J. Jlol. Biol. (1986) 190, 593-604
`
`Phosphocholine Binding Immunoglobulin Fab McPC603
`An X-ray Diffraction Study at 2·7 A
`
`Yoshinori Satowt, Gerson H. Cohen, Eduardo A. Padlan and David R. Davies
`
`Laboratmy of Molecular 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
`refinement and molecular modeling. The overall structure remains as previously reported,
`with an elbow bend angle between the variable and constant modules of 133°. Some
`adjustments have been made in the structure of the loops as a result of the refinement. The
`hypervariable loops are all visible in the electron density map with the 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.
`
`I. Introduction
`Direct information about the three-dimensional
`structure 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); �ew, a human myeloma
`protein that binds to a vitamin Kl derivative
`(Amzel et al.. 1974; Saul et a.Z., 1978); and )foPC603,
`a mouse plasmacytoma protein specific for phospho­
`choline (Segal el al., 1974). Although the structures
`of several monoclonal antibodies with known
`antigen binding specificities are being investigated
`{�fariuzza et al., 1983; Silverton et al., 1984; Colman
`et al., 1981; Gibson et al., 1985; Amit et al., 1985),
`refined, high-resolution structures from them are
`not available.
`The three-dimensional structure for the Fab of
`McPC603 (IgA,x:) protein LhaL was previously
`reported {Segal et al., 1974) was an unrefined
`structure based on 3· I A diffractometer daLa 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
`oscillation photography,
`augmented by
`the
`knowledge of the complete amino acid sequence and
`with the help of interactive molecular graphics
`procedures (Lipscomb et al., 1981; Diamond, 1981).
`The crystal structure bas been refined using
`restra,ined least-squares procedures {Hendrickson &
`
`t Present address: Photon Factory, Kational
`Laboratory for High Energy Physics, Oho-machi,
`Tsukuba-gum, Ibaraki-ken, 305, Japan.
`
`0022-2836/86{160190-12 503.00/0
`
`Konnert, 1981). In this paper, we report the results
`of this investigation.
`
`2. Materials and Methods
`(a) Crysta/, structure analysis and prelirninary
`refinem.ent at 3· 1 A resolution
`Crystals were prepared from concentrated solutions of
`ammonium sulfate as described (Rudikoff el al., 1972).
`After preparation, the crystals were transferred
`into
`stabilizing
`solutions consisting of
`50%
`saturated
`ammonium sulfate (pH 7·0), 0·21'>1-imidazole or O·l M­
`sodium cacodylate. Isornorphous heavy-atom derivatives
`were prepared using TmCl3 (American Potash &. Chemical
`Corp.) K2Pt(CNS)6 (K&K Laboratories, Inc.), and Kl
`(Fisher Scientific Co.). The thulium and platinum
`derivatives were prepared by soaking crystals in solutions
`containing cacodylate and 30 nrn-TmCl3 or 0·4 mll­
`K2Pt(CNS)6. respectively, for 3 to 4 weeks. An iodine
`derivative was prepared by soaking crystals in 50 rru-1-KI,
`2·5 mM-Chloramine T (Eastman Kodak Co.) for 2 weeks,
`after which the iodinated crystals were waahed with
`stabilizing solution to remove excess iodine. Double and
`triple derivatives were prepared using these heavy-a.tom
`compounds. The native and
`iodinated crysta.Js were
`prepared using imidazole buffer; all other derivatives
`were prepared in cacodyla.te buffer.
`:iiost of the 3·1 A data were collected using a Picker
`FAr..S-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
`thulium and platinum derivatives were obtained from
`difference Patterson syntheses at 4·5 A resolution (Pad.Jan
`el 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. (Lonrlon) Ltd.
`
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`

`594
`
`Y. Saiow et al
`
`atom refinement and phase calculation (Dickerson et al.,
`I 96l} were then computed using local versions of the
`programs of Busing et al. (1962) and �fatthews (1966).
