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`15 AUGUST 1986
`VoL. 233 • PAGEs 693-816
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`BIOEPIS EX. 1059
`Page 1
`
`

`

`AMERICAN
`ASSOCIATION FOR THE
`ADVANCEMENT OF
`SCIENCE
`
`SciENCE ISSN 0036-8o7s
`
`15 AUGUST 1936
`VOLUME 233
`NUMBER4765
`
`699
`
`MIIHIUQm- 701
`Mk§@ill§lhif- 702
`MRDII4i@IJ41111- 704
`Letters
`
`707
`
`Resean·h Artides
`
`Rep<H'b
`
`712
`
`713
`715
`717
`
`720
`
`722
`
`723
`
`727
`
`734
`
`740
`
`747
`
`755
`
`758
`
`•
`
`•
`
`This Week in Science
`
`The United States and the IIASA Connection
`
`Structural Basis for Antigen-Antibody Recognition: R. HUBER
`
`A Novel Strain of Recklessness • Rifkin Against the World
`
`Nuclear Waste: D. F. UTTER; A.M. WEINBERG; P. T. VERNIER; R . A. PALMER;
`P. WILSON; K. ANDERSON
`
`U .S., Japan Reach Truce in Chips War
`
`Computers in Class at the Awkward Age
`
`The Chesapeake Bay's Difficult Comeback
`Briefing: Air Force to Mothball Vandenberg, Reduce Reliance on Shuttle •
`Research Fares Well in New French Budget • Saving the Whales Faces New
`Hazard-Research Whaling • NY Bar Calls for Overhaul of R&D Enterprise •
`Graham Nomination Still in Limbo
`
`New Fossil Upsets Human Family
`
`Mathematicians Recognize Major Discoveries
`
`Depression Research Advances, Treatment Lags • Manic Depression and
`Creativity
`
`Metals and DNA: Molecular Left-Handed Complements: J_ K. BARTON
`
`Conservation in South America: Problems, Consequences, and Solutions:
`M . A. MAREs
`
`Cell Recognition by Neuronal Growth Cones in a Simple Vertebrate Embryo:
`J. Y. KUWADA
`Three-Dimensional Structure of an Antigen-Antibody Complex at 2.8 A
`Resolution: A. G. AMrr, R. A. MARIUZZA, S. E. V . PHILLIPS, R. J. POLJAK
`
`The Predicted Structure of Immunoglobulin Dl.3 and Its Comparison with the
`Crystal Structure: C. CHOTHIA, A. M . LESK, M. LEVITT, A. G. AMrr,
`R . A. MAR!UZZA, S. E. V. PHILLIPS, R. J. POLJAK
`
`Cambrian River Terraces and Ridgetops in Central Australia: Oldest Persisting
`Landforms? : A. J. STEWART, D. H . BLAKE, C. D. OLLIER
`
`SCIENCE Ia published weekly on Friday, except the laat week In December, and with a plua luue In * Y,!!!:
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`SCIENCE, VOL.
`
`BIOEPIS EX. 1059
`Page 2
`
`

`

`SciENCE
`
`(Upper) Three-dimensional structure of an antigen-antibody complex.
`COVER
`The antigen is Iyzozyme (green, with a protruding residue, glutamine-121 in red).
