Structure, Function, and Genetics
`
`PROTEINS
`
`
`
`Alan R. Liss, Inc, New York
`
`PFIZER EX. 1086
`
`Page 1
`
`PFIZER EX. 1086
`Page 1
`
`

`

`PROTEINS
`
`Structur~ Function, and Genetics
`Editor-in-Chief
`Cyrus Levinthal
`Department of Biological Sciences • Columbia University • New York, New York
`Executive Editor
`George D. Rose
`Department of Biological Chemistry • Hershey Medical Center, Pennsylvania State University • Hershey, Pennsylvania
`Associate Editors
`David Davies
`Section of Molecular Structure, N.I.A.M.D.D., National
`Institutes of Health, Bethesda, Maryland
`William DeGrado
`Central Research and Development Department, E.l.
`du Pont de Nemours and Company, WilminglOn,
`Delaware
`
`Thomas E. Creighton
`Medical Research CouncH Laborarory of Molecular
`Biology, Cambridge, England
`
`James Wells
`Department of Biological Chemistry, Genentech, Inc.,
`Soudl San Francisco, California
`
`john Abelson
`Division of Biology, California Institute of
`Technology, Pasadena, California
`Robert L. Baldwin
`Department of Biochemistry, Sun ford University
`School of Medicine, Stanford, California
`Hermanj.C. Berendsen
`laborarory of Physical Chemistry, Universit y of
`Groningen, Groningen, The Netherlands
`David Botstein
`Genentech Inc., South San Francisco, California
`Ralph Bradshaw
`Department of Biological Chemistr y, Universit y of
`California, Irvine, California
`jean-Michel Claverie
`Institute Pasteur, Unite d'Informatique Scientifique ,
`Paris, France
`David Eisenberg
`Molecular Biology Institute, Universit y of California,
`Los Angeles, California
`Donald M. Engelman
`Department of Molecular Biophysics and
`Biochemistry, Yale Universit y, New Haven,
`Connecticut
`S. Walter Englander
`Department of Biochemjstry and Biophysics,
`Universit y of Pennsylvania School of Medicine
`Philadelphia, Pennsylvania
`Richard M. Fine
`Department of Biological Sciences, Columbia
`University, New York, New York
`Robert). Fletterick
`Department of Biochemistry and Biophysics,
`University of California at San Francisco, San
`Francisco, California
`Lila M. Gierasch
`Department of Pharmacology, University of Texas
`Southwestern Medical Center, Dallas, Texas
`Walter Gilbert
`Biological Laboratories, Harvard University,
`Cambridge, Massachusetts
`Nobuhiro Go
`Department of Chemistry, Faculty of Science, Kyoto
`University, Kyoto, japan
`jonathan Greer
`Computer Assisted Molecular Design Group,
`Pharmaceutical Products Division, Abbott
`Laboratories, Abbott Park, Illinois
`
`Editorial Board
`Arnold T. Hagler
`The Agouron Institute, La jolla, California
`Jan Hermans
`Department of Biochemistry, University of North
`Carolina, Chapel Hill, North Carolina
`Barry Honig
`Department of Biochemistry and Molecular
`Biophysics, College of Physicians and Surgeons,
`Columbia University, New York, New York
`Leroy Hood
`Division of Biology, California Institute of
