of the antibody combining site is based on
`the crystal structures of three Fab species: Kol, a
`human myeloma. protein with unknown specificity
`(Marquart et al., 1980); New, a human myelorna
`protein that binds to a vitamin Kl derivative
`(Amzel et al., 1974; Saul et al., 1978); and McP0603,
`a mouse plasmacytoma protein specific for phospho(cid:173)
`choline (Segal et al., 1974). Although the structures
`of several monoclonal antibodies with known
`antigen binding specificities are being investigated
`(Mariuzza et al., 1983; Silverton et al., 1984; Colma.n
`et al., 1981; Gibson et al., 1985; Amit et al. , 1985),
`refined, high-resolution structures from them are
`not available.
`The three-dimensiona.J structure for the Fab of
`McPC603 (lgA, K) protein
`that was previously
`reported (Segal et al., 1974} was an unrefined
`structure based on 3· l A diffractometer data and
`with only partial sequence information. This work
`has now been extended through 'the collection of a
`complete set of 2·7 A diffraction data using
`augmented by
`oscillation photography,
`the
`kuow l~dge of Lhe complete amino acid sequence and
`with the help of interactive molecular graphics
`procedures (Lipscomb et al., 1981; Diamond, 1981).
`The crystal structure has been refined using
`restrained least-squares procedures (Hendricltson &
`
`t Present address: P hotQn FactQry, National
`Laboratory for High Energy Physics, Oho-machi,
`Tsukuba-gum, I baraki-ken, 305, Japan.
`
`0022-2836/86/160190- 12 $03.00/0
`
`K.onnert, 1981). In this paper, we report the results
`of this investigation.
`
`2. Materials and Methods
`
`(a) Crystal structure analysis and preliminary
`refinement at 3· 1 A resolution
`Crystals were prepared from concentrated solutions of
`ammonium sulfate as described (Rudikoff ei al., 1972).
`After preparation, the crystals were transferred into
`stabilizing solutions consisting of 50%
`saturated
`ammonium sulfate (pH 7·0), 0·2 M-imidazole or O· l M(cid:173)
`sodium cacodylate. Isomorphous heavy-atom derivatives
`were prepared using TmC1 3 (American Potash & Chemical
`Corp.) K.2Pt(CNS)6 (K &K Laboratories, Inc.), a.nd KI
`(Fisher Scienti6c Co.). The
`thu lium and pla tinum
`derivatives were prepared by soaking crystals in solutions
`cont aining cacodylate and 30 mlll-TmCl3 or 0·4 m111-
`K2Pt(CNS)6, respectively, for 3 to 4 weeks. An iodine
`derivative was prepared by soaking crystals in 50 mM-KI,
`2·5 mM-Chloramine T (Eastman Kodak Co.) for 2 weeks,
`after which the iodinated crystals were washed with
`stabilizing solution to remove excess iodine. Double and
`triple dcrivo.tivoo were prepared using these heavy atom
`compounds. The native and iodinated crystals were
`prepared using imidazole buffer; all other derivatives
`were prepared in cacodylate buffer.
`J\!Iost of the 3· l A data were colJected using a Picker
`FACS-I diffractometer (Segal et al., 1974); the TmCl3
`derivative data set was collected
`from precession
`photographs. Co-ordinates for the heavy-atom sites in the
`t.hulium and platin um derivatives were obtained from
`difference Patterson syntheses at 4·5 A resolution (Padlan
`et al., 1973). Those for the iodine derivative were obtained
`from a difference Fourier synthesis with phases computed
`using the first 2 derivatives. Alternating cycles of heavy-
`593
`
`© 1986 Academic Press Inc. (London) Ltd.
`
`1 of 12
`
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`

