This issue completes Volume 190
`
`'
`
`UNIVtRS\\1 Gr m,.,.i!N'tll'<A
`
`SEP1719S6
`
`l
`~/
`~J ourna of us«•a~
`.. ~
`h.,.
`OLECVLAR
`BIOLOGYs
`
`Editors in Chief
`J. C. KENDREW S. BRENNER
`
`Volume 190
`
`Number 4
`
`20 August 1986
`
`London
`Toronto
`
`San Diego
`Orlando
`Mont real
`Sydney
`
`New York
`Tokyo
`
`JMOBAK 190 (4) 519- 65 1
`ISSN 0022-2836
`
`PFIZER EX. 1085
`Page 1
`
`

`

`Journal of Molecular Biology
`
`Editors-in-Chief
`J . C. Kendrew . 7 All Saints Passage , Cambridge CB2 3LS, England
`H. Brenner, M.R .l'. Laboratory of Molecular Biology. University Postgraduate Medical School
`Hills Road. Cambridge CB2 2QH . England
`
`Editors
`
`}
`
`Gene structure
`GPne modifi cation
`Gene expression
`Gene regulation
`
`( 'el/8.·
`
`Cell deve lopment
`Cell fun ction
`
`Organelle structures }
`Macromolecular
`assemblies
`
`Molecules :
`
`Macromolecular
`structure
`
`Physical chemistry
`
`Letter8 to the
`Editor:
`
`C:eneral
`Preliminary X -ray data
`
`S . Brenner (address above ).
`P. Charnbon, Laboratoire de Genetiq ue Moleculaire des Eucaryote du
`CNRS. Institut de Chimie Biologique . Faculte de Medicine. II Rue
`Humann. 67085 Strasbourg Cedex. France.
`1l1. Gottesman , Institute of Cancer Research , College of Physicians &
`Surgeons of Columbia Uni\•ersity. 701 \\'. I 68th Street. New York.
`NY 10032, U., .A.
`P. von Hippel , Institute of Molecular Biology, University of Oregon,
`Eugene, OR 97403- 1229, U.S.A.
`B. Mach . Departement de Microbiologie. C.M.ll .. 9 av . de Champel , CH -
`1211 Geneve 4. Switzerland .
`K . . '11at8ubara , Institute for Molecular and Cellular Biology. Osaka
`l ' niversity . Yamada-oka. Suita, Osaka 565. Japan .
`H. E . Huxley , M.R.C. Laboratory of Molecular Biology.
`l ' niversitv
`Postgraduate Medical School. Hills Road . Cambridge CB2 2QH.
`England .
`A . Klug . M. R.C. Laboratory of Molecular Biology , University
`Postgraduate Medical School. Hills Road, Cambridge CB2 2QH.
`England .
`
`{
`{
`{ R. Hube·r. Max-Plan ck -Jnstitut fiir Biochemie. 8033 Martinsried bei
`
`Miinchen . Germany.
`J. C. Kendrew (address ~bove) .
`G. A . Gilbert , Department of Biochemistry. University of Birmingham,
`P .O . Box 363. Birmingham Bl5 2TT. England .
`S . Brenner (address above) .
`R . Huber (address above) .
`J. C. Kendrew (address above) .
`
`Associate Editors
`
`( '. N . f 'anlor . De partment of Hum a n C: e nPtil's and Dpvelopnwnt . C'ollegp of Physicians Nurgeo ns of Columbia
`l ' nivPrsity . 701 West IGH NtrPet. Hoom 1()02 . 1'\ew York . KY 100:12. LTXA .
`.-1 . H. Fnshl . ()ppartme nt of ('lwmi str.\·. [mpe ri a l College of Nc· iencc & T Pthnology . Nouth Kensington. London
`NW7 2:\Y .
`/. fft. rsk01cil:; . ()ppa rtnwnt of Bi oc lw mistry and Biophysic·s. HC' hool of ~l edi t in e. L' niv ersity of Ca lifo rnia . Na n Fra ncisco.
`C'A !H l43 . l'XA .
`H . /,askey . ( '. R.C'. :'llolec·ular Embryology Group . ()ppa rtnw nt of Zoology . Downing NtrPet. ('a mbri dge C' BZ 3EJ .
`Eng la nd .
`1'. /, u:;::.ali . C'Pntre de (:{• netique :'ll o iPe ul a in·. Ce ntre Xa ti onal de Ia HeC' lw rche Ncientifique. 91 C:if-sur-Ynotte. France .
`./.II . . 1/i//Pr. l)ppartm ent of Bi ology . L' nive r:sity of Californi a. 40;) Hilga rd ~h e nu e. Los Ange les. C'A 900:24. l' . ~ . :\ .
`.1/. F. .1/oody . Nchool of Pharm ac·y , l ' ni\'(•rsit y ·o f London, 29/39 Brunswi ck Squ a re. London \YC' I~ I A X . Engla nd .
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`
`PFIZER EX. 1085
`Page 2
`
`