`Technology, Pasadena, California
`Wayne L. Hubbell
`Jules Stein Eye Institute, University of California
`School of Medicine, Los Angeles, California
`Michael N.G.James
`Department of Biochemistry, University of Alberta,
`Edmonton, Alberta, Canada
`Alwyn jones
`Department of Molecular Biology, Uppsala, Sweden
`Arthur Karlin
`Departments of Biochemistry and Neurology,
`Col lege of Physicians and Surgeons, Columbia
`University, New York, New York
`Martin Karplus
`Department of Chemistry, Harvard University,
`Cambridge, Massachusetts
`William Konigsberg
`Department of Molecular Biophysics and
`Biochemistry, Yale University, School of Medicine,
`New Haven, Connecticut
`joseph Kraut
`Department of Chemistry, University of California at
`San Diego, La jolla, Californla
`Robert Langridge
`Computer Graphics Laboratory, Department of
`Pharmaceutical Chemistr y, Universit y of California,
`San Francisco, California
`Eaton E. Lattman
`Department of Biophysics, johns Hopkins School of
`Medicine, Baltimore, Maryland
`Dale L. Oxender
`Center for Molecular Genetics, University of
`Michigan, Ann Arbor, Michigan
`Alexander Rich
`Department of Biology, Massachusetts Institute of
`Technology, Cambridge, Massachusetts
`
`jane S. Richardson
`Department of Biochemistry, Duke University
`Medical Center, Durham, North Carolina
`Michael G. Rossmann
`Department of Biological Sciences, Purdue
`Universit y, West Lafayette, Indiana
`
`Chris Sander
`Biocomputing Programme, European Molecular
`Biology Laboratory, Heidelberg, Federal
`Republic of Germany
`
`Robert Sauer
`Department of Biology, Massachusetts Institute of
`Technology, Cambridge, Massachusetts
`
`Robert Schleif
`Department of Biology, johns Hopkins University,
`Baltimore, Maryland
`
`David Shortie
`Department of Biological Chemistry, johns HopkiDs
`University School of Medicine, Baltimore, Maryland
`
`Paul Sigler
`Department of Molecular Biophysics and Biochcmillry.
`Yale Universit y, New Haven, Connecticut
`
`john A. Smith
`Departments of Molecular Biology and Pathology,
`Massachusetts General Hospital, Boston,
`Massachusetts
`Thomas A. Steitz
`Department of Molecular Biophysics and
`Biochemistry, Yale Universit y, New Haven,
`Connecticut
`
`Lubert Stryer
`Department of Cell Biology, Stanford University
`School of Medicine, Stanford, California
`
`J. Craig Venter
`Section of Receptor Biochemistry and Molecular
`Biology, National Institute of Neurological and
`Communicative Disorders and Stroke, Natiorul
`Institutes of Health, Bethesda, Maryland
`
`Don Wiley
`Department of Biochemistry and Molecular BioloiJ.
`Harvard University, Cambridge, Massachusetts
`
`Kurt Wuthrich
`lnstitut fur Molekularbiologie und Biophysik,
`EidgenOssiche Technishc Hochschule, ZUrich,
`Switzerland
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`PFIZER EX. 1086
`Page 2
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`