`(Lower) The antigen and the antibody have been pulled apart to reveal their
`complementary contacting surfaces. See pages 747 and 755. [A. G. Amit et al.,
`Institut Pasteur, Paris, France]
`
`761
`
`Equatorial Pacific Seismic Reflectors as Indicators of Global Oceanographic
`Events: L. A. MAYER, T. H . SHIPLEY, E. L. WINTERER
`
`765
`
`Two Magnetoreception Pathways in a Migratory Salamander: ]. B. PHILLIPS
`
`767 Molecular Cloning of the Chicken Progesterone Receptor: 0 . M. CoNNEELY,
`W. P. SULLIVAN, D . 0 . ToFT, M. BIRNBAUMER, R. G. CooK, B. L. MAxwELL,
`T. ZARUCKI-SCHULZ, G. L. GREENE, w. T. SCHRADER, B. w. O'MALLEY
`770 Distinct Pathways of Viral Spread in the Host Determined by Reovirus S1 Gene
`Segment: K. L. TYLER, D . A. McPHEE, B. N. FIELDS
`
`774
`
`776
`
`Psychotornimesis Mediated by K Opiate Receptors: A. PFEIFFER, V. BRANTL,
`A. HERZ, H . M. EMRICH
`
`Transplantation of Fetal Hematopoietic Stem Cells in Utero: The Creation of
`Hematopoietic Chimeras: A. W. FLAKE, M. R. HARRISON, N . S. ADziCK,
`E. D. ZANJANI
`
`778 Amplification of an Esterase Gene Is Responsible for Insecticide Resistance in a
`California Culex Mosquito: C. MoucHES, N . PASTEUR,]. B. BERGE,
`0 . HYRIEN, M. RAYMOND, B. RoBERT DE SAINT VINCENT et al.
`
`780 Occult Drosophila Calcium Channels and Twinning of Calcium and
`Voltage-Activated Potassium Channels: A. WEI and L. SALKOFF
`
`783 A Protein Induced During Nerve Growth (GAP-43) Is a Major Component of
`Growth-Cone Membranes: ] . H . P. SKENE, R . D. ]ACOBSON, G. ]. SNIPES,
`C. B. McGUIRE,]. ]. NORDEN,]. A. FREEMAN
`Chromosome Y -Specific DNA Is Transferred to the Short Arm of X
`Chromosome in Human XX Males: M. ANDERSSON, D . C. PAGE,
`A. DE LA CHAPELLE
`
`786
`
`788 Active Human-Yeast Chimeric Phosphoglycerate Kinases Engineered by Domain
`Interchange: M. T. MAs, C. Y. CHEN, R. A. HITZEMAN, A. D . RIGGS
`
`793 Annual Meeting: Call for Contributed Papers
`
`794 A Cycle of Outrage, reviewed by H . Molotch • Les Fourrnis et les Plantes,
`R. C. BucKLEY • Geology of Sedimentary Phosphates, V. E. McKELVEY •
`Some Other Books of Interest • Books Received
`
`797 Benchtop Gas Chromatograph Mass Spectrometer • Mathematics Software for
`Personal Computer • Electrophoresis Blotting Apparatus • Bibliographic
`Software • Peptide Synthesizer • Programmable Viscometer • Database for
`Chemists • Literature
`
`Robert McC. Adams
`Robert W. Berliner
`Floyd E. Bloom
`Mary E. Clutter
`Mildred S. Dresselhaus
`Donald N. Langenberg
`Dorothy Nelkin
`Unda S. Wilson
`William T. Golden
`Treasurer
`William D. Carey
`Executive Officer
`
`Edttort.l Board
`David Baltimore
`William F. Brinkman
`Ansley J. Coale
`Joseph L. Goldstein
`James D. Idol, Jr.