`

`J. Mol . 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
`
`Laboratory 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 McPC603, 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
`hypervaria.ble 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.
`
`1. 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); New, a human myeloma
`protein that binds to a vitamin K1 derivative
`(Amzel et at., 1974; Saul et al., 1978); and McPC603,
`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; 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
`(lgA,K} protein that was previously
`reported (Segal et al., 1974) was an unrefined
`structure based on 3·1 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
`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 has been
`refined using
`restrained least-squares procedures (Hendrickson &
`
`t Present address: Photon Factory, National
`Laboratory for High Energy Physics, Oho-machi,
`Tsukuba-gum , Ibaraki-ken, 305, Japan.
`
`0022- 2836/86/ 160190- 12 $03.00/0
`
`Konnert, 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 et 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 0·1 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.), and KI
`(Fisher Scientific Co.). The
`thulium and platinum
`derivatives were prepared by soaking crystals in solutions
`containing cacodylate and 30 mM-TmC1 3 or 0·4 mM(cid:173)
`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 derivatives 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.
`Most of the 3·1 A data were collected using a Picker
`FACS-I diffractometer (Segal et al. , 1974); the TmCI 3
`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 (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.
`
`PFIZER EX. 1085
`Page 3
`
`

`

`594
`
`Y. Salow et a l.
`
`atom refinement and phase calculation (Dickerson et al.,
`1961) were then computed using local versions of the
`programs of Busing et al. (1962) and Matthews (1966).
`Table l shows the refined heavy-atom parameters. A
`" best" Fourier synthesis (Blow & Crick, 1959) was
`co mputed and a Kendrew skeletal model was fitted to the
`electron density using an optical comparator (Ri chards ,
`1968). The model was improved using t he computer
`graphics system GRIP at the Department of Computer
`Science, niversity of North Carolin a (Tsernoglou et al ..
`1977) and was then subjected to restrained least-squares
`refinement (Konnert, 1976; H endrickson & Konnert,
`the TI-ASC computer at the U.S . Naval
`1981 ) on
`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 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
`point, the fit of the model to a 2F0 -Fc map was
`examined
`using BILDER
`(Diamond ,
`1981 ),
`as
`implemented on a PDP 11/70 under the RSX-llM
`operating system (G. H. Cohen, unpublished results). In
`the second stage of refinement , the protein geometry was
`initially
`less
`restrained and subsequently
`tightened ,
`y ielding a final residual of0·24. The r.m.s. total shift from
`the atom ic positions at the start of the second stage of
`refinement was 0·42 A. A su lfate ion , that had been
`located in the hapten binding cavity (Padlan et al. , 1973;
`Segal et al. , 1974), was
`included
`throughout
`the
`refinement.
`The regions corresponding to residues 101 to 108 in the
`heavy chain and residues 31 to 35 in the light chain were
`not clearly defined
`in
`the original electron density
`fun ction based on the heavy-atom phases. These regions
`remained poorly defined after the preliminary refinement.
`
`(b) Crystal structure analysis and refinement
`at 2·7 A resolution
`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
`to 2·7 A were
`stabilizing solutions.
`Intensity data
`collected by rotation photography (Arndt & Wonacott,
`1977) with Kodak No-Screen Medical X-ray films (3 in a
`pack) using Ni-filtered CuKcx radiation from an Elliott
`GX-6 rotating anode X-ray generator operated at 40 kV
`and 40 rnA. 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 osci llation about the 2 crystal axes, b and c, with an
`
`t Abbreviations used: r.m .s., root-mean-square; F 0 ,
`observed structure factor amplitude; Fe, calculated
`tructure factor 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 , l st, 2nd and
`3rd CDR of the light chain; Hl , H2 and H3 , l st, 2nd
`and 3rd CDR of the heavy chain.
`
`Table 1
`Heavy-atom parameters
`
`Co-ordinates
`
`Heavy-ato m
`compound
`
`X
`
`y
`
`Occupancyt
`
`K2Pt(CNS) 6
`TmC1 3
`Iodine-I
`lodine-2
`lodine-3
`lodine-4
`
`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
`
`0·02 18
`0·0245
`0·0119
`0·0184
`0·0125
`0·0044
`
`Thermal
`factor
`(A2)
`
`19·6
`34·8
`28·3
`7·5
`6·7
`6·8
`
`t The site occupancy is on an arbitrary scale in which the
`average structure amplitude of the native protein is 14·3.
`
`osci llation range of 1·25° and an overlap of 0·25°. The
`time for each exposure was 14 to 21 h.
`The films were scanned at 100 Jlm 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 & M.A.
`Navia (unpublished results). Intensities from each pack
`were further processed through intra-film-pack scaling,
`post refinement, scaling and averaging using programs
`specially written
`for
`these
`purposes
`(Y. Sa tow,
`unpublished results). Intensities in a single pack were
`scaled by refining non-linear response correction factors
`(Matthews et al., 1972) and absorption factors for the film
`base and emulsion, then were corrected 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
`factors
`for
`films . This program
`follows closely
`the
`individual
`formalism of the established algorithms of Hamilton et al.
`(1965) and Rossmann et al. (1979). Post refinement of the
`crystal orientations, lattice constants and rocking curve
`parameters were done as proposed by Winkler et al.
`(1979) and Rossmann et al. (1979). The total num ber
`of 147 ,706
`intensities,
`in cluding partially
`recorded
`reflections, were finally scaled and symmetry averaged ,
`yielding 24,235 unique reflections for the re olution range
`of 10 A to 2·7 A. The agreement factor:
`N.,
`R = L L llhj-I--;.1/L NJh ,
`
`h
`h
`j
`where the intensity ]hi for reflection h was measured Nh
`times , was 0·077 or 0·054 for
`the structure factor
`amplitudes Fhj· 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 taken from
`the 3·1 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 su lfate ion located in the 3·1 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-factor was 0·41. After 5 cycles of
`positional parameter
`refinement with
`an overall
`temperature factor , the value was reduced to 0·33.
`Individual temperature factors for the atoms were used in
`the succeeding cycles. The program restrains the values of
`
`PFIZER EX. 1085
`Page 4
`
`