`Table I shows the refined heavy-atom para.meters. A
`·'best" Fourier synthesis
`(Blow & Crick, 1959) was
`computed and a Kendrew skeletal model was fitted to the
`electron density using an optical compa.rator (Richards,
`1968). The model was improved using the computer
`graphics system GRIP at the Department of Computer
`Science, University of '.'forth Carolina (Tsernoglou et al ..
`1977) and was then subjected to restrained least-squares
`refinement (Konnert, 1976; Hendrickson & Konnert,
`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. This was
`reduced to 0·30 after 5 cycles with a single overall
`temperature
`factor and tight structural restraints.
`Further refinement using indi,,idual 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
`point, the fit of the model to a 2F0-F. map was
`examined using BILDER
`(Diamond,
`as
`1981),
`implemented on a PDP 11 /70 under the RSX-llM
`operating system (G. II. Cohen. unpublished 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
`located in the hapten binding cavity (Padlan et al., 1973;
`Segal el al.. 1974), was
`included
`throughout the
`refinement.
`The regions correspondjng to residues 101 to 108 in the
`heavy chain and residues 31 to 35 in the light cha.in were
`not clearly defined
`in the original electron density
`function based on the heavy-atom phases. These regions
`remained poorly defined after the preliminary refinement.
`
`(b} Crystal stmclure analy1ti8 and refinement
`at 2·7 A re8olution
`A new set of crystals was prepared for the higher
`resolution, 2·7 A, phase of this study. The imidazole
`hnffar P.mployP.rl rl11ring cryetallization was replaced b y
`transferred to
`cacodylate when
`the crystals were
`stabilizing solutions. Intensity data to 2·7 A were
`c.:u!Jt:cLe<l by mtation photography (Arndt & Wonacott.
`1977) with Kodak Ko-Screen Medical X-ray films (3 in a
`pack) using Xi-filtered CuKtX radiation from an Elliott
`GX-6 rotating anode X-ray generator operated at 40 k\.
`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-Non.ius 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 use<l: 1".m.1>., ruuL-mt:as1-1;quare; F0•
`observed structure factor amplitude; F .. calculated
`structure fact.or amplitude; w, weight; CDR.
`complementarity determining region (Kabat et al., 1983);
`VL, light chain variable domain; VH, heavy chain
`variable domain; CL. light chain constant domain; CHI,
`first constant domain of heavy chain; m.i.r., multiple
`isomorphous replacement; Ll, L2 and L3, 1st, 2nd and
`3rd CDR of the light chain; HI, H2 and H3, 1st, 2nd
`and 3rd CDR of the heavy chain.
`
`Table 1
`Heavy-atom pararn.eters
`
`Co-ordinates
`
`Heavy-atom
`compound
`
`K2Pt(CNS)6
`TmCl3
`Iodine-I
`lu<liue·2
`Tocline-3
`lodine-4
`
`x
`
`y
`
`z
`
`Occupancyt
`
`0·3807
`0·3427
`0·2336
`0·2409
`0·2328
`0·1241
`
`0·3936
`0·6617
`0·7083
`0·6794
`0·6328
`0·7017
`
`0·5422
`0·2523
`0·5569
`0·5695
`0·6404
`0·4838
`
`0·0218
`0·0245
`0·0119
`0·0184
`0·0125
`0·0044
`
`Thermal
`factor
`(A')
`
`19·6
`34·8
`28·3
`7·5
`6·i
`6·8
`
`t The site occupancy is on an arbitrary scale in which the
`average structure amplitude of the native protein is 14·3.
`
`oscillation range of l ·25° and an overlap of 0·25°. The
`time for each exposure was 14 to 21 h.
`The films were scanned at 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 & �LA.
`�avia. (unpublished results). Intensities from each pack
`were further processed through i.ntra-film-pack scaling.
`post. refinement, scaling and averaging using programs
`(Y. Satow.
`specially \\Titten
`fol'
`these purposes
`unpu blishecl results). Intensities in a single pack were
`scaled by refining non-linear response correction factors
`(.:\latthews et al., 1972) and absorption factors for the film
`base and emulsion. then were col·rected for Lorentz and
`polarization factors. The measured intensities from the
`films were merged. reduced to a unique set and processed
`by a scaling and averaging program, which refines
`relative scale and exponential fall-off fact-0rs for
`individual films. This program follows closely
`the
`formalism of the established algorithms of Hamilton e.l al.