`

`PROTEINS: Structure, Function, and Genetics 5:271-280 (1989)
`
`Three-Dimensional Structure of a Fluorescein-Fab
`Complex Crystallized in 2-Methyl-2,4-pentanediol
`James N. Herron, 1 Xiao-min He, 1 Martha L. Mason, 1 Edward W. Voss, Jr.,2 and Allen B. Edmundson1
`1Department of Biology, University of Utah, Salt Lake City, Utah 84112 and 2Department of Microbiology,
`University of Illinois, Urbana-Champaign, Illinois 61081
`
`ABSTRACT
`The crystal structure of a flu-
`orescein-Fab (4-4-20) complex was determined
`at 2. 7 A resolution by molecular replacement
`methods. The starting model was the refined 2. 7
`A structure of unliganded Fab from an au(cid:173)
`toantibody (BV04-01) with specificity for single(cid:173)
`stranded DNA. In the 4-4-20 complex fluores(cid:173)
`cein fits tightly into a relatively deep slot
`formed by a network of tryptophan and ty(cid:173)
`rosine side chains. The planar xanthonyl ring of
`the hapten is accommodated at the bottom of
`the slot while the phenylcarboxyl group inter(cid:173)
`faces with solvent. Tyrosine 37 (light chain) and
`tryptophan 33 (heavy chain) flank the xantho(cid:173)
`nyl group and tryptophan 101 (light chain) pro(cid:173)
`vides the floor of the combining site. Tyrosine
`103 (heavy chain) is situated near the phenyl
`ring of the hapten and tyrosine 102 (heavy
`chain) forms part of the boundary of the slot.
`Histidine 31 and arginine 39 of the light chain
`are located in positions adjacent to the two
`enolic groups at opposite ends of the xanthonyl
`ring, and thus account for neutralization of one
`of two negative charges in the haptenic dianion.
`Formation of an enol-arginine ion pair in a re(cid:173)
`gion of low dielectric constant may account for
`an incremental increase in affinity of 2-3 orders
`of magnitude in the 4-4-20 molecule relative to
`other members of an idiotypic family of mono(cid:173)
`clonal antifluorescyl antibodies. The phenyl
`carboxyl group of fluorescein appears to be hy(cid:173)
`drogen bonded to the phenolic hydroxyl group
`of tyrosine 37 of the light chain. A molecule of
`2-methyl-2,4-pentanediol (MPD), trapped in the
`interface of the variable domains just below the
`fluorescein binding site, may be partly respon(cid:173)
`sible for the decrease in affinity for the hapten
`inMPD.
`
`Key words: antifluorescyl monoclonal anti(cid:173)
`body, high-affinity binding site, ef(cid:173)
`fects of MPD on hapten binding
`
`INTRODUCTION
`The antifluorescein system is well suited for
`studying the molecular basis of antigenic specificity
`because it offers both a wide range of binding affin(cid:173)
`ities (105- 1010 M - 1), and a variety of experimental
`
`© 1989 ALAN R. LISS, INC.
`
`techniques for correlating antigen binding affini(cid:173)
`ties, kinetics, and thermodynamics.1- 9 Further(cid:173)
`more, it has been possible to develop a family of
`idiotypically cross-reactive antibodies in which indi(cid:173)
`vidual monoclonals vary in affinity over a 1000-fold
`range. 9 Amino acid sequences recently determined
`for eight of these antibodies in one of our laborato(cid:173)
`ries (E.W.V.) show that at least six were derived
`from the same germline variable genes. Thus, the
`antifluorescein idiotype family can be used to fur(cid:173)
`ther the understanding of both idiotypy and affinity
`maturation. In this report, we describe the three(cid:173)
`dimensional structure of a complex of dianionic flu(cid:173)
`orescein with the antigen-binding fragment from
`the antibody (4-4-20) with the highest affinity in
`this idiotype family.
`The 4-4-20 monoclonal is an IgG2a (K) antibody
`that binds fluorescein with an association constant
`of 3.4 x 1010 M - 1 in aqueous solution. This affinity
`decreases 300-fold
`in 47% (v/v) 2-methyl-2,4-
`pentanediol (MPD), the solvent used for cocrystalli(cid:173)
`zation of the 4-4-20 Fab with fluorescein hapten. 5
`The antibody is a highly specialized molecule that
`does not cross-react with rhodamine compounds. 1•6
`In the formation of a complex with the 4-4-20 anti(cid:173)
`body, fluorescein satisfied criteria for a site-filling
`ligand, with the xanthonyl ring behaving as the
`"immunodominant" moiety.6 Despite its relatively
`large size and distinctive chemical features , fluores(cid:173)
`cein induces a diverse immune response when in(cid:173)
`jected as a conjugate with keyhole limpet hemocya(cid:173)
`nin (KLH). 1•7- 9 Interpretation of binding studies
`have sometimes been ambiguous with heteroge(cid:173)
`neous populations of antibodies. It therefore seemed
`appropriate to determine the mode of binding in a
`single molecular species, particularly one with high
`affinity for fluorescein.
`This crystal system affords a rare opportunity to
`consider both the structural features responsible for
`high-affinity binding and the effects of solvent in
`lowering that affinity. The complex will also be ex-
`
`Received January 20, 1989; revision accepted March 24,
`1989.
`Address reprint requests to A.B. Edmundson, Department of
`Biology, University of Utah, Salt Lake City, UT 84112.
`James N . Herron's present address is Department of Phar(cid:173)
`maceutics, University of Utah , Salt Lake City, UT 84112.
`
`PFIZER EX. 1086
`Page 3
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`