`Leon Knopoft
`Seymour Upset
`WaMer Massey
`Oliver E. Neison
`Allen Newell
`Ruth Patrick
`David V. Ragone
`Vera C. Rubin
`Howard E. Simmons
`Solomon H. Snyder
`Robert M. Solow
`
`Board of Reviewing
`Edttora
`Qais AI-Awqati
`James P. Allison
`Luis W. Alvarez
`Don L. Anderson
`C. Paul Bianchi
`Elizabeth H. Blackburn
`Floyd E. Bloom
`Char1es A. Cantor
`James H. Clark
`Biuce F. Eldridge
`Stanley Falkow
`Theodore H. Geballe
`Roger I. M. Glass
`
`Stephen P. Goft
`Robert B. Goldberg
`Patricia S. Goldman-Rakic
`Corey S. Goodman
`Richard M. Held
`Gloria Heppner
`Eric F. Johnson
`Konrad B. Krauskopf
`Kar1 L. Magleby
`Joseph B. Martin
`John C. McGift
`Mon Meister
`Mortimer Mishkin
`Peter Otson
`Gordon H. Orians
`John S. Pearse
`Yeshayau Pocker
`Jean Paul Revel
`
`Frederic M. Richards
`James E. Rothman
`Thomas C. Schelling
`Ronald H. Schwartz
`Stephen M. Schwartz
`Otto T. Solbrig
`Robert T. N. Tjian
`Virginia Trimble
`Geerat J. Vermeij
`Martin G. Weigert
`Irving L. Weissman
`George M. Wh~esides
`Owen N. Witte
`William B. Wood
`Harriet Zuckerman
`
`TABLE OF CONTENTS 697
`
`BIOEPIS EX. 1059
`Page 3
`
`

`

`Three-Dimensional Structureoof an ¥tigen(cid:173)
`) Antibody Complex at 2.8 A Resolution
`A. G. AMIT, R. A. MA.ruuzzA, S. E. V. PHILLIPS, R. J. PoLJAK
`
`The 2.8 A resolution three-dimensional structure of a
`complex between an antigen (lysozyme) and the Fab
`fragment from a monoclonal antibody against lysozyme
`has been determined and refined by x-ray crystallographic
`techniques. No conformational changes can be observed
`in the tertiary structure of lysozyme compared with that
`determined in native crystalline forms. The quaternary
`structure ofFab is that of an extended conformation. The
`antibody combining site is a rather flat surface with
`protuberances and depressions formed by its amino acid
`side chains. The antigen-antibody interface is tighdy
`packed, with 16 lysozyme and 17 antibody residues
`making close contacts. The antigen contacting residues
`belong to two stretches of the lysozyme polypeptide
`chain: residues 18 to 27 and 116 to 129. All the comple(cid:173)
`mentarity-determining regions and two residues outside
`hypervariable positions of the antibody make contact with
`the antigen. Most of these contacts ( 10 residues out of 17)
`are made by the heavy chain, and in particular by its third
`complementarity-determining region. Antigen variability
`and antibody specificity and affinity are discussed on the
`basis of the determined structure.
`
`T HE BINDING OF FOREIGN ANTIGENS TO COMPLEMENTARY
`
`structures on the surface of B and T lymphocytes represents
`the initial step in the sequence of events leading to activation
`of the immune system. The receptor molecule on the surface of B
`lymphocytes responsible for antigen recognition is membrane
`immunoglobulin. A mature B cell produces and inserts into its
`plasma membrane only limited amounts of a single kind of immuno(cid:173)
`globulin. Contact with antigen results in the expansion of B cell
`clones specific for that antigen and in their differentiation into
`plasma cells capable of producing and secreting large amounts of
`antibody of the same specificity (monoclonal antibody) .
`Antibody molecules of the immunoglobulin G (IgG) class, the
`most abundant in normal serum, are composed of two identical light
`(L) and two identical heavy (H ) polypeptide chains. The amino
`terminal regions of the H and L chains, termed V H and V L, are each
`about 110 amino acids long and have variable (and homologous)
`amino acid sequences. The constant (C) half of the L chain, CL, and
`the constant regions CH 1, CH2, and CH3 of the H chain, each about
`100 amino acids long, have homologous sequences that belong to
`one of a few classes (K and A for L chains; JJ.., B, -y, e, and a for H
`chains). The V H and V L regions each contain three hypervariable or
`complementarity-determining regions (CDR1, CDR2, and CDR3)
`responsible for antigen recognition. These are flanked by less
`variable (FR1, FR2, FR3, and FR4) "framework" regions (1) .
`Present understanding of the three-dimensional structure of
`
`antibody combining sites is based on x-ray diffraction studies of
`myeloma immunoglobulins as reviewed (2). These have shown that
`the conformation of combining sites is determined by the amino
`acid sequences, unique to each different antibody, of the CDR's.