`

`594
`
`Y. Satow et al.
`
`atom refinement and phase calculation (Dickerson et al.,
`1961) were then computed using local versions of the
`program$ of ~using et at. (11)62) and Matthews (1966).
`Table l shows the refined heavy-atom parameters. A
`"best " Fourier synthesis (Blow & Crick, 19n9) waR
`computed and a Kendrew skeletal model was fitted to the
`electron density using an optical compa.rator (Richa.rds,
`1968). The model wa:s impruvt'tl u~iHg Lht' cumputer
`graphics system GRIP at the Department of Computer
`Science, University of North Carolina (Tsernoglou et al.,
`1977) and was then subjected to restrained least-squares
`refinement (Konnert, 1976; Hendrickson & Koonert,
`1981) on the TI -ASC computer at the U.S. Naval
`Research Laboratory. The
`initial value of
`the
`conventional crystallographic R-factor was 0·41. 'l'his was
`reduced to 0·30 after 5 cycles with a single overall
`temperature factor and
`tight structural restraints.
`Further refinement using individual temperature factors
`for the atoms reduced the residual to 0·27. The r.m.s.t
`total shift from the original position was 0·76 A. At this
`poin t, the fit of the model to a 2F0 -F0 map was
`examined using BILDER
`(Diamond,
`1981),
`as
`implemented on a PDP 11/70 under the RSX-llM
`operating system (G. H. Cohen, un published results). In
`the second stage of refinement, the protein geometry was
`initially
`less restrained and subsequently tightened,
`yielding a final residual of0·24. The r.m.s. total shift from
`the atomic positions at the start of the second stage of
`refinement was 0 ·42 A. A sulfate ion, that had been
`loca.ted in the hapten binding cavity (P adlan et al., 1973;
`Segal et al., 1974), was
`included
`throughout
`the
`refinement.
`The regions corresponding to residues 10 1. to 108 in the
`heavy chain and residues 31 to 35 in the light chafo were
`not clearly defined in the original electron density
`function based on the heavy-atom phases. These regions
`remained poorly defined after the p reliminary refinement.
`
`(b) Crystal structure analysis and refinement
`at 2·7 A resoluticm
`A new set of crystals was prepared for the higher
`resolution, 2·7 A, phase of this study. The imidazole
`buffer employed during crystallization was replaced by
`cacodylate when
`the crystals were
`transferred
`to
`Intensity data to 2·7 A were
`stabilizing solutions.
`oo!lecwd l!y rotation photography (Arndt & Wo:nacott,
`1977) with Kodak No-Screen Medical X-ray films (3 in a
`pa.ck) using Ni-filtered CuKa radiation from an Elliott
`GX-6 rotating anode X -ray generator operated a.t 40 kV
`and 40 mA. A Franks double bent mirror .system
`(Harrison, 1968) purchased from Brandeis University was
`used to focus the X-ray beam. Data were recorded on an
`Enraf-Nonius Arndt--Wonacott rotation camera with a
`nominal 87 mm crystal-to-film distance. The data
`consisting of 88 film packs were collected from 26 crystals
`by oscillation about the 2 crystal axes, b and c, with an
`
`t Abbreviations used: r .m.s., root-mean-square; F 0 ,
`observed structure factor amplitud.e; F0 , calculated
`structure factor amplitude; w, weight; CDR,
`complementarity determining region (Kabat et rd., 1983);
`VL, light cha.in variable domain; VH, hea.vy chain
`variable domain; CJ~, light chain constant domain; CHl,
`first constant domain of heavy chain; m.i.r., multiple
`isomorphous replacement; L l, L2 and L3, lst, 2nd and