`272
`
`J. N. HERRON ET AL.
`
`amined to assess the influence of the carrier protein
`(KLH) on the location and orientation of the hapten
`in the combining site. In the preparation of the im(cid:173)
`munogen, molecules of the isothiocyanate derivative
`of fluorescein amine (isomer I) were coupled to
`KLH, with the principal reactions presumed to in(cid:173)
`volve the e-amino group of lysine side chains. The
`4-4-20 hybridoma was obtained by fusion of hyper(cid:173)
`immunesplenocytes stimulated with the fluorescein(cid:173)
`KLH conjugate.
`The present work adds to previous crystallo(cid:173)
`graphic studies of complexes in which small mole(cid:173)
`cules were diffused into crystals of human and mu(cid:173)
`rine immunoglobulin fragments with binding sites
`shaped like cavities or shallow depressions. 10-15
`These studies have helped define the molecular ba(cid:173)
`sis of low- and medium-affinity interactions and
`have provided the background to broaden our under(cid:173)
`standing of antigenic specificity. Block-end types of
`interactions over large external surfaces have been
`studied in cocrystals of Fab molecules and protein
`antigens
`like
`lysozyme and
`influenza neur(cid:173)
`aminidase.16-19 Antibodies binding DNA have
`grooves as potential combining sites, and cocrystals
`of Fabs and oligodeoxynucleotides are currently be(cid:173)
`ing subjected to X-ray analyses.20-23 The structure
`of an unliganded Fab (J539) with specificity for ga(cid:173)
`lactan in carbohydrates has also been determined. 24
`A model for the binding of the ligand was proposed
`on the basis of solution studies and the structure of
`the combining site.
`Recently, a single-chain antigen-binding pro(cid:173)
`tein25 was constructed to simulate the Fv fragment
`of the 4-4-20 antibody (an Fv fragment consists of
`the "variable" domains of the heavy and light
`chains). The three-dimensional structure of the 4-
`4-20 Fab should prove very useful in assessing the
`properties and applications of the single chain pro(cid:173)
`tein.
`
`MATERIALS AND METHODS
`Preparation of Fluorescein-Fab Complex
`Procedures for the isolation and purification of the
`4-4-20 monoclonal antibody and fluorescein-Fab
`complex were described in a previous article. 5 In
`outline the Fab fragments were prepared by hydro(cid:173)
`lysis of fluorescein-IgG complexes with papain.
`Liganded Fabs were purified by chromatofocusing
`on 15 ml Pharmacia PBE 94 columns, with a linear
`pH gradient of 9 to 6 formed with Pharmacia Poly(cid:173)
`buffer 96. Because of potential destructive effects of
`dye-sensitized photooxidation by free fluorescein , it
`was necessary to protect the liganded protein from
`light at all stages of the procedures. Columns, tubes,
`and vessels used in affinity chromatography, chro(cid:173)
`matofocusing, dialysis, enzymatic hydrolysis, and
`crystallization trials were all covered with alumi(cid:173)
`num foil. All manipulations and transfers were car(cid:173)
