`
`Three-Dimensional Structure of a Fluorescein-Fab
`Complex Crystallized in 2-Methyl-2,4-pentanediol
`James N. Herron,’ Xiao-min He,’ Martha L. Mason,’ Edward W. Voss, Jr.? and Allen B. Edmundson’
`‘Department of Biology, University of Utah, Salt Lake City, Utah 841 12 and ‘Department of Microbiology,
`University of Illinois, Urbana-Champaign, Illinois 61 081
`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-
`toantibody (BV04-01) with specificity for single-
`stranded DNA. In the 4-4-20 complex fluores-
`cein fits tightly into a relatively deep slot
`formed by a network of tryptophan and ty-
`rosine side chains. The planar xanthonyl ring of
`the hapten is accommodated at the bottom of
`the slot while the phenylcarboxyl group inter-
`faces with solvent. Tyrosine 37 (light chain) and
`tryptophan 33 (heavy chain) flank the xantho-
`nyl group and tryptophan 101 (light chain) pro-
`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-
`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-
`clonal antifluorescyl antibodies. The phenyl
`carboxyl group of fluorescein appears to be hy-
`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-
`sible for the decrease in affinity for the hapten
`in MPD.
`
`techniques for correlating antigen binding affini-
`ties, kinetics, and thermodynamic^.'-^ Further-
`more, it has been possible to develop a family of
`idiotypically cross-reactive antibodies in which indi-
`vidual monoclonals vary in affinity over a 1000-fold
`range.g Amino acid sequences recently determined
`for eight of these antibodies in one of our laborato-
`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-
`ther the understanding of both idiotypy and affinity
`maturation. In this report, we describe the three-
`dimensional structure of a complex of dianionic flu-
`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 IgG2, (K) antibody
`that binds fluorescein with an association constant
`of 3.4 x 10” Mpl in aqueous solution. This affinity
`decreases 300-fold in 47% (viv) 2-methyl-2,4-
`pentanediol (MPD), the solvent used for cocrystalli-
`zation of the 4-4-20 Fab with fluorescein h a ~ t e n . ~
`The antibody is a highly specialized molecule that
`does not cross-react with rhodamine compounds.’S6
`In the formation of a complex with the 4-4-20 anti-
`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-
`cein induces a diverse immune response when in-
`jected as a conjugate with keyhole limpet hemocya-
`nin (KLH).’z7-’ Interpretation of binding studies
`have sometimes been ambiguous with heteroge-
`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-
`
`Key words: antifluorescyl monoclonal anti-
`body, high-affinity binding site, ef-
`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-
`ities (105-1010 M-’), and a variety of experimental
`
`0 1989 ALAN R. LISS, INC.
`
`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-
`maceutics, University of Utah, Salt Lake City, UT 84112.
`
`PETITIONER'S EXHIBITS
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`Exhibit 1086 Page 1 of 10
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`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-
`munogen, molecules of the isothiocyanate derivative
`of fluorescein amine (isomer I) were coupled to
`KLH, with the principal reactions presumed to in-
`volve the €-amino group of lysine side chains. The
`4-4-20 hybridoma was obtained by fusion of hyper-
`immune splenocytes stimulated with the fluorescein-
`KLH conjugate.
`The present work adds to previous crystallo-
`graphic studies of complexes in which small mole-
`cules were diffused into crystals of human and mu-
`rine immunoglobulin fragments with binding sites
`shaped like cavities or shallow
`These studies have helped define the molecular ba-
`sis of low- and medium-affinity interactions and
`have provided the background to broaden our under-
`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-
`aminida~e.’~-~’ Antibodies binding DNA have
`grooves as potential combining sites, and cocrystals
`of Fabs and oligodeoxynucleotides are currently be-
`
`ing subjected to X-ray a n a l y ~ e s . ~ ~ - ~ ~ The structure
`of an unliganded Fab (5539) with specificity for ga-
`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-
`teinZ5 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-
`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 a r t i ~ l e . ~ In
`
`outline the Fab fragments were prepared by hydro-
`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-
`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-
`matofocusing, dialysis, enzymatic hydrolysis, and
`crystallization trials were all covered with alumi-
`num foil. All manipulations and transfers were car-
`ried out in reduced light. Under such precautions
`
`the liganded Fab was eluted from the chromatofo-
`cusing column as a single component with a pZ 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-
`perature (12-14°C) control. Graded aliquots of MPD
`were added to 40 p1 samples of the liganded protein
`in flat-bottomed glass vials. Crystals appeared in 2
`days with final MPD concentrations of 38-60%
`(viv), the optimum being -47%. Bladed crystals
`suitable for X-ray analysis grew to dimensions of 0.6
`x 0.35 x 0.3 mm (1 x w x d) in 1-2 months. Green
`fluorescence, attributable to the dissociation of flu-
`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; ci = 82.1, p = 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 a t
`12-14°C. Temperature instability of the complex in
`MPD had also been noted in solution.’ For example,
`irreversible increases in the standard free-energy
`changes (AGO) in the liganded IgG and Fab mole-
`cules occurred in 40% MPD at relatively low tem-
`peratures (transition temperature of 30°C).