`The structures of two complexes of antigen-binding fragments
`(Fab) of myeloma immunoglobulins with small ligands have also
`been determined (3, 4). Although these studies resulted in useful
`models for ligand-antibody interactions, they are insufficient to
`establish unequivocally the precise size and shape of antibody
`combining sites, the nature and extent of antigen-antibody interac(cid:173)
`tions, and the occurrence of possible conformational changes (if
`any) in the antibody after antigen binding. In addition, the precise
`structure of antigenic determinants on protein molecules remains to
`be determined (5) . Equally important are questions concerning the
`nature of possible conformational changes in the complexed antigen
`and the effect of single amino acid substitutions on antigenic
`specificity and antigen recognition by the antibody.
`We have recently determined the three-dimensional structure of
`an antigen-antibody complex, one between lysozyme and the Fab
`fragment of a monoclonal antibody to hen egg white lysozyme, at 6
`A resolution (6). We have since extended the resolution of the x-ray
`structure determination to 2.8 A, and now present a complete
`description of antigen-antibody interactions in the complex.
`Structure determination. The production of hybrid cell lines
`secreting murine monoclonal antibody to hen egg white lysozyme,
`and the purification, crystallization (7), and 6 A resolution crystal
`structure determination ( 6) of the complex between Fab D 1.3 and
`lysozyme have been described. Crystals grown from solutions
`containing 15 to 20 percent polyethylene glycol 8000 at pH 6.0 are
`monoclinic, space group P2~, with a= 55 .6, b = 143.4, c = 49.1
`A, 13 = 120.5°, and one molecule of complex per asymmetric unit.
`Three heavy atom isomorphous derivatives were prepared with
`(NH4)2PtC4, K3F5U02, and p-hydroxymercuribenzenesulfonate.
`X-ray intensities were measured to 2.8 A resolution with the use of a
`four-circle automatic diffractometer. Heavy atom sites were refined
`in alternate cycles of phasing and refinement (8); isomorphous
`phases, including anomalous scattering contributions (9), were
`calculated. The mean figure of merit (10) to 2.8 A resolution was
`0.47 for 15592 reflections. The electron density map calculated
`from these data was not readily interpretable, presumably because of
`lack of isomorphism of the heavy atom derivatives affecting phase
`determination at high resolution. The phases were further refined by
`a density modification technique (11) with a molecular envelope
`traced from the Fab-lysozyme model determined at 6 A resolution
`(6). The resulting phases depend only on the observed data and the
`overall shape and position of the complex, but are independent of
`the detailed conformation of the previous model ( 6). The resulting
`
`A. G. Amit, R. A. Mariuzza, and R. ]. Poljak are in the Departemenr d'Immunologie,
`Instirut Pasteur, 75724 Paris Cedex 15, France. S. E. V. Phillips is in the Astbury
`Department of Biophysics, Universiry of Leeds, Leeds, United Kingdom.
`
`15 AUGUST 1986
`
`RESEARC H ARTICLES 74 7
`
`BIOEPIS EX. 1059
`Page 4
`
`

`

`Fig. l. Stereo diagram of the Ca skeleton of the
`complex. Fab is shown (upper right) with the heavy
`and light chains with thick and thin bonds, respec(cid:173)
`tively. The lysozyme active site is the cleft containing·
`the label HEL. Antibody-antigen interactions are
`most numerous between lysozyme and the heavy
`chain CDR loops.
`
`CHI
`
`CHl
`
`electron density map was much improved, and an atomic model was
`fitted to it on an Evans and Sutherland PS300 interactive graphics
`system with the use of the program FRODO (12). The amino acid
`sequence ofFab Dl.3 was derived from the corresponding light and
`heavy chain complementary DNA (eDNA) sequences (13). Of the
`562 amino acid residues in the complex, 24 of those in the constant
`regions could not be located in the initial map. The atomic
`coordinates were submitted to alternate cycles of restrained crystal(cid:173)
`lographic least-squares refinement (14) and model building. The
`model was checked in the later stages of refinement by sequentially
`omitting segments of the polypeptide chain (up to 20 percent of the
`total) and rebuilding them in maps phased from the remainder of
`the structure in combination with isomorphous replacement data
`(15) . All residues have now been located, and the current crystallo(cid:173)
`graphic R factor is 0.28 for all data in the 20 to 2.8 A resolution
`range. (R = :£ I IFoi-IFcl I I :£1Fol, where Fo, Fe are the observed
`and calculated structure factors of x-ray reflections_) No attempt was
`made to locate solvent molecules. Two isotropic temperature factors
`were used for each residue, one for the main chain atoms, and
`another for the side chain atoms. Stereochemical restraints were
`adjusted to give a standard deviation in c-c bonds of ±0.03 A. No
`restraints were applied between residues across the antibody-antigen
`interface. Atomic coordinates will be deposited at Brookhaven Protein
`Data Bank after higher resolution and crystallographic refinement.