`(1965) and Rossmann et al. (1979). Post refinement of the
`crystal orientations, lattice constants and rocking cun·e
`parameters were done a& proposed by Winkler et al.
`(1979) and Rossmann et al. (1979). The total number
`147,706
`inrlu<ling p11.rti11lly
`intensities,
`of
`rec0rded
`reflections, were finally scaled and symmetry averaged.
`yielding 24,235 unique reflections for the resolution range
`of 10 A to 2·7 A. The agn:t:rut:nt fal'tor:
`N•
`R =LL 11 h;-l,,l/L xJh.
`h j
`h
`where the intensity lhJ for reflection h was measured s.
`times. was 0·077 or 0·054 for the structure factor
`amplitudes Fhf" Within a 3 ,\ 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 mocfol for the 2·7 A work WM taken from
`the 3· l A results. It was subjected to a number of cycles
`of least-squares refinement (Hendrickson & Konnert.
`1981) with periodic exalllinatiuu and rebuilding of the
`model using BILDER 011 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
`squares refinement. With an O\•erall temperature factor.
`the initial value of the R-factor was 0·41. After 5 cycles of
`positional parameter
`refinement with an
`overall
`temperature factor, the value was reduced to 0·33.
`Indindual temperature factors for the atoms were used in
`the succeeding cycles. The program restrains the values of
`
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`
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`

`Plwsphoclwline Bin.ding lmmunoglobitlin Fab McPC603
`
`595
`
`3. Results and Discussion
`Table 3 shows an estimate of the quality of the
`stereochemica.l parameters for the final model. It is
`expressed in te rm s of the r.m.s. deviations of the
`various classes of parameters from accepted values
`(Sielecki et al., 1979). The </>,t/I plot for the main
`chain is shown in Figure l. There are a few residues
`that have "forbidden" </>,ijl values. The quality of
`the map in t,hese regions together with the low
`
`Table 3
`Summary of stereochemical criteria (Hendrickson &
`Konnert, 1981; Siele.cki et al., 1979; Cohen et al.,
`1981)
`
`the B-factors so that each is influenced by the B-factors
`similarity of these pairs of domains using the program
`of the atoms t-0 which it is bound as well as the atoms 1
`(G. H. Cohen, unpublished results), which
`ALIGN
`removed along a chain. In the final stages of least-squares
`iteratively rotates one set of atoms to another set to
`refinement, particular care was taken to ensure that the
`optimize their fit while preserv ing the order of the linear
`sequences of the 2 se ts. The program uses the algorithm
`that
`stercochemistry was kept
`reasonable
`and
`of �eedleman & Wunsch ( 1970) to identify the structural
`w(IF01-IF,1)2 was approximately constant over the range
`of the data used (8·0 A through 2·7 A).
`homology while accounting for insertions and deletions.
`constantly
`The main-chain
`stereochemistry was
`Interdomain
`and
`intermolecular
`contacts were
`monitored by the program GEOM (G. H. Cohen,
`calculated with the aid of the program CONTAX (E. A.
`Padlan, unpublished results). Two at-Oms are defined to
`unpublished results) and points of significant departure
`be in contact if their co-ordinates lie within the sum of
`from expected stereochemistry were examined and
`their van der Waals radii plus l ·0 A. Tntramolecular
`corrected via interactive comput er graphics. In addition
`main-chain hydrogen bonds were caloulated by the
`to o. sulfate
`ion, 138 water moleoulea of variable
`program EREF (M. Levitt, personal communication).
`occupancy were identified from examination of l!:.F and
`2F0-Fc maps and refined with the protein molecule.
`As sequence data beca.me available. the '·working"
`sequence was updated appropriately. The VL and VH
`sequences are listed (sequences 12, p. 45 and I, 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 VL (Rudikoff, unpublished results).