`ried out in reduced light. Under such precautions
`
`the liganded Fab was eluted from the chromatofo(cid:173)
`cusing column as a single component with a pi of
`7.2.
`After dialysis against 50 mM sodium phosphate,
`pH 7 .2, the solution of the fluorescein-Fab complex
`was concentrated to 25 mg/ml by ultrafiltration (the
`acceptable range for crystallization was 10-30
`mg/ml). The complex was crystallized by a batch
`method in an environment with strict light and tem(cid:173)
`perature (12-14°C) control. Graded aliquots ofMPD
`were added to 40 t.J..l samples of the liganded protein
`in flat-bottomed glass vials. Crystals appeared in 2
`days with final MPD concentrations of 38-60%
`(v/v), the optimum being - 47%. Bladed crystals
`suitable for X-ray analysis grew to dimensions of0.6
`x 0.35 x 0.3 mm (l x w x d) in 1-2 months. Green
`fluorescence, attributable to the dissociation of flu(cid:173)
`orescein from the complex in MPD, was observed in
`each crystallization tube.
`
`Collection of X-Ray Diffraction Data
`The fluorescein-Fab complex crystallized in the
`triclinic space group P1, with a = 58.3, b = 43.9,
`and c = 42.5 A; o: = 82.1, 13 = 87.3, and-y = 84.6°.5
`Crystals disintegrated at temperatures > 22°C, but
`were mechanically stable even in the X-ray beam
`when the ambient temperature was maintained at
`12-14°C. Temperature instability of the complex in
`MPD had also been noted in solution.5 For example,
`irreversible increases in the standard free-energy
`changes (LlG0
`) in the liganded IgG and Fab mole(cid:173)
`cules occurred in 40% MPD at relatively low tem(cid:173)
`peratures (transition temperature of 30°C).
`A single crystal was used to collect X-ray diffrac(cid:173)
`tion data to 2.7 A resolution with a Nicolet P21 dif(cid:173)
`fractometer operated at 40 kV and 35 rnA (CuKa r a(cid:173)
`diation). The data set included 11,116 unique
`reflections, of which 9120 (82.0%) were observed at
`intensity levels > 1.5 standard deviations (based on
`counting statistics).
`
`Determination of the Three-Dimensional
`Structure of the Liganded Fab
`The fluorescein-Fab complex crystallized in the
`same space group (P1) as the unliganded Fab of the
`BV04-01 IgG2b autoantibody, with specificity for
`single-stranded DNA.5·26 Unit cell dimensions for
`the two crystals were nearly identical and one of
`us (X-M.H. ) found by molecular replacement meth(cid:173)
`ods27-30 that the proteins were in the same orienta(cid:173)
`tions in these unit cells. With the refined 2.7 A
`structure of the BV04-01 Fab as starting model, the
`orientation of the 4-4-20 Fab was determined more
`accurately with rotation function programs.
`Crystallographic refinement31·32 of the structure
`was initiated with the X-ray diffraction data5 for the
`4-4-20 Fab and the atomic model of the BV04-01
`Fab.22·23 After 30 cycles of refinement with 2.7-6.0
`A data (8304 reflections), the amino acid sequences
`
`PFIZER EX. 1086
`Page 4
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`