`3rd CDR of Litt: ligbL i.:111.iiu; Hl, H2 11.nd H3, l st, 2nd
`and 3rd CDR of the heavy chain.
`
`Table 1
`Heavy-atom parameters
`
`Co-ordinates
`
`;t
`
`y
`
`z
`
`Occupancyt
`
`Thermal
`fact-Or
`(A'l
`
`Heavy-atom
`compound
`
`0-0218
`0-3936 0·5422
`03807
`10·6
`K 2Pt(CN8)6
`34-8
`TmCl3
`0·0245
`0·6617 0·2523
`0·3427
`Iodine-I
`28·3
`0·0119
`0·7083 0·5569
`0·2336
`7-5
`Iodine-2
`0·0184
`0·2409 0·6794 0·5695
`6 ~ .,
`0·0125
`0·6328 0·6404
`Iodine-3
`0·2328
`0·1241
`0·7017
`0·4838
`6·8
`0·0044
`Iodine-4
`t The site occupancy ia on an arbitrary scale in which the
`average structure amplitude of t he native protein is 14·3.
`
`oscillation range of l ·25° a11d an overlap of 0·25°. The
`time for each exposure was 14 to 21 h.
`The films were scanned a.t 100 µm steps on an
`Optronics P -1000 film scanner. The initial film processing,
`which included preliminary refinement of the crystal
`orientations and successive evaluation of the integrated
`intensities, was made on a PDP 11/70 computer using the
`rotation program package written by G. Cornick & jj_ A
`~avia (unpubli11hed. result.<s). Intensities from each pack
`were further processed through intra-film-pack scaling,
`post refinement, scaling and averaging using programs
`specially writte11
`for
`these purposes
`(Y. Satow.
`unpublished results). I ntensities in a single pack were
`scaled by refining non-linear respo·nse correction factor~
`(Matthews et al., 1972) and absorption factors for the film
`base a.nd emulsion, then were corrected for Lorentz and
`polal'ization factors. The measured intensities from the
`films were merged, reduced to a unique set and processed
`by a scaling and averaging program, which l'efines
`fall-off factors
`for
`relative scale and exponential
`films. This program
`follows closely
`the
`individual
`formalism of the established algorithms of Hamilton el. al.
`(1965) and Rossmann et al. (1979). P ost refinement of the
`crystal orientations, lattice constants and rocking curve
`para.meters were done as proposed by Winkler et al.
`(1979) and Rossmann et al. (1979). The total n umber
`of J 47 ,706
`intensities,
`including partially recorded
`reflections, were finally scaled and symmetry averaged,
`yielding 24,235 uniq ue reflections for the resolution range
`of 10 A w 2•7 A. The u.greerue11t faetol':
`R =LL 11.;-I.11L. ,\~[h,
`j
`h
`h
`where the intensity /hi for reflection h was measured .Vh
`times, wos 0·077 01• 0·054 for
`the structure factor
`amplitudes Fhi· Within a 3 A sphere, 95% of the data
`were retained; within the complete 2·7 A sphere, 93% were
`retained. The earlier diffractometer data were not
`included in this final set of 2·7 A film data.
`The initial model for the 2·7 A work was ta.ken from
`the 3· l A results. It was subjected to a number of cycles
`of least-squares refinement (Hendrickson & Konnert,
`1981) with periodic examination and rebuilding of the
`model using BILDER on a VAX 11/780 (R. Ladner,
`unpublished results). The sulfate ion located in the 3· l A
`analysis was not included in the early stages of least(cid:173)
`squares refinement. With an overall temperature factor,
`the initial value of the R-fact-Or was 0·41. After 5 cycles of
`positional parameter
`refinement with an overall
`temperature factor, the value was reduced to 0·33.
`Indi vidu~I temperature factors for the atoms were used in
`the succeeding cycles. The program restrains the values of
`
`Nh
`
`2 of 12
`
`Celltrion, Inc., Exhibit 1085
`
`