`The CHI sequence given by Auffray et al. (1981),
`obtained by translation of the nucleotide sequence of
`cDKA complementary to alpha-chain mRNA from J558
`tumor oolls, (sequence 60, p. 175, Kabat et al., 1983) was
`used. This sequence differs in 4 places from that obtained
`by Tucker et al. (1981) from BALB/c genomic DNA,
`whiJe they both differ in 2 of these positions from the
`sequence reported by Robinson & Appella (1980) for
`MOPC5 l l, as quoted by Kabat el al. (1983). Examination
`of the final electron density map at these positions did
`not permit a clear distinction to be made between these
`sequences. The sequence ofMOPC21(sequence23, p. 167,
`Kabat et al., 1983 from Svasti & Milstein, 1972) was used
`for the CL domain of 11cP0603.
`Throughout this paper, the amino acid numbering is
`serial, starting from number I with the lst residue of each
`chain of the molecule. The correspondence between our
`numbering scheme and that of Kabat et al. (1983) is
`presented in Table 2.
`As noted by Segal et al. (1974), the molecule possesses 2
`approximate local 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 tliat of Kabat et al. (1983)
`
`Light chain
`
`Heavy chain
`
`This work
`
`Kabat d al.
`
`This work
`
`Kabat et. al.
`
`1-27
`28-33
`34-220
`
`1-27
`27a-27f
`28-214
`
`1-52
`1-52
`52a-52c
`53-55
`53-82
`56-85
`86-88
`82a-82c
`89-106
`83-100
`107-109
`IOOa-IOOc
`101-130
`110-139
`133-135
`140-142
`137-157
`143-163
`164-Jil
`162-169
`171-183
`172-184
`185-200
`185-200
`202-206
`201-205
`208-218
`206-216
`220-225
`217-222
`
`R = LllF.1-IFcll
`'[IPJ
`Average l:i.P
`Jnt.cratomic distances (A)
`1-2
`1-3
`1-4
`Planarity (A}
`Chiral volnmes (A3)
`�on-bonded contacts (A)
`1-4
`Other
`Angles (deg.)
`w, Xs (Arg)
`z •. . . . , l.4
`Temperature factors (A2)
`Main chain 1-2
`:\lain chain 1-3
`Side chain 1-2
`Side chain 1-3
`
`Final mod el
`
`Target u
`
`0·225
`136
`
`t
`
`0·020
`0·040
`0·037
`0·027
`0·257
`
`0·015
`0·020
`0·025
`0·020
`0·150
`
`0·24
`0·34
`
`0·50
`0·50
`
`27·0
`5·0
`
`15·0
`5·0
`
`0·5
`l·O
`0-4
`0·7
`
`0·5
`0·7
`0·5
`0·7
`
`The standard groups dictionary used is specified in Table 2 of
`Sielecki et al. (1979). In this Table, F, re fers to the observed
`structure fact-Or, E'c is the calculated structure factor and /l.F' is
`the quantit.y IJF0f-IFcll· The target u rept't!sents
`lhe iuvcnse
`square-root of the weights used for the parameters listed. The
`
`
`''alue.� given are the r.m.s. deviations from the respective ideal
`values.
`t The weight chose n for the structure factor refinement, the
`"target u., of l:i.i', was m odeled by the function w = (l/d}'
`";th:
`d = 40-500(sin(0)/.l-l/6).
`
`By this means, w(AP) was approximately constant over all of
`the dab (w re presenting the weight for a given reflection). Other
`details of this Table are explained fully by Cohen et al. (1981).
`
`3 of 12
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`

`596
`
`Y. Satow et al.
`
`-�5. oo
`
`o.oo
`
`�5. 00
`
`.. 0
`c !!!
`
`0 0 (!)
`c
`"'
`
`0
`0
`�
`
`0
`
`;
`
`0
`C!I
`
`x
`
`a
`
`(!)
`
`0
`
`0
`x
`•
`
`oC\
`x 0
`ox
`0 C!I
`0
`.,,
`0
`0 0
`_.,
`C!I
`C!I 0 'B) �
`<!I
`·········· ... ·-·····o····-·'o �-
`�o
`··-····
`0
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`0
`C!l e
`f
`0 0 0 0
`(!) C!l(!)tio 1i o
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`lt
`(!)