`

`STRUCTURE OF FLUORESCEIN-FAB COMPLEX
`
`273
`
`TABLE I. Refinement Data
`
`Average 11F*
`R-factor
`< B > in A2t
`No. of reflections, I > 1.5 CJ (l):j:
`Root mean square (rms) deviations
`From ideal distance (A )
`Bond distance
`Angle distance
`Planar 1-4 distance
`Hydrogen bond distance§
`rms deviation from planarity (A )
`rms deviation from ideal chirality CA3)
`rms deviation from permitted contact distances CA)
`Single torsion contacts
`Multiple torsion contacts
`Possible hydrogen bond
`rms deviation from ideal torsion angles (0
`For prespecified <J>, ljJ angles
`For planar group (0 or 180)
`For staggered group (± 60 or 180)
`For orthonormal group (± 90)
`
`)
`
`Actual rms deviation
`46.6
`0.215
`15.5
`8304
`
`0.028
`0.055
`0.032
`0.132
`0.014
`0.210
`
`0.280
`0.400
`0.409
`
`Target
`value
`
`0.03
`0.04
`0.03
`0.05
`0.025
`0.150
`
`0.500
`0.500
`0.500
`
`15.0
`3.0
`15.0
`15.0
`*The weight for the structure factor refinement was obtained from the equation w = (lla)2 , where a =
`40 - 278 x [sin(S)IA - 116] .
`t Average temperature factor.
`+Resolution limits of 2.7-6.0 A.
`§Explicit hydrogen bonds for J3-pleated sheets.
`
`30.8
`6.3
`26.9
`28.1
`
`of the light and heavy chains were altered to corre(cid:173)
`spond to those of the 4-4-20 Fab.34 The model of the
`4-4-20 Fab was improved through alternating cycles
`of refinement and interactive model building on an
`Evans and Sutherland PS300 graphics system with
`the FRODO program.35·36 Polypeptide backbones
`and amino acid side chains were fitted to 2F0 - Fe
`maps, in which F 0 and Fe were observed and calcu(cid:173)
`lated structure factors. Fluorescein was located in a
`t1F (F0 - Fel map, for which the phase angles were
`calculated from the refined atomic model of the pro(cid:173)
`tein (atomic coordinates for fluorescein were omit(cid:173)
`ted). The ligand-protein complex was subjected to
`additional cycles of refinement until the R factor
`(~II Fo 1-1 Fe II /~ I Fo I) began to plateau at its cur(cid:173)
`rent value of 0.180 (before idealization of bond
`lengths and angles; 0.215 after).
`
`RESULTS
`Description of the Three-Dimensional
`Structure of the Fluorescein-Fab Complex
`
`The results of the crystallographic refinement of
`the complex are presented in Table I. This structure
`could be refined very quickly because of the great
`similarities with the structure of the unliganded
`BV04-01 Fab.
`Figure 1 contains the three-dimensional "cage"
`electron density to which a skeletal model of fluo(cid:173)
`rescein was fitted by interactive computer graphics.
`The torsion angle measured between the xanthonyl
`and benzoyl rings was 73° in the bound hapten.
`
`An o:C skeletal model of the 4-4-20 Fab is shown
`as a stereo pair in Figure 2, with a model of fluores(cid:173)
`cein codisplayed in the binding site. Tracings of the
`o:C chains of the 4-4-20 and BV04-01 Fabs are su(cid:173)
`perimposed in Figure 3. Details of the structures of
`the ligand and combining site are presented in Fig(cid:173)
`ure 4 (skeletal models). Solvent-accessible sur(cid:173)
`faces37 are illustrated in Figure 5. Amino acid
`sequences34 for the hypervariable regions are listed
`in Figure 6.
`Polypeptide chains could be traced unambigu(cid:173)
`ously in both the light and heavy chains of the 4-
`4-20 complex. Significantly, the third hypervariable
`loop, which was difficult to follow in the heavy chain
`of the BV04-01 Fab, was well defined in the fluores(cid:173)
`cein-Fab complex. In the presence ofligand, constit(cid:173)
`uents of this loop were found to have small temper(cid:173)
`ature factors (B values), characteristic of regions
`with low mobility.
`
`Comparison of the Structures of the 4-4-20 and
`BV04-01 Fabs
`
`The 4-4-20 Fab is an extended molecule in which
`the pseudotwofold axes between the pairs of variable
`and constant domains are nearly colinear (i.e., the
`measured "elbow bend" angle between the two
`pseudodiads is 171 °). Except for the third hypervari(cid:173)
`able loops, in which the 4-4-20 heavy chain is
`shorter than the BV04-01 sequence by three resi(cid:173)
`dues, the o:C tracings are remarkably similar in the
`two proteins. This similarity is readily understand-
`
`PFIZER EX. 1086
`Page 5
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`