`

`Phosphocholine Binding lmmunoglobulin Fab McPC603
`
`595
`
`the B-factors so that each is influenced by the B-factors
`of the atom.s to which it is bound as well as the atoms 1
`removed along a chain. In t he final stages of least-squares
`refinement, particular care was taken to ensure that the
`st.e1'eochemist.ry wn.<;
`t.h:..t.
`kio.pt.
`rP.n.<;nn:..blA
`R.nil
`w(IF0l-IF0 1)2 was approximately constant over the range
`of the data used (8·0 A through 2·7 A).
`The main-chain
`stereochemistry was constantly
`the program GEOM (G. H. Cohen,
`monitored by
`unpublished results) and points of significant departure
`from expected stereochemistry were examined and
`corrected via interactive computer graphics. In a<ldition
`t-0 a sulfate
`ion, 138 water molecules of variable
`occ.upancy were id entified from examination of !!Ji' and
`2F"-F0 maps and refined with the protein molecule.
`As sequence <la.ta became available, the " working"
`sequence was updated appropriately. The VL and VH
`sequences are listed (sequences 12, p. 45 and 1, p. 128,
`respectively) by Kabat et al.
`(1983), quoted from
`Rudikoff et al. (1981) and Rudikoff & Potter (1974), with
`the addition of the tetrapeptide Leu-Glu-Ile-Lys, which
`occurs at the end of V J, (Rudikoff, unpublished results).
`The CHl sequence given by Auffray et al.
`(1981),
`obtained by translation of the nucleotide sequence of
`cDNA complementary to alpha-chain mRNA from J 558
`tumor cells, (sequence 60, p. 175, Kabat et al., 1983) was
`used. This sequence differs in 4 p laces from that obtained
`by Tucker et al. (198 1) from BALB/c genomic DNA,
`while they both d iffer in 2 of these positions from the
`sequence reported by Robinson & Appella (1980) for
`MOPC51 l , as quoted by K abat et al. (1983). Examination
`of the final electron density map at these positi-0ns did
`not permit a clear distinction to be made between these
`seq uences. The sequence ofMOPC21 (sequence23, p. 167,
`Ka.bat et al., 1983 from Svasti & Milstein, 1972) was used
`for the CL domain of McPC603.
`Throughout this paper, the amino acid numbering is
`serial, starting from number I with the 1st residue of each
`chain of the molecule. The correspondence between our
`numbering scheme and that of Kabat et al. (11.983) is
`presented in Table 2.
`As noted by Segal et al. (1974), t he molecule possesses 2
`approximate loca.l dyad symmetry axes, which relate the
`pair of variable domains and the pair of constant domains
`to each other . We examined the relationship and the
`
`Table 2
`Correspondence between the numbering scheme used
`here and that of Kabat et al. (1983)
`
`L.ight chain
`
`Heavy chain
`
`This work
`
`Kabat et al.
`
`This work
`
`Kabat et al.
`
`1- 27
`28- 33
`34-220
`
`1- 27
`27a-27f
`28- 214
`
`l-52
`53- 55
`56- 85
`86-88
`89-106
`107-109
`lJ()- 139
`140- 142
`143- 163
`164-171
`l 7:2-184
`18..5-200
`201- 205
`206-216
`217- 222
`
`1-52
`52a- 52c
`53-82
`82a- 82c
`83- 100
`IOOa-lOOc
`101- 130
`133- 135
`137- 157
`162-169
`171-183
`185-200
`202-206
`208-218
`220-225
`
`similarity of these pairs of domains using the program
`ALIGN
`(G. H. Cohen, unpublished
`results), which
`iteratively rotates one set of atoms to another set t-0
`optimize their fit while preserving the order of the Hnear
`seqnen~.es of the 2 .~et.s. The program uges the algorithm
`of Needleman & Wunsch (1970) to identify the structural
`homology while accounting for insertions and deletions.
`Interdomain
`and
`intermolecular
`contacts were
`calculated with the aid of the program CONTAX (E. A.
`Padlan, unpublished results). Two at-Oms are defined to
`be in oontact if their co-ordinate8 lie within the sum of
`their van der Waals radii plus l ·O A. Intramolecular
`main-chain hydrogen bonds were calculated by the
`program EREF (M- Levitt, personal communication).
`
`3. Results and Discussion
`Table 3 shows an estimate of the qu ality of the
`stereochemical parameters for the final model . I t is
`expressed in terms of t he r.m.s. deviations of the
`various classes of parameters from accepted values
`(Siclccki et (tl., 1\)70). The </>,l/t plot for the main
`cha in is shown in Figure 1. There are a few residues
`t hat have " forbidden" </>,t/I values. The quality of
`t he map in these regions together with the low
`
`Table 3
`Summary of stereochemical criteria (Hendrickson &
`Konnert, 1981; Sielecki et al., 1979; Cohen et al.,
`1981)
`
`Final model
`
`Target u
`
`R= IllF.l - IF.11
`IIF.,1
`Average l!i.F
`Interatomic distances (A)
`1- 2
`1- 3
`1-4
`Planarity (A)
`Chiral volumes (A3 )
`~on-bonded contacts (A)
`1-4
`Other
`Angles (deg.)
`w , Xs (Arg)
`Xv · · ·, X4
`Temperature factors (A2 )
`Main chain 1- 2
`Main chain 1- 3
`Side chain l-2
`Side chain 1- 3
`
`0·225
`136
`
`0·020
`0·040
`0·037
`0·027
`0·257
`
`0 ·24
`0·34
`
`27·0
`5·0
`
`0·5
`l·O
`0·4
`0·7
`
`t
`
`0·015
`0·020
`0·025
`0·020
`0·150
`
`0·50
`0·50
`
`15·0
`5·0
`
`0·5
`0·7
`0·5
`0·7
`
`The standard groups dictionary used is specified in Table 2 of
`Sielecki et al. (1979). ln this Table, F0 refers to the observed
`structure factor, F0 is the calculated structure factor and l!i.F is
`the quantity llF.1-1.li',ll. The target a represents the inverse
`square-root of the weights used for the parameters listed. The
`value~ given are the r.m.s. deviations from the respective ideal
`valaes.
`t The weight chosen for the structure factor refinement, the
`"target er" of l!i.F, was modeled by the function w = (1/d)2 with:
`d = 40-500(sin(O)/A.- l/6).
`By this means, w(l!i.F) was approximately constant over all of
`tbe data (w representing the weight for a given reflection). Other
`details of this Table are explained fully by Cohen et al. ( 1981).
`
`3 of 12
`
`Celltrion, Inc., Exhibit 1085
`
`