`(!)GI/, 0 C!X'l
`(!) 0 (!)
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`0 0
`)(
`c ., I
`0 0
`ui
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`0 0
`(!)
`c .,
`)(
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`
`1 35. 00
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`Xx
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`
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`
`x
`X' x
`
`x (!)
`
`x
`
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`p 0
`0
`
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`
`0
`0
`0
`
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`;,, 0
`
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`x
`
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`
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`
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`' ...
`)( x !"
`g
`
`u
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`
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`
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`llOX (!)
`·llO. OD
`
`- s.oo
`
`(!)
`
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`
`o. oo
`PHI
`Figure I. A plot of the dihedral a.ngles at ca.ch alpha-carbon atom for the refined co-ordinates . .-\sterisk. Pro; cross,
`Gly: circle, any other residue.
`
`the assignment of more
`thwarted
`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-fo.ctor
`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 l'lfoPC603 have been deposited in the Protein
`Data Bank at Brookhaven National Laboratory
`(Bernstein eJ, al., 1977).
`
`(a) General structure of the Fab
`Several changes were observed in the molecule as
`a consequence of this refinement, although the
`overall structure remained similar to that described
`earlier (Segal eJ, al., 1974). Most of these changes are
`to be found in tbe details of the loops, in particular
`Ll and H3. The region of LI is poorly defined in the
`electron density maps, even with the higher­
`resolution data. We have now
`rebuilt
`the
`neighboring H3, whose density has become less
`ambiguous and, concurrently, have altered the
`conformation of LI. Jn Figure 2, LI may be noted to
`protrude from the general surface of the molecule.
`The direction of this protrusion is reasonably
`correct but the electron density is too weak to
`define the positions of the three outermost amino
`acid residues.
`
`Three other residues have tentative placement:;
`due to problems in maintaining proper stereo­
`chemistry while fitting the electron density: Glnl62
`and Asn 163 (residues 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
`corresponding to the stronger density was chosen
`for the model.
`The 442 residues of �lcPC603 Fab include 25
`proline residues. Of these, five are cis-proline:
`residues 8 and 101 in VL, 147 in CL, and 143 and
`155 in CHJ. The assignment of the configuration of
`all five ci�-proline residues was unambiguous. The
`structurally homologous Pro8 and Pro95 of Rei
`(Huber & Steigemann, 1974), and Prol47 (CL) and
`Prol55 (CHI) of Kol (Marquart et al., 1980), aJso
`have been found in the cis conformation. Prol43
`(CHI) of McPC603 has no counterpart in previously
`report.ed antibody structures.
`
`(b) Crystal packing
`The McPC603 Fab crystallizes in the space group
`P63 (Rudikoff et al., 1972), with a = 162·53 A,
`c = 60·72 A. The molecules are situated in clusters
`
`4 of 12
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`

`Phosphocholine Binding lmmitnoglobulin Fab McPC603
`
`597
`
`Figure 2. A stereo drawing of the alpha-carbon skeleton of McPC603. Continuous lines denote the heavy chain. The
`
`
`filled circles show the complementarity-determining residues (Kabat el 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 v-ia 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 1, 26, 27, 100,
`102, 104, 105 108 and 110 of the VH of the
`neighboring molecule while residues 18, 66, 67, 69,
`
`skeletons of 4 unit cells and
`the ab plane, illustrating the large channels
`Figure 3. A projection of the alpha-carbon
`
`
`
`
`that run parallel to the c axis through the crystal. 'l'he heavy chains have been drawn bolder in the 3 molecules that are
`
`cell. The 3-fold axes are indicated by the symbol .&:
`
`3-fold axis of the lower left-hand clustered about a crystallographic
`2-fold screw axes are located midwa.y 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. Satow et al.
`
`structure viewed per pendicular to the ac plane, illustrating the effect of the dyad screw axis.
`Figure 4. The projected
`The plane of projection is along the long diagonal of the unit cell (see Fig. 2). It contains the two 3-fold axes and the 2-fold
`
`
`screw midway between them. For clarity, only the 4 molecules closest to the plane have been drawn. The heavy line
`
`corresponds to the heavy chain of the molecule.