`

`Phosphocholine Binding Immunoglobulin Fab McPC603
`
`595
`
`t he B-factors so that each is influenced by the B-factors
`of the atoms to which it is bound as well as the atoms 1
`removed along a chain. In the final stages of least-sq uares
`refinement, particular care was taken to ensure that the
`stereochemistry was
`kept
`reasonable
`and
`that
`w(IF.I-1Fcll 2 was approximately constant over the range
`of the data used (8·0 A through 2·7 A).
`constantly
`The main-chain
`stereochemistry was
`monitored by
`the program GEOM
`(G. H. Cohen,
`unpublished results) and points of significant departure
`from expected stereochemistry were examined and
`corrected via interactive computer graphics. In addition
`to a sulfate
`ion, 138 water molecules of variable
`occupancy were identified from examination of t1F and
`2F0 - Fe maps and refined with the protein molecule.
`As sequence data 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
`R.udikoff et al. (1981) and Rudikoff & Potter (1974) , with
`the addition of the tetrapeptide Leu-Glu -IIe-Lys, which
`occurs at the end of VL (Rudikoff, unpublished results).
`The CHI sequence given by Auffray et al.
`(198 1),
`obtained by translation of the nucleotide sequence of
`eDNA complementary to alpha-chain mRNA from J558
`tumor cells, (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 BALBJc genomic DNA,
`while they both differ in 2 of these positions from the
`sequence reported by Robinson & Appella (1980) for
`MOPC51 1, as quoted by Kabat et 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 of MOPC2 1 (sequence 23, p. 167,
`Kabat 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 l with the 1st 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 that of Kabat et al. (1983)
`
`Light 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
`
`1-52
`53-55
`56-85
`86-88
`89-106
`107- 109
`110-139
`140- 142
`143- 163
`164- 171
`172- 184
`185- 200
`201- 205
`206- 216
`217- 222
`
`1-52
`52a-52c
`53-82
`82a-82c
`83-100
`100a- 100c
`101- 130
`133- 135
`137- 157
`162-169
`171- 183
`185-200
`202-206
`208-218
`220-225
`
`si milarity of these pairs of domains using the program
`ALIGN
`(G. H. Cohen, unpublished
`results) , which
`iteratively rotates one set of atoms to another set to
`optimize their fit while preserv ing the order of the linear
`sequences of the 2 sets. The program uses the algorithm
`of Needleman & Wunsch (1970) to identify the stru ctural
`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 atoms are defined to
`be in contact if their co-ordinates lie within the sum of
`their van der Waals radii plus 1·0 A. Intramolecular
`the
`main-chain hydrogen bonds were calculated by
`program EREF (M. Levitt, personal communication).
`
`3. Results and Discussion
`Table 3 shows an estimate of the quality of the
`stereochemical parameters for the final model. It is
`expressed in terms of the r.m .s. deviations of the
`various classes of parameters from accepted values
`(Sielecki et al ., 1979). The </>,1/1 plot for the main
`chain is shown in Figure 1. There are a few residues
`that have " forbidden" </>,1/1 values. The quality of
`the map in these regions together with the low
`
`Table 3
`Summary of stereochemical criteria (Hendrickson &
`Konnert , 1981 ; Bielecki et al., 1979; Cohen et al. ,
`1981)
`
`Final model
`
`Target u
`
`R = l:IJF.I-JFcll
`l:IF.I
`Average llF