`

`596
`
`Y. Satow et al.
`
`-135. 00
`
`-90. 00
`
`-QS. 00
`
`0 . 00
`
`QS.00
`
`90. 00
`
`135. 00
`
`(!)
`
`(!)
`
`0
`0
`0
`"'
`
`0
`0
`~
`
`x
`
`"'
`
`0
`- o
`._o
`V> •
`
`t!> o
`
`(!)
`
`····· o ·······i
`(!)
`
`"'
`
`(!)
`
`t!> Cj.
`
`x
`
`(!)
`
`t!> x
`(!)
`~ (!) Cl
`(!)
`
`(!)·t!>@···
`
`"'e
`
`t!> t!>f"'
`
`Xx
`
`x
`
`l!)
`
`l!)
`
`(!)
`
`(!)
`
`(!)
`
`(!)
`
`x
`
`~
`
`<!> (!)
`
`(!)
`x
`
`x
`
`(!)
`
`x
`
`.,('.
`
`x
`
`x
`
`"' '" 0
`
`0
`
`ID
`p
`
`0
`0
`
`"'
`'"
`
`0
`0
`
`p
`0
`0
`
`Q
`~
`"'
`'
`
`~
`
`Q
`Q
`
`C>
`
`'!'
`
`0
`C>
`
`~
`
`0
`Cl
`0
`.,
`0
`x
`7
`- 180 .00
`
`t!>
`
`~
`
`(!)
`
`(!)
`x
`
`0
`
`e
`
`i~ ~
`Cl(!)(!) ~(!) lc (!)
`
`<ID(!)
`
`(!)
`
`(!)
`
`C!I!>
`(!)
`'1J!J (!>(!)
`
`(!)
`
`(!)
`
`x
`
`Ill
`
`(!)
`
`Cl
`
`llE O X
`
`(!)
`
`x
`
`x
`
`(!)
`
`x
`
`x
`
`x
`
`'
`"'
`~
`g
`
`I
`ID
`0
`Q
`0
`
`'
`w
`~
`0
`0
`
`(!)
`x
`
`x
`
`x
`
`-l35. 00
`
`- 90. 00
`
`- 5 .00
`
`0.00
`PHI
`
`ijS. 0 0
`
`90. 00
`
`135. 00
`
`18 0 . 00
`
`Figure I. A plot of the dihedral angles at each alpha-carbon at om for t he refu1ed co-ordinates. Asterisk, Pro; cross,
`Gly; circle, any other residue.
`
`the assignment of more
`thwa.rted
`resolution
`satisfactory geometry. The estimate of error in this
`model is 0·3 A as determined by the method of
`Luzzati (1952). The final crystallographic R-factor
`is 0·225 for 23,737 reflections in the range of 8·0 A
`to 2·7 A. The diffraction data, co-ordinate data,
`temperature factors and solvent occupancies for the
`Fab of McPC603 have been deposited in the Protein
`Data Bank at Brookhaven N ational Laboratory
`(Bernstein et al., 1977).
`
`(a) General structure of the Pab
`Several changes were observed in t he molecule as
`a consequence of this refinement, although the
`overall structure remained similar to that described
`earlier (Segal et al., 1974). Most of these changes are
`to be found in the details of the loops, in particular
`Ll and H 3. The region of LI is poorly defined in the
`electron density maps, even with the higher(cid:173)
`rebuilt
`the
`resolution data. We have now
`neighboring H3, whose density has become less
`the
`am biguous and, concurrently, have altered
`conformation of L l . In Figure 2, L I may be noted to
`protrude from the general surface of the molecule.
`The direction of t his protrusion
`is reasonably
`correct but the electron density is too weak to
`define the positions of th e t h ree ou termost a mino
`acid residues.
`
`Three other residues have tentative placements
`due to problems in maintaining proper stereo(cid:173)
`chemistry while fitting the electron density: Glnl62
`and Asnl 63 (resid.ues 156 and 157 in the numbering
`used by Kabat et al., 1983) of CL and Glu202
`(residue 203, Kabat et al., 1983} of CHI . In these
`cases, we permitted the stereochemical constraints
`to dictate
`the
`final placement. Also, several
`disordered side-chains were observed within the
`entire molecule. In all t.hese cases, the configuration
`corl'esponding to the st.ronger density was chosen
`for the model.
`The 442 residues of :\1.cPC603 Fab include 25
`proline residues. Of
`these, fi ve a.re cis-proline:
`residues 8 and JO I in VL, 147 in CL , and 143 and
`l55 in CHI. The assignment of the configuration of
`all fi ve ci.s-proline residues was unambiguous. The
`structurally homologous Pro8 and Pro95 of Rei
`(Huber & Steigemann, 1974), and Prol 47 (CL) and
`Prol 55 (CHI ) of K ol (Marquart et al., 1980), also
`have been found in the cis conformation. Prol43
`(CH l ) of McPC603 has no counterpart in previously
`reported antibody structures.
`
`(b) Crystal padcing
`The McPC603 Fab crystallizes in the space group
`(Ru d ikoff et al., 1972), with a= 162-53 A,
`P63
`c = 60·72 A. The molecules are situated in clusters
`
`4 of 12
`
`Celltrion, Inc., Exhibit 1085
`
`