`Conformation of the complexed antigen and of the Fab. The
`overall structure of the complex at 2.8 A resolution (Fig. 1) confirms
`the results of the 6 A resolution study (6). The assignment of the H
`and L polypeptide chains of Fab is unchanged. The closely packed 13
`sheets are seen in Fab as are the helical and p-sheet structures
`surrounding the active site in lysozyme. The Fab appears in an
`almost fully extended conformation, with a definite" separation
`between the variable (V) and constant (C) domains. With the
`exception of this difference in quaternary structure, Fab Dl.3
`compares closely to other known Fab's (4, 16), except in the CDR
`loops. Predicted structures for D 1.3 (17) based on other Fab's also
`agree well with the determined structure in the framework 13-sheet
`regions and in some, but not all, of the CDR loops. The relative
`disposition of the variable subunits of the H chain (V H) and of the L
`chain (V L), is unaltered, indicating no change in quaternary struc(cid:173)
`ture in the V domain resulting from antigen binding. Since the
`crystal structure of the unliganded Fab Dl.3 has not been deter(cid:173)
`mined, detailed changes in antibody conformation remain to be
`verified. However, the similarity with other Fab structures suggests
`
`that possible conformational changes would be small. This observa(cid:173)
`tion is in agreement with that made by nuclear magnetic resonance
`(NMR) on the unliganded and hapten-liganded (dinitrophenol)
`mouse myeloma protein MOPC315 (18) .
`A least-squares fit of Co: atoms of lysozyme in the complex and
`native lysozyme refined at 1.6 A in its tetragonal crystal form (19 ) gives
`a root-mean-square (rms) deviation of 0.64 A between the two (see
`Fig. 2). Since the error in atomic positions in the complex can
`be estimated (20) to be approximately 0.6 A, the difference is not
`significant. Furthermore, the largest changes (up to 1.6 A) occur in
`regio.ns remote from antibody contacts. Similar comparisons of
`native tetragonal lysozyme with other crystal forms gave rms
`deviations of 0.88 A with triclinic lysozyme refined from x-ray and
`neutron diffraction data (21) and 0.46 A for orthorhombic lysozyme
`determined at physiological temperature (22). Some differences in
`side chain conformation are observed between tetragonal and
`complexed lysozyme, but close examination with computer graphics
`revealed these to be similar to differences observed between different
`crystal structures of native lysozyme. Thus, complex formation with
`antibody Dl.3 produces no more distortion of the structure of
`lysozyme than does crystallization.
`The antigen-antibody interface. The interface between antigen
`and antibody extends over a large area with maximum dimensions of
`about 30 by 20 A (Figs. 3 and 4). The antibody combining site
`appears as an irregular, rather flat surface with protuberances and
`depressions formed by the amino acid side chains of the CDR's of
`V H and V L· In addition, there is a small cleft between the third
`CDR's ofVH and VL, corresponding to the binding site character(cid:173)
`ized in hapten-antibody complexes (3, 4 ). The cleft accepts the side
`chain Gin 121 of lysozyme although this is not the center of the
`antigen-antibody interface (Fig. 3).