`A single crystal was used to collect X-ray diffrac-
`tion data to 2.7 A resolution with a Nicolet P21 dif-
`fractometer operated at 40 kV and 35 mA (CuK, ra-
`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 (Pl) as the unliganded Fab of the
`BV04-01 IgGzb autoantibody, with specificity for
`single-stranded DNA.’,26 Unit cell dimensions for
`the two crystals were nearly identical and one of
`us (X-M.H.) found by molecular replacement meth-
`ods27-30 that the proteins were in the same orienta-
`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 r e f i r ~ e m e n t ~ l l ~ ~ 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
`
`PETITIONER'S EXHIBITS
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`STRUCTURE OF FLUORESCEIN-FAB COMPLEX
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`273
`
`TABLE I. Refinement Data
`
`Target
`value
`
`Actual rms deviation
`46.6
`0.215
`15.5
`8304
`
`Average AF*
`R-factor
`< B > in
`No. of reflections, Z > 1.5 u (I)$
`Root mean square (rrns) deviations
`From ideal distance (A)
`Bond distance
`Angle distance
`Planar 1-4 distance
`Hydrogen bond distanceg
`rms deviation from planarity (A)
`rms deviation from ideal chirality (A3)
`rms deviation from permitted contact distances (A)
`Single torsion contacts
`Multiple torsion contacts
`Possible hydrogen bond
`rms deviation from ideal torsion angles (“1
`For prespecified 4, JI angles
`15.0
`30.8
`6.3
`For planar group (0 or 180)
`3.0
`26.9
`For staggered group (+ 60 or 180)
`15.0
`15.0
`28.1
`For orthonormal group (2 90)
`*The weight for the structure factor refinement was obtained from the equation w = (lie)', where u =
`40 - 278 x [sin(O)/h - 1161.
`tAverage temperature factor.
`$Resolution limits of 2.7-6.0 A.
`$Explicit hydrogen bonds for P-pleated sheets
`
`0.028
`0.055
`0.032
`0.132
`0.014
`0.210
`
`0.280
`0.400
`0.409
`
`0.03
`0.04
`0.03
`0.05
`0.025
`0.150
`
`0.500
`0.500
`0.500
`
`of the light and heavy chains were altered to corre-
`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 ~ r o g r a m . ~ ~ . ~ ~ Polypeptide backbones
`and amino acid side chains were fitted to 2F,-Fc
`maps, in which F, and F, were observed and calcu-
`lated structure factors. Fluorescein was located in a
`A F (F,-F,) map, for which the phase angles were
`calculated from the refined atomic model of the pro-
`tein (atomic coordinates for fluorescein were omit-
`ted). The ligand-protein complex was subjected to
`additional cycles of refinement until the R factor
`(Z /I F, 1-1 F, I/ /Z I F, I ) began to plateau at its cur-
`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-
`rescein was fitted by interactive computer graphics.
`The torsion angle measured between the xanthonyl
`and benzoyl rings was 73” in the bound hapten.
`
`An aC skeletal model of the 4-4-20 Fab is shown
`as a stereo pair in Figure 2, with a model of fluores-
`cein codisplayed in the binding site. Tracings of the
`aC chains of the 4-4-20 and BV04-01 Fabs are su-
`perimposed in Figure 3. Details of the structures of
`the ligand and combining site are presented in Fig-
`ure 4 (skeletal models). Solvent-accessible sur-
`
`f a c e ~ ~ ~ are illustrated in Figure 5. Amino acid
`sequences34 for the hypervariable regions are listed
`in Figure 6.