`

`Phosphocholine Binding l mmunoglobulin Fab llfcPG603
`
`597
`
`Figure 2. A stereo drawing of the alpha-carbon skeleton of ..\IcPC603. Continuous lines denote t he heavy chain. The
`filled circles show t he complementarity-determining residues (Kabat et al., 1983).
`
`of three at each of the crystallographic 3-fold axes
`of the unit cell (Fig. 3). Each cluster is related to
`neighboring clusters via the 2-fold screw situated
`midway between the 3-fold axes (Figs 3 and 4). The
`clusters of three are maintained principally by a set
`
`of hydrogen-bond and van der Waals contacts
`between neighboring molecules. Residues 14 to 20
`and 71 of VL interact with residues l , 26, 27, 100,
`102, 104, 105 108 a.nd llO of the VH of the
`neighboring molecule while residues 18, 66, 67, 69,
`
`Figure 3. A projection of the alpha-carbon skeletons of 4 unit cells and t he ab plane, illustrating t he large channels
`that nm parallel to the c axis through the crystal. The heavy chains have been drawn bolder in the 3 molecules t hat are
`clustered about a crystallographic 3-fold axis of the lower left-hand cell. The 3-fold axes a re indicated by t he symbol ..&:
`2-fold screw axes are located midway between each adjacent pair of 3-fold axes; a 63 axis is located at each corner of
`each cell .
`
`5 of 12
`
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`
`

`

`598
`
`Y. SaJ.ow et al.
`
`Figure 4. The projected structure viewed perpendicular to the ac plane, illustrating the effect of the dyad screw a.xis.
`The plane of projection is along the long diagonal of the unit cell (see Fig. 2). It contains the two 3-fold axes and Lhc 2-fold
`screw midway between them. For clarity, only t be 4 molecules closest to the plane have been drawn. Tho heavy line
`corresponds to t he heavy chain of the molecule.
`
`73 and 82 of the same VL meet the VL of the
`neighboring molecule at residues 35, 55 and 61 to
`63. This pattern repeats three times around the
`axis. Other intermolecular contacts are found
`between VH of one molecule and CL and CH 1 of a
`second molecule related to the first bv t he 2-fold
`screw axis. Several additional contacts are found
`between VL and CHI of a neighbor via the z unit
`cell translation. It has been noted (Padlan et al. ,
`1973) that roughly 70% of Lhe cell volume is
`occupied by solvent, thus permitting the diffusion
`of hapten to t he molecule in the crystal. The long
`axis of
`t he molecule makes an angle of
`appro~'imately 50° with t he xy plane of the unit ceU
`(Fig. 4).
`
`( c) I ntramolecular pseudosymmetry
`Analysis of t he approximate 2-fold axes relating
`pairs of domains yielded the following results: for
`the VL/VH relationship, we find a 173° rotation
`with a r.m.s. deviation of l ·3 A (3·5 A for poorest
`agreement ) between 385 matched pairs of main(cid:173)
`chain atoms out of 456 atoms. A number of the
`residues of the hypervariable loops align quite well,
`particularly at t he beginning and end of each loop.
`The departure from a more exact 2-fold symmetry
`is probably due to the fact t hat t he interface
`residues can be described as fitting a cylinder
`containing four strands from the light chain and
`five strands from the hell-vy chain (Novotny et n.l.,
`1983). This asymmetry of t he VH/VL interaction
`distinguishes it from most of t he VL/ VL inter(cid:173)
`actions observed in Bence- Jones dimers which, in
`many cases, display exact 2-fold symmetry. The
`dya<l symmetry of the CL/CHI pair is not as close.
`The best alignment for the constant modules uses
`
`358 of the possible 400 pairs of main-chain atomis to
`achieve a r.m.s. deviation of 2·0 A (5· l A for poorest
`agreement) between t he pairs of matched atoms.
`The best fit between CL and CH l
`involves a
`rotation of 169° together with a t ranslation of
`2·6 A a long t he rotation axis. A comparison of t hese
`two axes of rotation yields an elbow bend of 133°,
`essentially unchanged from the earlier report (Segal
`et al. , 1974).
`
`( d) I ntramolecular contacts
`As noted elsewhere (reviewed by Davies &
`Metzger, 1983; Amzel & Poljak, 1979), VL/ VH
`contacts involve hypervariable residues as well as
`framework residues. The boxed regions of Table 4
`indicate hyperva.riable
`to hy_µervaria.ble residue
`interactions, while other regions of the Table
`involve framework residues. Nearly half (46 in 105)
`of t he interactions between VL and VH involve
`only hypervariable residues, with most of these
`being located at the upper end of the interface
`(Fig. 2) in the vicinity of the combining site and
`with the framework interactions at the other end. A
`number of good hydrogen-bonded contact,s occur,
`notably two between t he highly conserved residues
`Gln44L and Gln39H, between Gln39H and Tyr93L,
`between t he CDR residues Asp97L and Asn I 0 I H ,
`and between the hydroxyl group of TyrlOOL and
`Glu35H . The latter interaction appears to be
`import.a.nt in ms\..intaining the integrit.y of t.he
`phosphocholine binding pocket (Rudikoff et al.,
`1981) as it has been observed (Rudikoff et at., 1982)
`that a. mutation of Glu35H to Ala results in a loss of
`phosphocholine binding ability.
`Other interdomain, intramolecular conLacts are
`presented in Tables 5 to 7. There are no interactions
`
`6 of 12
`
`Celltrion, Inc., Exhibit 1085
`
`