`Interatomic distances (A)
`1- 2
`1- 3
`1- 4
`P lanarity (A)
`Chiral volumes (A3 )
`Non-bonded contacts (A)
`1- 4
`Other
`Angles (deg.)
`w , Xs (Arg)
`x, , ... , X4
`Temperature factors (A 2
`Main chain 1- 2
`Main chain 1- 3
`Side chain 1- 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
`1·0
`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) . In this Table, F. refers to the observed
`structure factor, Fe is the calculated structure factor and !lF is
`the quantity IIF.I-IFcll- The target u represents the inverse
`square-root of the weights used for the parameters listed. The
`values given are the r.m.s. deviations from the respective ideal
`values.
`t The weight chosen for the structure factor refinement, the
`" target u" of !lF , was modeled by the function w = (1 /d) 2 with :
`
`d = 40-500(sin(£l)/J.-1 /6).
`By this means, w(llF) was approximately constant over all of
`the data (w representing the weight for a given reflection). Other
`details of this Table are explained fully by Cohen et al. (1981).
`
`PFIZER EX. 1085
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`596
`
`Y. Satow et a!.
`
`0
`0
`
`(!)
`
`(!)
`
`(!)
`
`(!)
`
`(!)
`
`~
`
`(!)(!)
`
`X
`
`(!)
`
`X
`
`(!)
`
`X
`
`X
`
`X
`
`X
`
`X'-
`
`X
`
`(!)
`X
`
`X
`
`X(!)
`
`X
`
`X
`
`00
`
`(!)
`
`X
`
`(!)
`
`(!)
`
`ClCj.
`
`X
`
`(!)
`
`(!)
`
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`
`(!)
`
`(!)X
`C!!!,
`
`0
`0
`
`0
`X
`CD
`1
`- 80.00
`
`Figure 1.· A plot of the dihedral angles at each alpha-carbon atom for the refined co-ordinates. Asterisk , 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 (I952). 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 National Laboratory
`(Bernstein et al., I977).
`
`(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 et al., I974) . Most of these changes are
`to be found in the details of the loops, in particular
`LI and H3. The region of LI is poorly defined in the
`electron density maps, even with
`the higher(cid:173)
`resolution data. We have now
`rebuilt
`the
`neighboring H3, whose density has become less
`ambiguous and, concurrently, have altered the
`conformation of Ll. In 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 placements
`due to problems in maintaining proper stereo(cid:173)
`chemistry while fitting the electron density : Glni62
`and Asni63 (residues 156 and I57 in the numbering
`used by Kabat et al. , I983) of CL and Glu202
`(residue 203, Kabat et al., I983) 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 these cases, the configuration
`corresponding to the stronger density was chosen
`for the model.
`The 442 residues of McPC603 Fab include 25
`proline residues. Of these, five are cis-proline:
`residues 8 and lOI in VL, I47 in CL, and I43 and
`I55 in CHI. The assignment of the configuration of
`all five cis-proline residues was unambiguous. The
`structurally homologous Pro8 and Pro95 of Rei
`(Huber & Steigemann, I974) , and Proi47 (CL) and
`Proi55 (CHI) of Kol (Marquart et al. , I980) , also
`have been found in the cis conformation. Prol43
`(CHI) of McPC603 has no counterpart in previously
`reported antibody structures.
`
`(b) Crystal packing
`The McPC603 Fab crystallizes in the space group
`(Rudikoff et al., 1972), with a= I62·53 A,
`P6 3
`c = 60·72 A. The molecules are situated in clusters
`
`PFIZER EX. 1085
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`