`The lysozyme antigenic determinants recognized by Dl.3 are
`made up of two stretches of polypeptide chain, comprising residues
`18 to 27 and 116 to 129, distant in the amino acid sequence but
`adjacent on the protein surface. All six CDR's interact with the
`antigen and in all, 16 antigen residues make close contacts with 17
`antibody residues (Tables 1 and 2). Two antibody contacting
`residues, V L Tyr 49 and V H Thr 30, are just outside segments
`commonly defined as CDR's [sequence numbers are as in Kabat et
`a/.. (1) except for VH CDR3; see Tables 2 and 3]. VH Thr 30 is a
`constant or nearly constant residue in mouse H chain subgroups I
`and II, as is V L Tyr 49 in mouse kappa chains. While the interaction
`of V L Tyr 49 with antigen is relatively weak (one van der Waals
`
`SCIENCE, VOL. 233
`
`BIOEPIS EX. 1059
`Page 5
`
`

`

`Fig. 2. TheCa skeleton of lysozyme in the complex
`(thick trace) superimposed by least squares on that of
`native lysosyrne in the tetragonal crystal form (thin
`trace). The interface to Fab is at the top, and no
`significant conformation change is apparent in this
`region. Greater differences, although still not signifi(cid:173)
`cant at this resolution, occur at the bottom of the
`·
`molecule.
`
`described above, a tightly packed interface which mostly excludes
`solvent.
`.
`Although the antigen-antibody interface involves all six CDR's of
`the Fab, there are more interactions with VH than with VL CDR's,
`and with VH CDR3 in particular (Tables 1 to 3). The geometrical
`
`:2
`5
`0
`
`"' lf
`ty
`0
`
`VI
`e
`n
`11!
`
`I~
`).
`JJ
`·e
`3
`11
`
`contact between its aromatic side chain and Cct of Gly 22 of
`lysozyme), there is a strong hydrogen bond between the hydroxyl
`group of VH Thr 30 and the carbonyl oxygen of Lys 116 of
`lysozyme. This specific interaction involving an invariant antibody
`residue demonstrates
`that the functional distinction between
`"framework" (FR) and CDR residues, although largely maintained,
`is not absolute. The interacting surfaces are complementary, with
`protruding side chains of one lying in depressions of the other (Fig.
`3) in common with other known protein-protein interactions (23) .
`There are many van der Waals interactions interspersed with
`hydrogen bonds. This is most striking for the side chain of Gin 121,
`which penetrates deeply into the Fab, surrounded by three aromatic
`side chains, V L T yr 32 and Trp 92 and V H T yr 101 (Figs. 3, 4, and
`5). Its amide nitrogen forms a strong, buried hydrogen bond to the
`main chain carbonyl oxygen ofV L Phe 91 (Fig. 5 and Table 3). The
`adjacent V H Tyr 101 extends to the surface of lysozyme, its terminal
`hydroxyl group forming hydrogen bonds to the main chain nitro(cid:173)
`gens of Val120 and Gin 121 , and to Oo1 of Asp 119. Many
`hydrogen bonds occur between the side chains of the antigen and
`the main polypeptide chain of the antibody, and vice versa (Table
`3). Hydrogen bonds between main polypeptide chain atoms, similar
`to those in f3-sheet structures, occur between Lys 116 of lysozyme
`and VH Gly 31 , and between Gly 11 7 and VH Gly 53, where the
`lack of side chains allo ws close approach. There are many side chain(cid:173)
`side chain close interactions forming, together with the ones
`
`Fig. 3. Space filling representation of Fab Dl.3 and lysozyme. (A) Antigen(cid:173)
`antibody complex structure as determined in this work. The antibody H
`chain is shown in blue, the L chain in yellow, lysozyme in green, and
`Gin 121 in red. (B) The Fab and lysozyme models have been pulled apart to
`indicate protuberances and depressions of each fit in complementary surface
`features of the other. Compare with (A) above. At the top of the interface,
`protruding V L residues His 30 and Tyr 32 fit into a depression in lysozyme,