`

`Phosphocholine Binding Immunoglobulin Fab M cPC603
`
`599
`
`Table 4
`Contacts between residu,e,s of V L and V H
`
`Y33 E35 Q39 R44
`
`IA5 W47 A50 E61 Y97 NlOI Yl03 8 105 Tl06 W107 Y l 08 Fl09 Wll2 Gl l3 All4
`
`I:
`
`4
`
`Q95 D
`
`I .
`'----~
`
`I.
`
`K36
`F38
`Y42
`Q44
`P49
`P50
`152
`Y55
`G5G
`Y93
`
`~~~
`
`P!Ol
`Ll02
`Fl04
`GJ05
`Al06
`
`5
`
`3
`
`5
`
`2
`
`3
`
`2
`
`l
`9
`
`4 D l ._____
`
`2
`
`3
`
`1
`1
`6
`
`4
`4
`
`8
`
`_
`
`10 -2 ;
`
`Residues listed in the left column are from VL; the residues listed abo\'e the columns are from VH.
`The numbers in the Table correspond to the number of pairs of atoms from 2. residues that are within potential van der Waals
`contact distance, as calculated by the program CONTAX (see Materials and Methods, section (b)). A dot indicates no contacts.
`The boxed regions delineate possible hypervariable/hypervariable interactions.
`
`involving VL with CHI or VH with CL. Tables 6
`and 7 show that the number of interdomain
`intrachain contacts, i.e. VL with CL or VH with
`CHI, is small.
`
`(e) Domain structure
`The structures of the four domains of McP C603
`are illustrated by Figure 5, which shows also the
`location of the hydrogen bonds between t he main(cid:173)
`chain amide groups. The general tertiary structure
`of the domains and the distribution of the hydrogen
`
`bonds are very like those of other antibody classes
`and subgroups. Thus, the o: heavy-chain doma ins of
`McPC603 Fab resemble
`those of Lhe y chain
`domains of the human proteins New and Kol, and
`the K chain domai n~ of McPC:~O~ resemble the A.
`chain domains of New, Kol and Meg, as well as the
`K chains of various human VL dimers (Padlan &
`Davies, 1975; Padlan, 1977). A significant difference
`from the y chain structure occurs in CHI , where
`there
`is an additional disulfide bond formed
`between residues 198 and 222 (residues 198 and 225,
`in Kabat et al., 1983). The Cys222 residue should
`therefore be regarded as forming the end of CHI
`
`Table 5
`Contacts between residues of CHI and CL
`
`YJ31 PJ32 Ll33 Tl34 L t:l5 Pl36 Il45 Vl73 Fl75 P l 76 A178 8188 QI90
`
`ll23
`Fl24
`8127
`El29
`Q;l 30
`S l33
`Vl39
`Fl41
`Nl43
`Ll66
`s !(18
`W l69
`Tl70
`8 180
`Ml 81
`8182
`R217
`
`2
`5
`
`4
`
`5
`
`3
`3
`
`14
`l
`
`2
`2
`
`3
`
`2
`4
`
`l
`2
`
`3
`
`.l
`3
`5
`
`Residues list.ed in the left columns a re from CHl ; the residues listed above the columns a.re
`from CL.
`The numbers are as in Table 4.
`
`7 of 12
`
`Celltrion, Inc., Exhibit 1085
`
`

`

`600
`
`Y. Satow et al.
`
`Table 6
`Contacts between residW3s on V L and CL
`
`Table 7
`Contacts between residW3s of VH and CHI
`
`Yl46 Dl7l Ql72 8174 Kl75 Dl76 8177 Yl79
`
`P46
`A86
`L89
`Kl09
`El 11
`Il l 2
`Kll3
`Rll4
`
`2
`I
`6
`
`2
`
`7
`
`4
`
`3
`5
`
`6
`
`5
`
`The residues listed in the left column are from VL; those listed
`at the column heads are from CL.
`The numbers are as in Table 4.
`
`EJ23 8124 Al2-.5 Fl54 Pl55 0157 T l 58 D21l 8212
`2
`
`2
`
`2
`
`4
`
`3
`
`3
`
`G9
`GlO
`Lil
`Tll7
`Tll9
`8121
`
`The residues listed in the left column are from VH; those listed
`at the column heads are from CHl.
`The numbers are as in Table 4.
`
`( c )
`
`\ d)
`Figure 5. Schematic drawings of the (a) VH, (b) VL, (c) CHl and (d) CL domains illustrating the hydrogen
`bonds between t he main-chain amide residues. The circles marking hypervariable residues in V H and VL are drawn
`with a double line.
`
`8 of 12
`
`Celltrion, Inc., Exhibit 1085
`
`