`Phosphocholine Binding I mmunoglobulin Fab McP C603
`
`597
`
`Figure 2. A st ereo drawing of t he a lpha-carbon skeleton of McPC603. Continuous lines denote the heavy chain. The
`filled circles show t he complementarity-determining residues (K abat et al. , 1983).
`
`of three at each of t he cryst allographic 3-fold axes
`of the uni t cell (Fig. 3). Each cluster is related t o
`neighboring clusters via t he 2-fold screw situated
`midway between t he 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. R esidues 14 to 20
`and 71 of VL interact with residues 1, 26, 27 , 100,
`102, 104, 105 108 and 110 of t he VH of the
`neighboring molecule while residues 18, 66, 67 , 69,
`
`Figure 3. A p rojection of the alpha-carbon skeletons of 4 unit cells and t he ab plane, illustrating t he large cha1mels
`t hat ru n parallel to t he c ax is t hrough t he crystal. T he heavy chains have been drawn bolder in t he 3 molecules that are
`clustered about a crystallographic 3-fold axis of t he lower left-hand cell . The 3-fold axes are indicated by the symbol .A.:
`2-fold screw axes are located midway between each adj acent pair of 3-fold axes; a 63 axis is located at each corner of
`each cell.
`
`PFIZER EX. 1085
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`598
`
`Y. Satow et al.
`
`Figure 4. The projected structure viewed perpendicular to the ac plane, illustrating the effect of the dyad screw axis.
`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
`neighboring molecule at residues 35, 55 and 6I to
`63 . This pattern repeats three times around the
`axis. Other
`intermolecular contacts are found
`between VH of one molecule and CL and CHI of a
`second molecule related to the first by the 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.,
`I973) that roughly 70% of the cell volume is
`occupied by solvent, thus permitting the diffusion
`of hapten to the molecule in the crystal. The long
`the molecule makes
`an
`angle of
`axis of
`approximately 50° with the xy plane of the unit cell
`(Fig. 4).
`
`(c) Intramolecular pseudosymmetry
`Analysis of the approximate 2-fold axes relating
`pairs of domains yielded the following results: for
`the VLJVH relationship, we find a 173° rotation
`with a r.m.s. deviation of 1·3 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 the beginning and end of each loop.
`The departure from a more exact 2-fold symmetry
`is probably due to the fact that the interface
`residues can be described as fitting a cylinder
`containing four strands from the light chain and
`five strands from the heavy chain (Novotny et al. ,
`1983). This asymmetry of the VHJVL interaction
`distinguishes it from most of the VLJVL inter(cid:173)
`actions observed in Bence- Jones dimers which, in
`many cases, display exact 2-fold symmetry. The
`dyad symmetry of the CLJCHl pair is not as close.
`The best alignment for the constant modules uses
`
`358 of the possible 400 pairs of main-chain atoms to
`achieve a r .m.s. deviation of 2·0 A (5·1 A for poorest
`agreement) between the pairs of matched atoms.
`The best fit between CL and CHI involves a
`rotation of I69° together with a translation of
`2·6 A along the rotation axis. A comparison of these
`two axes of rotation yields an elbow bend of I33°,
`essentially unchanged from the earlier report (Segal
`et al. , I974) .
`
`(d) Intramolecular contacts
`As noted elsewhere (reviewed by Davies &
`Metzger, 1983; Amzel & Poljak, 1979), VLJVH
`contacts involve hypervariable residues as well as
`framework residues. The boxed regions of Table 4
`indicate hypervariable to hypervariable residue
`interactions, while other regions of the Table
`involve framework residues. Nearly half (46 in 105)
`of the 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 contacts occur,
`notably two between the highly conserved residues
`Gln44L and Gln39H, between Gln39H and Tyr93L,
`between the CDR residues Asp97L and AsnlOJH ,
`and between the hydroxyl group of TyrlOOL and
`to be
`Glu35H. The latter interaction appears
`important in maintaining the integrity of the
`phosphocholine binding pocket (Rudikoff et al.,
`1981) as it has been observed (Rudikoff et al. , 1982)
`that a mutation of Glu35H to Ala results in a Joss of
`phosphocholine binding ability.
`Other interdomain , intramolecular contacts are
`presented in Tables 5 to 7. There are no interactions
`
`PFIZER EX. 1085
`Page 8
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`