`
`73 and 82 of the same VL meet the VL of the
`358 of the possible 400 pairs of main-chain atom� to
`
`
`
`achieve a r.m.s. deviation of 2·0 A (5· l A for poorest
`neighboring molecule at residues 35, 55 a.nd 61 lo
`agreement) between the pairs of matched atoms.
`63. This pattern repeats three times around the
`The best fit between CL and CHI involves a
`
`axis. Other intermolecular contacts are found
`between VH of one molecule and CL and CHI of a
`
`rotation of 169° together with a translation of
`second molecule related to t.he first bv the 2-fold
`2·6 A along the rotation axis. A comparison of these
`screw axis. Several additional contacts are found
`two axes of rotation yields an elbow bend of 133°,
`between VL and CHI of a neighbor via the z unit
`essentially unchanged from the earlier report (Segal
`et al., 1974).
`
`cell translation. lt has been noted (Padlan et al.,
`1973) that roughly 70% of the cell volume is
`occupied by solvent, thus permitting the diffusion
`
`of hapten to the molecule in the crysta l. The long
`axis of the molecule makes an angle of
`approximately 50° with the xy plane of the unit cell
`(Fig. 4).
`
`(d) Intramolecular
`contacts
`As noted elsewhere (reviewed by Davies &
`;\fetzger, 1983; Amzel & Poljak, 1979), VL/VH
`contacts involve hypervariable residues as well as
`
`framework residues. The boxed regions of Table 4
`
`
`indicate hypervariable to hypervariable residue
`( c) I ntramolecular
`pseudosymmetry
`interactions, while other regions of the Table
`Analysis of the approximate 2-fo ld axes relating
`
`
`involve framework residues. Nearly half (46 in 105)
`
`pairs of domains yielded the following results: for
`of the interactions between VL and VH involve
`
`
`only hypervariable residues, with most of these
`
`the VL/VH relationship, we find a 173° rotation
`being located at the upper end of the interface
`
`with a r.m.s. deviation of 1 ·3 A (3·5 A for poorest
`agreement) between 385 matched pairs of main­
`(Fig. 2) in the vicinity of the combining site and
`chain atoms out of 456 atoms. A number of the
`with the framework interactions at the other end. A
`residues of the hypcrvariable loops align quite well,
`number of good hydrogen-bonded contacts occur,
`particularly at t.he beginning and end of each loop.
`notably two between the highly conserved residues
`Gln44L and Gln39H, between Gln39H and Tyr93L,
`
`
`The departure from a more exact 2-fold symmetry
`
`is probably duo to the fact that the interface between the CDR residues Asp97L and Asn I 0 I H,
`residues can be described as fitting a cylinder
`and between the hydroxyl group of TyrlOOL and
`containing four strands from the light chain and
`latter interaction appears to be
`Olu35H. The
`five strands from the heavy chain (Novotny et al.,
`important in mii.intaining t.he integrity of the
`
`phosphocholine binding pocket (Rudikoff et al.,
`1983). This asymmetry of the VH/VL interaction
`
`distinguishes it from most of the VL/VL inter­
`1981) as it has been observed (Rudikoff et al., 1982)
`
`that a mutation of Glu35H to Ala results in a loss of
`actions observed in Bence-Jones
`dimers which, in
`many cases, display exact 2-fold symmetry. The
`phosphocholine
`binding ability.
`
`
`Other interdomain, intramolecular contacts are
`dyad symmetry of the CL/CHl pair is not as close.
`
`presented in Tables 5 to 7. There are no interactions
`The best alignment for the constant modules uses
`
`6 of 12
`
`BI Exhibit 1085
`
`

`

`Phosplwcholine Binding Immunogwbulin Fab McPC603
`
`599
`
`Table 4
`Contacts between residues of V L and V H
`
`Y33 E35 Q39 R44 IA5 W47 A50 E61 Y97 NlOI YI03 8105 TIOO W107 YlOS FI09 WI 12 GI 13 Al 14
`5
`
`3
`
`l
`9
`
`l
`
`5
`
`2
`
`3
`
`2
`
`I:
`
`4
`
`I.