`

`Phosphocholine Binding lmmunoglobulin Fab .ll1cPC603
`
`601
`
`8:0
`
`7·0
`
`6·0
`
`4·0
`
`3·0
`
`2·0
`
`1·0
`
`f YME ..... QPPIKRLEW I AASllN KGNKYTTEYS ASVKGRF I VS ROISQS I L YLQMNALRAEDT A I YYCAANYYGSTWYFDYWSAQTTVTVSS
`E VKL VUG GGL YQPIGSlR LSCA TSGFT FSD
`o 1vr.1roSPs~i:sYSA8ERV!M1C•ss osL -!NSGNOKNFL~wvoaiCrsoP!: KLL IVGA
`sr~ESGVP oRFr§SGSG riiFn_!1 ssvoiEDL!vmai<oHs
`!PLTF&AGTK~EI KR
`90
`50
`20
`40
`70
`30
`100
`10
`60
`80
`110
`( g)
`
`-
`
`-
`
`- -
`
`-
`
`- ----- ----
`
`-
`
`~ - - - -
`
`~
`
`~ - - - 1-
`
`-
`
`-
`
`8-0
`
`~o
`
`...
`
`5-0
`
`+o
`
`3·0
`
`-
`
`...
`2·0
`
`...
`1·0
`
`8·0
`
`7·0
`
`6·0
`
`5·0
`
`4 ·0
`
`3·0
`
`2·0
`
`1·0
`
`8-0
`
`~o
`
`&O
`
`+o
`
`-3·0
`
`-
`2·0
`
`1·0
`
`1, 1111
`11
`11111
`l1l1
`111
`..
`KSGKO .!. TIVNFPPAL~SGG RYTlllSNQL TLPAVECP E_GESVKCSVQ!!OS NP VOELO~llC
`ESARN P'T ,!.YPI. TLPPAL§_ SOPV I I GCL.!,HOYFl'SGTM!!YTWG
`AOAAP!VS I FPPSSEQLTSGGASVV~FLNNFYPK 0 .!. NVKWKI DGSE_RO NGVLNSW!OODSKOSTY§_MSSTL TL TK!1:EYERH NSYT£EATH KTSTSf'. I VKSFllRNE£
`120
`130
`140
`150
`160
`170
`180
`190
`200
`210
`220
`
`1 u I
`
`I
`
`"I
`
`Figure 6. The separation (in A) of corresponding alpha-carbon a-toms of the superimposed domains of (a) VH an d VL,
`and (b) CHI and CL. The upper sequence is t hat of the heavy chain and the lower sequence is of the light chain. Every
`10th letter in each sequence is underlined. The numbers below the sequences refer to t he residue number in the light
`cha.in _ Where the structural alignment has matched identical residue types, the corresponding letters are shown in bold
`face. Gaps occur when there are no corresponding residues for comparison. The horizontal broken lines indicate the
`r.m.s. separation.
`
`( b)
`
`9 of 12
`
`Celltrion, Inc., Exhibit 1085
`
`

`

`602
`
`Y. Satow et al.
`
`rather than the beginning of the hinge, consistent
`with the gene structure obser\1ed by Tucker et al.
`(1981).
`The structures of the light and heavy chain
`domains of McP C603 are compared in Figure 6(a)
`and (b). The sequence identity derived from t he
`structural alignment is 25% for the variable pair
`and 22% for the constant pair, excluding gaps. In
`the variable domains,
`the framework residues
`superimpose rather well, except for the region from
`residues 60 to 74. Large differences are observed in
`the CDRs, but these are frequently t he result of
`differences in the lengths of the hypervariable loops.
`In the constant domains, the differences between
`the light and heavy chains are comparable to those
`found between the variable domains.
`
`(f) Bound carbohydrate
`Robinson & Appella (1979) noted the presence of
`carbohydrate attached to Asn l55 (CH I) during
`their sequence analysis of t he heavy chain of MOPC
`47A. In electron density maps of McPC603 there is
`suggestive density in the vicinity of the homologous
`Asnl60 that indicates the p ossibility of carbo(cid:173)
`hydrate here as well. This density is, however,
`insufficiently defined to permit fitt ing of any
`carbohydrate ch ain. It appears, therefore, that
`carbohydrate does not occupy a fixed position on
`the surface of the molecule. T he lack of any nearby
`contacts from neighboring domains could also
`contribute t o the delocalization of the carbohydrate
`moiety. An analogous poorly defined trace of
`density is found in the electron density map of the
`Fab of J539 (Suh et al., unpublished results).
`
`(g) 'l'he sulfate ion in the wmbining sit