`

`274
`
`J . N. HERRON ET AL.
`
`1
`
`2
`
`Fig. 1. Three-dimensional electron density (orange) corre(cid:173)
`sponding to fluorescein (green) in the combining site of the 4-4-20
`Fab. This electron density was obtained in a difference Fourier
`map after crystallographic refinement of the ligand-protein com(cid:173)
`plex. A skeletal model of fluorescein was fitted to the electron(cid:173)
`density by interactive computer graphics.
`
`Fig. 2. Stereo diagram of a C tracings of the light (blue) and
`heavy (red) chains of the 4-4-20 Fab, with fluorescein (green) 1n
`the combining site. The "variable" domains are at the top and the
`"constant" domains are at the bottom. Disulfide bonds are repre(cid:173)
`sented by yellow bars.
`
`PFIZER EX. 1086
`Page 6
`
`

`

`STRUCTURE OF FLUORESCEIN-FAR COMPLEX
`
`275
`
`Fluorescein Binding Site
`Fluorescein fits into a slot lined by constituents of
`bo_th the light and heavy chains (see Figs. 4 and 5),
`and participates in a network~of interacting aro(cid:173)
`matic groups with the protein. There are 68 pairs of
`atoms of the ligand and protein separated by < 4 A.
`Of the potential interactions, 42 _are associated with
`the xanthonyl moiety ~nd 26 with thE! benzoyl group
`of fluorescein. This distribution is consistent with
`- the assignment of the xanthonyl moiety as the "im-
`-niunodominant" portion of the hapten.
`_ Tryptophan L101 forms the bottom-of the slot and
`tryptophan H33 and tyrosine L37 provide the sides.
`The width of the slot, as measured from the distance
`between the centroids of the rings oftryptophlln H33
`and tryosine L37, is about 9 A. The phenolic ring of
`tyrosine L37 is stacked with the xantbonyl moiety of
`fluorescein. Tryptophan H33 interacts with the hap-
`- ten to produce an aromatic pair of the type described
`by Burley and Petsko.38 One enolic group (oxyg~n 1)
`on the xanthonyl ring is only 3.0 A from an imid(cid:173)
`azolium nitrogen of histidine L31. The other enolic
`group (oxygen 3) is sufficiently close (2.8 A) to the
`guanido group of arginine L39 for an electrostatic
`interaction and is also within hydrogen bonding dis(cid:173)
`tance of serine L96. Thus one of the .ligand's. two
`negative charges is ;eutralized by the antibody. Ox(cid:173)
`ygen 2, in the ether linkage at the top-ef the middle
`ring of the xanthonyl moiety, does not participate in
`.intenictions-with the protein.
`-
`-
`Surprisingly, the second negative charge on fluo(cid:173)
`rescein (the. carboxyLic acid) is not formally neutral(cid:173)
`ized by- a protein substitue~t. Instead, this group
`faces the solvent at an open end of the slot. Partial
`charge compensation is achieved by the formation of
`a hydrogen bond between the phenolic hydroxyl
`group of tyrosine L37-and one of the carbo~ylic acid
`oxygen atoms (the electron density in a 2F0 - Fc map
`is continuous between the two sets of atoms).
`Tyrosine 103 extends- toward the phen.yl ring of (cid:173)
`fluorescein ~nd tyrosine 102 contributes to the
`_structural integrity of the binding site-\Yithout ac-
`- tua1ly being in contact with the hapten (see Fig. 4). _
`There is a clear sol; ent channer for. a lysine side
`- chain to be connected to an isothiocyanate group in
`the para position of the phenyl ring, as would be
`expected for a hapten coupled to the KLH carrier
`protein in the immunogen. This solvent channel is
`shown in Figure ·5.
`Glutamine H50, which replaces an important
`arginine residue in the putative binding site of the
`BV04-01 Fab, is mostly buried in the presence of
`fluorescein in the 4-4-20 Fab. However, the polar
`end of the side chain (the carbonyl oxygen of the
`amide group) is within hydrogen bonding distance of
`the indole nitrogen of tryptophan H33. Arginine
`H52; another key residue in the BV04-01 binding
`region, is salt bridged to glutamic acid H59 (an ala(cid:173)
`nine in BV04-01).
`
`Fig. 3. Superimposed aC tracings of the 4-4-20 and BV04-01
`Fabs, the latter ·having specificity for single-straneed DNA. The
`two chains of the 4-4-20 Fab are colored red (heavy) and blue
`(light). The 4-4-20 Fab was cocrystallized with fluorescein in 2-
`methyl-2,4-pentanediol and the BV04-01 Fab was crystallized
`without ligand in ammonium sulfate. Yet the two crystals were
`nearly isomorphous and the structures of the Fabs were very
`similar.
`
`able in the light chains, which differ in only six
`amino · acid positions. 34 However, there are 42 -re(cid:173)
`placements in the variable domain of the 4-4-20
`heavy chain. Substitutions that appear to be critical
`for the binding of fluorescein are arginine 39 for
`histidine and tryptophan 101 for leucine in the light
`chain; tryptophan 33 for alanine and glutamine 50
`for arginine in the heavy chain. Because of differ(cid:173)
`ences in the lengths of the third hypervariable loops,
`tyrosine 102 .and 103 have nQ direct counterparts in
`the BV04-01 Fab. Interestingly, arginine 39 is re(cid:173)
`placed by histidine in other members of the antiflu(cid:173)
`orescyl idiotype family and by glutamine in the Meg
`-Bence-Janes dimer. This glutamine is located out(cid:173)
`side the regions available for ligand binding.12
`
`PFIZER EX. 1086
`Page 7
`
`