`
`D
`
`I
`I
`6
`
`2
`
`3
`
`4
`4
`4
`
`K36
`F38
`Y42
`Q#
`P49
`P50
`L52
`Y55
`056
`Y93
`Q95
`D97
`YIOO
`PIOI
`Ll02
`Fl04
`GJ05
`Al06
`
`Residues listed in the left column are from VL; the residues Listed above 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 \'&n 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 hypcrv&riable/hypervariable interactions.
`
`bonds are very like those of other antibody classes
`
`involving VL with CHI or VH with CL. Tables 6
`and subgroups. Thus, the a heavy-chain domains of
`and 7 show that the number of interdomain
`restlmble tho� of the y chain
`cont.ac�, i.e. VL wiLh CL or VH with
`McPC603 Fab
`intraehairi
`domains of the huma.n proteins �ew and Kol, and
`CHl, is small.
`the K cha.in domains of :.\IcPC'Ai03 resemhle the 1
`chain domains of New, Kol and Meg, as well as the
`IC chains of various human VL dimers (Padlan &
`(e) Domain sU-ucture
`Davies, 1975; Pad.Ian, 1977). A significant difforence
`from the y chain structure
`The structures of the four domains of )JcPC603
`occurs in CHl, where
`there is an additional disulfide bond formed
`
`are illustrated by Figure 5, which shows also the
`between residues 198 and 222 (residues 198 and 225,
`location of the hydrogen bonds between the main­
`chain amide groups. The general tertiary structure
`in Kabat et al., 1983). The Cys222 residue should
`be regarded as forming the end of CHl
`of the domains and the distribution
`therefore
`of the hydrogen
`
`Table 5
`Contacts between residues of CHI and CL
`
`YJ31 PJ32 l.133 'T'l34 T.l:lfi Pl'.16 Tl4..'> Vl7'.l Fl75 Pl76 Al78 8188 Ql90
`2
`5
`
`5
`
`4
`
`3
`3
`
`14
`1
`
`2
`2
`
`2
`4
`
`l
`2
`
`3
`
`I
`3
`5
`
`3
`
`1123
`Fl24
`St27
`El29
`QJ30
`$133
`\1139
`Fl4l
`l\143
`L166
`8168
`WI69
`Tl70
`S!80
`M18l
`$182
`R217
`
`Roodues listed in the left columns are from CHl; the residues listed above the columns are
`from CL.
`The numbers are as in Table 4.
`
`7 of 12
`
`BI Exhibit 1085
`
`

`

`600
`
`P46
`A86
`L89
`Kl09
`Elll
`Ill2
`K113
`Rll4
`
`¥146 0171 Ql72 8174 Kl75 Dl76 8177 Yl79
`2
`
`7
`
`4
`
`3
`5
`
`2
`I
`6
`
`6
`
`5
`
`E123 8124 A125 FJ54 Pl55 Gl57 Tl58 D2ll 8212
`2
`
`2
`
`2
`
`4
`
`3
`
`3
`
`G9
`GIO
`Lil
`Tlli
`Tll9
`8121
`
`Th e residues listed in the left column are from VH: those listed
`at the column heads are from CHI.
`The numbers are as in Table 4.
`
`Y. Satow et al.
`
`Table 6
`Contacts between residues on VL and CL
`
`Table 7
`Contacts between residues of V H and CHI
`
`The residues listed in the left column are from YL; those listed
`at the column heads are from CL.
`The numbers are as in Table 4.
`
`(o)
`
`( c }
`
`\d)
`Figure 5. Schematic drawings of the (a) VH, (b) VL, (c) CHl and (d) CL domains illustrating the hydrogen
`amide residues. The circles
`residues in VH and VL are drawn
`bolids between the main-chain
`marking hypervariable
`''ith a double !foe.