`

`Phosphocholine Binding Immunoglobulin Fab McPC603
`
`599
`
`Table 4
`Contacts between residues of V L and V H
`
`Y33 E35 Q39 R44
`
`IA5 W47 A50 E6l Y97 NlOl Yl03 Sl05 Tl06 Wl07 Yl08 Fl09 Wll2 Gll3 All4
`
`5
`
`3
`
`5
`
`2
`
`3
`
`2
`
`l
`9
`
`4
`
`2
`
`3
`
`l
`l
`6
`
`6,·
`
`K36
`F38
`Y42
`Q44
`P49
`P50
`L52
`Y55
`G56
`Y93
`
`~~:X,
`
`PlOI
`LI02
`Fl04
`Gl05
`AJ06
`
`Q95 0
`
`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 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 hypervariablefhypervariable 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 McPC603
`are illustrated by Figure 5, which shows also the
`location of the hydrogen bonds between the main(cid:173)
`chain amide groups. The general terti!;try structure
`of the domains and the distribution of the hydrogen
`
`bonds are very like those of other antibody classes
`and subgroups. Thus, the a. heavy-chain domains of
`McPC603 Fab resemble
`those of the y chain
`domains of the human proteins New and Kol, and
`the K chain domains of McPC603 resemble the A.
`chain domains of New, Kol and Meg, as well as the
`K chains of various human VL dimers (Padlan &
`Davies, I975; Padlan, I977). A significant difference
`from the y chain structure occurs in CHI, where
`there
`is an additional disulfide bond formed
`between residues I98 and 222 (residues 198 and 225,
`in Kabat et al., I983). The Cys222 residue should
`therefore be regarded as forming the end of CH I
`
`Table 5
`Contacts between residues of C H 1 and C L
`
`Yl3 1 Pl32 Ll33 Tl34 Ll35 Pl36 1145 Vl73 Fl75 Pl76 Al78 Sl88 Ql90
`
`2
`5
`
`4
`
`5
`
`3
`3
`
`14
`l
`
`2
`2
`
`2
`4
`
`l
`2
`
`3
`
`I
`3
`5
`
`3
`
`1123
`F l 24
`Sl27
`El29
`Ql30
`Sl33
`Vl39
`Fl41
`Nl43
`Ll66
`Sl68
`Wl69
`Tl70
`SISO
`Ml8l
`Sl82
`R217
`
`Residues listed in the left columns are from CHI; the residues listed above the columns are
`from CL.
`The numbers a