`Polypeptide chains could be traced unambigu-
`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-
`cein-Fab complex. In the presence of ligand, constit-
`uents of this loop were found to have small temper-
`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-
`able loops, in which the 4-4-20 heavy chain is
`shorter than the BV04-01 sequence by three resi-
`dues, the aC tracings are remarkably similar in the
`two proteins. This similarity is readily understand-
`
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`J. N. HERRON ET AL.
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`Fig. 1 . Three-dimensional electron density (orange) corre-
`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-
`plex. A skeletal model of fluorescein was fitted to the electron-
`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) in
`the combining site. The “variable” domains are at the top and the
`“constant” domains are at the bottom. Disulfide bonds are repre-
`sented by yellow bars.
`
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`STRUCTURE OF FLUORESCEIN-FAB COMPLEX
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`275
`
`Fluorescein Binding Site
`Fluorescein fits into a slot lined by constituents of
`both the light and heavy chains (see Figs. 4 and 5 ) ,
`and participates in a network of interacting aro-
`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 and 26 with the benzoyl group
`of fluorescein. This distribution is consistent with
`the assignment of the xanthonyl moiety as the “im-
`munodominant” portion of the hapten.
`Tryptophan LlOl 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 of tryptophqn H33
`and tryosine L37, is about 9 A. The phenolic ring of
`tyrosine L37 is stacked with the xanthonyl 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 (oxygen 1)
`on the xanthonyl ring is only 3.0 A from an imid-
`azolium nitrogen of histidine L31. The other enolic
`group (oxygen 3) is suficiently close (2.8 A) to the
`guanido group of arginine L39 for an electrostatic
`interaction and is also within hydrogen bonding dis-
`tance of serine L96. Thus one of the ligand’s two
`negative charges is neutralized by the antibody. Ox-
`ygen 2, in the ether linkage at the top of the middle
`ring of the xanthonyl moiety, does not participate in
`interactions with the protein.
`Surprisingly, the second negative charge on fluo-
`rescein (the carboxylic acid) is not formally neutral-
`ized by a protein substituent. 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 carboxylic acid
`oxygen atoms (the electron density in a 2F,-F, map
`is continuous between the two sets of atoms).
`Tyrosine 103 extends toward the phenyl ring of
`fluorescein and tyrosine 102 contributes to the
`structural integrity of the binding site without ac-
`tually being in contact with the hapten (see Fig. 4).
`There is a clear solvent channel 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-
`nine in BV04-01).
`
`Fig. 3. Superimposed (YC tracings of the 4-4-20 and BVO4-01
`Fabs, the latter having specificity for single-stranded 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 BVO4-01 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 position^.^^ However, there are 42 re-
`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-
`ences in the lengths of the third hypervariable loops,
`tyrosine 102 and 103 have no direct counterparts in
`the BV04-01 Fab. Interestingly, arginine 39 is re-
`placed by histidine in other members of the antiflu-
`orescyl idiotype family and by glutamine in the Mcg
`BenceJones dimer. This glutamine is located out-
`side the regions available for ligand binding.”
`
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`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-
`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 LlOl 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-
`uents of the 4-4-20 Fab were found to be involved in
`a large proportion (23 of the 26 examples in the sur-
`vey) of major packing interactions. The light chain
`is also prominent in crystal packing of the unli-
`ganded BV04-01. The close similarities in amino
`acid sequences and conformations of the two 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-
`vanced stages of the crystallographic refinement.