`

`276
`
`J. N. HERRON ET AL.
`
`Fig. 4. Stereo diagram of the hapten binding site in the 4-4-20
`Fab. Light (L) chain constituents are blue, heavy (H) chain com(cid:173)
`ponents are red, and fluorescein is green. The xanthonyl (three
`ring) group of fluorescein is flanked by tyrosine L37 on the left and
`tryptophan H33 on the right, with tryptophan L 101 forming the
`
`bottom of the slot. Enolic oxygen atoms on opposite corners of the
`xanthonyl group point toward histidine L31 (left) and arginine L39
`(right). The phenyl carboxyl group of fluorescein (single ring) is
`located below tyrosines H103 (left) and H102 (right).
`
`of the unliganded BV04-01 Fab. Light chain constit(cid:173)
`uents of the 4-4-20 Fab were found to be involved in
`a large proportion (23 of the 26 examples in the sur (cid:173)
`vey) of major packing interactions. The light chain
`is also prominent in crystal packing of the unli(cid:173)
`ganded BV04-0l. The close similarities in amino
`acid sequences and conformations of the t wo light
`chains are apparently reflected in the packing of the
`parent Fabs, despite the differences in crystallizing
`media.
`
`Sequestering of an MPD Molecule in the
`VL-VH Interface
`
`In the interface of the variable domains below the
`fluorescein binding site (closest distance ~4.6 A),
`there was a module of electron density that could not
`be assigned to either protein or hapten. This module
`had the size and shape expected for a molecule of
`MPD, and persisted in maps calculated after ad(cid:173)
`vanced stages of the crystallographic refinement.
`The cage electron density for the putative solvent
`molecule is shown with the surrounding protein con(cid:173)
`stituents in Figure 7. MPD was omitted from the
`model used to calculate the phases for the difference
`Fourier map. The binding site for MPD was lined by
`both polar and apolar residues, such as threonine 99,
`serine 101, and tryptophans 47 and 108 of the heavy
`chain, and arginine 39, tyrosine 41, and tryptophan
`101, and phenylalanine 103 of the light chain . Hy(cid:173)
`droxyl groups on threonine H99 and tyrosine L41
`were located within hydrogen bonding distances of
`the 2- and 4-hydroxyl groups of MPD.
`
`Fig. 5. Solvent-accessible surface dot representation37 of the
`hapten combining site, with a skeletal model of fluorescein super(cid:173)
`imposed. Note the free space above the para position of the phe(cid:173)
`nylcarboxyl group of fluorescein. In the immunogen used to elicit
`the production of the 4-4-20 antibody, a lysine side chain on a
`carrier protein would be linked to an isothiocyanate group in the
`para position of the hapten. Lysine would presumably occupy the
`free space when the hapten entered the antibody combining site.
`
`Crystal Packing Interactions in the 4-4-20 and
`BV04-0l Systems
`Analyses of packing interactions revealed struc(cid:173)
`tural features that are conducive to the formation of
`4-4-20 Fab crystals nearly isomorphous with those
`
`PFIZER EX. 1086
`Page 8
`
`

`

`STRUCTURE OF FLUORESCEIN- FAB COMPLEX
`
`277
`
`L I GHT CHA IN
`
`CDR 1
`
`C R S S 0 S LV H S N G NT Y L RW
`40
`25
`30
`35
`27 a b c d e 28 30
`25
`35
`
`HE AVY CHAIN
`
`CDR1
`
`SDYWMN
`* 30
`35
`t 30
`35
`
`CD R2
`Y K VSN RF SG
`* 55
`60
`t 50
`55
`
`CD R3
`CSOSTHV P W T
`*
`95
`100
`90
`95
`
`CDR2
`AOIRNKPYNYE TYYSDSVKG R
`* 50
`55
`60
`65
`t 50 52 a b c 53 55
`
`60
`
`65
`
`CDR3
`GSYYGMDYW
`* 100
`105
`IOOa b c d 101
`
`Fig. 6. Amino acid sequences of the hypervariable regions
`(complementarity-determining regions or CDR45
`) of the light and
`heavy chains of the 4-4-20 Fab. These sequences were deter-
`
`mined by Bedzyk et al.34 (*) The strict sequential numbering sys(cid:173)
`tem used in our computers and graphics terminals. (t) The more
`widely used numbering scheme of Kabat et al. 45
`
`DISCUSSION
`A simple, yet restricted combining site seems well
`suited for a specialized high-affinity antibody for
`fluorescein. Space available for binding in the site is
`limited. While geometrically optimized, the aro(cid:173)
`matic, electrostatic, and hydrogen bonding interac(cid:173)
`tions are relatively few in number. We suspect that
`the formation of such a tight complex was accompa(cid:173)
`nied by conformational adjustments in the protein.
`Recent immunological studies support this view. For
`example, antibodies elicited to the liganded site of
`the 4-4-20 antibody were not reactive with the non(cid:173)
`liganded, idiotypic state. 39 These studies suggest the
`generation of new epitopes on ligand binding as a
`consequence of induced conformational changes.
`It is interesting to ask what factors contribute to a
`high-affinity site. There is a red shift in the absorp(cid:173)
`tion spectrum when fluo