`
`8 of 12
`
`BI Exhibit 1085
`
`

`

`Plwsphodwline Binding Immunoglobulin Fab 11fcPG603
`
`601
`
`8·0
`
`7·0
`
`6·0
`
`5·0
`
`4·0
`
`3·0
`
`2·0
`
`1·0
`
`8:0
`
`7·0
`
`6·0
`
`5:0
`
`4·0
`
`3·0
`
`2·0
`
`1·0
`
`E VKL VEIG GGl Y DPIGSlRlSCAT SGf TFSD FYllEWVROP'IKRlEW IAASRNXG'NICYTTEYS ASVkGRf I VS AOTSQS I l YlOMHMJIAEDT Al YYCMJ<YYGSTWlfl1'WC
`AITTVTVSS
`D IVllTQIPS�LSWSA8EllY!M1CKSS OSl -lNSGllOK!IFL�OGKKD P!Kll
`ST!ESGYP DRfr§SGSG IDFTlll SSYOAEDl!YYYCoNoHS
`IYGA
`!Pi TFSAGHlElltll
`10 20
`30 40
`50
`60
`70
`BO
`90
`100 110
`( a )
`
`8·0
`
`.... 7·0
`
`6·0
`
`5·0
`
`4·0
`
`- - - -
`
`.... 3·0
`• I - - - - - - - -
`
`2·0
`
`- - -
`
`-
`
`- - - - - - - - - -
`
`8·0 -
`
`7·0 .
`
`6·0
`
`5·0
`
`4·0
`
`3·0
`
`-
`2·0
`
`1-0
`
`- 11
`
`
`
` 1111 111 I 111 11111
`
`1,1111 I "I
`
`l·O
`
`-
`RYTIISNQL TLPAYECP £GESYKCSYQj!OS NP YOELO.YllC
`ESAPHPT _!.YPL TLPPAl§_ SDPYllGCL_!.HOYFPSG™!YTWG KSGKO!TTYNFPPA�SGG
`
`AO.V.'!VS I FPPSSEl!LTIGGASYY£FLNNFYPK 0 .!. NYKWKI OGSiRO NGVUISW!DOOSKOSTl§_MSSTl TL TK�YERH NSYT£EATH KTSTS!'. I VKSFNRHE!i_
`120 130 140 150 160
`170 180
`190
`200
`210 220
`
`( b )
`Figure 6. The separation (in A) of corresponding alpha·carbon atoms of the superimposed domains of (a) VH and VL,
`and (b) CHI and CL. The upper sequence is that of the heavy chain and the lower sequence is of the light chain. Every
`10th letter in each sequence is underlined. The numbern below the sequences refer to the residue number in the light
`chain. 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.
`
`9 of 12
`
`BI Exhibit 1085
`
`

`

`602
`
`Y. Satow et al.
`
`rather than the beginning of the hinge, consistent
`with the gene structme observed by Tucker et al.
`(1981).
`The structures of the light and heavy chain
`domains of )foPC603 are compared in Figme 6(a)
`and (b). The sequence identity derived from the
`structural alignment is 25% for the variable pair
`and 22% for the constant pair, excluding gaps. In
`the variu.ble doma.ins, 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 the 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 Asnl55 (CHI) during
`their sequence analysis of the heavy chain of )IOPC
`47A. In electron dcmiity maps of MoPC603 there is
`suggestive density in the vicinity of the homologous
`Asnl60 that indicates the possibility of carbo­
`hydrate here as well. This density is, however,
`insufficiently defined to permit fitting of any
`carbohydrate chain. It appears, therefore, that
`carbohydrate does not occupy a fixed position on
`the surface of the molecule. The lack of any nearby
`contacts from neighboring domains could also
`contribute to 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) The sulfate ion in the combining site
`Figure 7
`illustrates the combining site of
`McPC603 as seen in these crystals. In addition to
`several water molecules, the m.i.r. density map
`contains a large peak that has been assigned to a
`sulfate ion (Padlan et al., 19'73). Thjs occupies the
`same location a$ the phosphate group of phospho­
`choline when the latter binds to the site, and its
`presence is presumably due to the high concentra­
`tion of ammonium sulfate (about 2 M) in the
`crystal. This sulfate ion is in contact with a number