`The cage electron density for the putative solvent
`molecule is shown with the surrounding protein con-
`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-
`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 repre~entation~~ of the
`
`hapten combining site, with a skeletal model of fluorescein super-
`imposed. Note the free space above the para position of the phe-
`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
`BV04-01 Systems
`Analyses of packing interactions revealed struc-
`tural features that are conducive to the formation of
`4-4-20 Fab crystals nearly isomorphous with those
`
`PETITIONER'S EXHIBITS
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`Exhibit 1086 Page 6 of 10
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`-
`-
`
`H E A V Y C H A I N
`
`CD R i
`S D Y W M N
`* 30
`35
`30
`35
`
`L I G H T C H A I N
`
`C D R I
`I
`I
`C R S S O S L V H S N G N T Y L R W
`*
`40
`35
`25
`30
`t
`2 7 0 b c d e 2 8 30
`25
`35
`
`-
`
`CDR2
`Y K V S N R F S G
`* 55
`60
`5 5
`50
`
`CDR3
`C ' S 0 S T H V P W ' l
`*
`I00
`95
`90
`95
`t
`
`STRUCTURE OF FLUORESCEIN-FAB COMPLEX
`
`277
`
`CDR2
`A ' Q I R N K P Y N Y E T Y Y S D S V K G ' R
`* 50
`6 5
`55
`60
`5 0 5 2 0 b c53 55
`
`6 0
`
`65
`
`CDR3
`G S Y Y G M D Y W
`* too
`105
`lOOa b c d 101
`t
`
`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-
`tem used in our computers and graphics terminals. (+) The more
`widely used numbering scheme of Kabat et
`
`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-
`matic, electrostatic, and hydrogen bonding interac-
`tions are relatively few in number. We suspect that
`the formation of such a tight complex was accompa-
`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-
`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-
`tion spectrum when fluorescein is bound to the
`4-4-20 and other monoclonal antifluorescyl anti-
`This shift has been attributed to hydro-
`phobic effects involving tryptophan side chains. The
`model of the complex supports such a view, since two
`trytophan residues are located in close juxtaposition
`to the ligand in the 4-4-20 combining site. However,
`strategically placed tryptophans are not sufficient to
`account for the high affinity of 4-4-20. For example,
`the 9-40 antifluorescyl antibody has tryptophan res-
`idues in homologous positions in its amino acid se-
`quence and a lower affinity for the ligand than
`4-4-20.41
`Quenching of fluorescence is characteristic of an-
`tifluorescyl antibodies. The maximum quenching
`constant for the 4-4-20 molecule is greater than the
`Q,,, values of some antibodies (e.g., 20-4-4), but
`slightly smaller than those for other antibodies (e.g.,
`
`20-20-3) with lower affinities for f l u o r e ~ c e i n . ~ ~ ~ ~
`Moreover, Qmax for 4-4-20 actually increases in
`MPD while the ligand affinity decrease^.^ These re-
`sults indicate that there is not a strong correlation
`between fluorescence quenching and high affinity.
`Interactions resulting in quenching appear to be
`very diverse. Tryptophan has been invoked as a pos-
`sible participant in such interactions and in one case
`(20-20-3) histidine was suggested to be involved in
`the protonation of
`the enolic group of bound
`fluorescein.2 The 4-4-20 active site contains both
`tryptophan and histidine residues in positions that
`should be favorable for fluorescence quenching.
`Model-based hypotheses seem to be especially use-
`ful
`in explaining deuterium fluorescence en-
`
`h a n ~ e m e n t ~ , ~ ~ and iodine quenching43 of bound
`ligand in antifluorescyl antibodies. The liganded 4-
`4-20 molecule shows about 188% fluorescence en-
`hancement in deuterium oxide (relative to water).
`Such experiments afford a measure of the degree of
`hydrogen bonding between ligand and protein. The
`enhancement for 4-4-20 was 10 times greater than
`values for four other antifluorescyl monoclonal an-
`tibodies and hydrogen bonding was therefore consid-
`ered to be very important for ligand binding.2 The
`structure of the complex is consistent with this pro-
`posal and can be used to identify the most prominent
`hydrogen bonds (enol with histidine L31 and phenyl
`carboxyl group with tyrosine L37). Only 2.5%
`quenching of bound fluorescein was noted when the
`liganded 4-4-20 IgG was titrated with potassium
`
`i ~ d i d e . ~ , ~ ~ This observation indicated that fluores-
`cein bound in the 4-4-20 active site was essentially
`inaccessible to iodide. Quenching was higher (8.4-
`15.5%) in four other antibodies with lower affinities
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1086 Page 7 of 10
`
`
`
`278
`
`J. N. HERRON ET AL.
`
`Fig. 7. Stereo diagram of the cage electron density (lavender)
`corresponding to a molecule of MPD in its binding site just below
`that of fluorescein in the V domain interface. Key residues of the
`light (blue) and heavy chains (red) are labeled with three-letter
`
`abbreviations. The hydroxyl groups of threonine H99 and tyrosine
`L41 may form hydrogen bonds with the 2-and 4-hydroxyl groups
`of MPD.
`
`for f l ~ o r e s c e i n . ~ Figures 4 and 5 show a very tight fit
`of the ligand in the site, and the presence of tyrosine
`102 and 103 further shields the bound ligand from
`additional reactants. These tyrosine residues are not
`present in the sequence of the 9-40 antibody, in
`which bound fluorescein is more accessible to
`i ~ d i d e . ~
`The interactions of tyrosine L37 with fluorescein
`appear to be important in orienting the ligand in the
`high-affinity site. The stacking of the phenyl group
`of tyrosine with the xanthonyl ring was further sta-
`bilized by a hydrogen bond between the phenolic hy-
`droxyl group and the phenylcarboxyl moiety of flu-
`orescein. The dual interaction of fluorescein with
`tyrosine also played a major role in fixing the tor-
`sion angle between the xanthonyl and phenyl rings,
`which would have greater freedom to rotate in solu-
`tion.
`Studies of the effects of pH on lifetimes of dissoci-
`ation indicated that ionizable groups (pK > 8.0) in
`
`the 4-4-20 active site contribute to the binding of
`f1uoresceim2 Lifetime maxima at pH 6.5-7.0 sug-
`gested that the xanthonyl moiety is negatively
`charged when bound to the 4-4-20 (the pK of the
`enolic group is 6.744). In the 3-D structure of the
`complex the two enolic groups of fluorescein are
`bracketed by the ionizable side chains of histidine
`L31 and arginine L39. At the pH used for crystalli-
`zation the histidine side chain would be expected to
`be in the uncharged form. The formation of an ion
`pair with arginine probably makes a very signifi-
`cant contribution to the binding energy, particularly
`in a region of low dielectric constant. In the 9-40
`
`antibody this arginine is replaced by h i ~ t i d i n e , ~ ~ a
`substitution that could partially account for the de-
`crease in affinity relative to 4-4-20.
`Rhodamine 110 and rhodamine B, structural an-
`alogs of fluorescein with amino (110) and diethyl-
`amino (B) groups substituted for the enolic oxygen
`atoms, do not bind to 4-4-20.' Acylamino groups
`
`PETITIONER'S EXHIBITS
`
`Exhibit 1086 Page 8 of 10
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`STRUCTURE OF FLUORESCEIN-FAB COMPLEX
`
`279
`
`would have formal positive charges a t relatively low
`pH values, ( 6 1 , a t which there would be electro-
`static repulsion by like charges on the histidine L31
`and arginine L39 side chains. At pH 8 the only
`charge on a rhodamine molecule would reside on the
`phenylcarboxyl group. A monoanionic ligand would
`be incompatible with the pH profile of binding,
`which indicates a strong dependence on the presence
`of a dianion. In the case of rhodamine B, there is an
`additional steric factor to consider. For example, tet-
`ramethyl rhodamine formed a tight complex with
`the Mcg Bence-Jones dimer, but the tetraethyl de-
`rivative (rhodamine B) could not be accommodated
`in the binding site. Exposure to rhodamine B led to
`destruction of a crystal of the Mcg dimer in 5
`hours. l3
`The crystal structure of the fluorescein-Mcg com-
`plex provides further insight into the minimal re-
`quirements for low-affinity binding of the ligand. As
`in 4-4-20, the ligand was accommodated in a net-
`work of aromatic residues. The xanthonyl ring of
`fluorescein was more deeply immersed in the cavity
`than the benzoyl moiety and participated in more
`interactions (14 vs 6) with the protein. A tyrosine
`side chain was wedged between the xanthonyl and
`benzoyl groups and largely dictated the torsion an-
`gle of 57” between these two rings. At the pH (6.2) of
`the crystals fluorescein was a monoanion and the
`charged group (phenylcarboxyl moiety) was oriented
`toward bulk solvent at the entrance of the binding
`cavity. In summary, the low-affinity site was more
`voluminous and there were fewer ligand-protein in-
`teractions. Aromatic rings did not stack, and poten-
`tial negative charges were not balanced by basic
`side chains on the protein.
`Thermodynamic studies indicated that the tem-
`perature stabilities of both the intact 4-4-20 anti-
`body and Fab were significantly decreased in the
`presence of 40% (v/v) MPD.5 These studies also sug-
`gested that the decrease in affinity for fluorescein in
`MPD was mainly attributable to conformational
`changes in the protein. Future comparisons of the
`present structure with that of the same complex
`crystallized in polyethylene glycol5 will hopefully
`provide greater understanding of the general effects
`of these solvents.
`Since MPD was added to the crystallization mix-
`ture after the formation of the hapten-Fab complex,
`we speculate that fluorescein would be dissociated
`from the protein prior to the admission of such a
`sizable solvent molecule to a deeper portion of the
`variable domain interface. Irrespective of the mode
`of entry of MPD, the local perturbations would be
`expected to have significant effects on the affinity
`constant of a hapten bound in a