`between residues Ilc 124 and Leu 129 (see also Table 1 ). Below the Gin 121,
`!n red, a protuberance of lysozyme consisting of residues around Thr 118 fits
`uuo a surface depression fo rmed by V H residues of CDR1 and CDR2 (V H
`Trp 52 can be seen at the bottom of this depression). (C) End-on views of
`the antibody combining site (left) and the antigenic markers of lysozyme
`recognized by antibody Dl.3, formed from (B) above, by rotating each of
`the molecules approximately 90° about a vertical axis. Contacting residues on
`the antigen and antibody arc shown in red, except for Gin 121 shown in light
`purple. L chain residues that contact the antigen are labeled 1 (His 30), 2
`(Tyr 32), 3 (Tyr 49), 4 (Tyr 50), 5 (Phe 91 ), 6 (Trp 92), and 7 (Ser 93). H
`chain residues that contact the antigen are labeled 8 (Thr 30), 9 (Gly 31 ), 10
`(Tyr 32), 11 (Trp 52), 12 (Gly 53 ), 13 (Asp 54), 14 (Arg 99), 15 (Asp 100),
`16 (Tyr 101), and 17 (Arg 102); see Table l. Lysozyme residues thatcontact
`the antibody are labeled 1 (Asp 18), 2 (Asn 19), 3 (Arg 21 ), 4 (Gly 22), 5
`(Tyr 23), 6 (Ser 24), 7 (Leu 25), 8 (Asn 27), 9 (Lys 116), 10 (Gly 117), 11
`(Thr 118), 12 (Asp 119), 13 (Val 120), 14 (Gin 121), 15 (Ile 124), and 16
`(Leu 129). Gin 121 fits into the antibody surface pocket surrounded by V L
`and VH residues 2, 5, 6, 7, and 16 (Table 1).
`
`15 AUGUST 1986
`
`RESEARCH ARTICLES 749
`
`----------------~~~=-~------
`
`BIOEPIS EX. 1059
`Page 6
`
`

`

`Fig. 4. Stereo diagram of the antibody-antigen interface in
`a similar orientation to Fig. l. All atoms are shown for
`those residues involved in the interaction. Heavy and light
`main chains are indicated by thick and thin bonds, respec(cid:173)
`tively, and hydrogen bonds by dotted lines. Lysozyme
`residues broadly lie below the diagonal from top left to
`lower right of the diagram.
`
`center of the surface lies near V H CD R3, and is occupied by the side
`chain of V H Asp 100, which forms H bonds to the side chains of
`Ser 24 and Asn 27 of lysozyme. Of the antibody hypervariable
`regions, V L CDR2 contributes the least to antigen binding. A large
`number of antibody side chains in the interface (9 out of 15 if we
`exclude Gly residues) are aromatic, thus presenting large areas of
`hydrophobic surface to the antigen; in addition, some of them such
`as V L Tyr 50 and V H Tyr 101 participate in hydrogen bonding with
`the antigen via their polar atoms. In all, 748 A2 or about ll percent
`of the solvent-accessible surface (24) of lysozyme is buried on
`complex formation, together with 690 A2 for the antibody.
`Antigen variability and antibody specificity. The fine specificity
`of monoclonal antibody Dl.3 for other avian lysozymes shows its
`ability to distinguish a single amino acid change in the antigen, at
`position 121. Fab Dl.3 binds hen egg white lysozyme with an
`equilibrium affinity constant of :4.5 X l07M- 1 (25). Bobwhitt: quail
`lysozyme, with four amino acid sequence differences (26) from hen
`lysozyme but none in the interface with Fab Dl.3, binds with
`sinlilar affinity (25). The binding of antibody D 1.3 to the lysozymes
`of partridge [three amino acid differences (26)], California quail
`[four amino acid differences (26)], Japanese quail [six amino acid
`differences (27)], turkey [seven amino acid differences (28)], and
`pheasant and guinea fowl [ten an1ino acid differences each (29)] is
`undetectable (KA < 1 x l05M - 1
`) with the enzyme-linked inlmu(cid:173)
`noabsorption assay used in our laboratory. These lysozymes differ
`from hen lysozyme in the anlino acid residue at position 121, which
`makes close contacts with the antibody. Except for Japanese quail
`and pheasant lysozymes, all have Gln replaced by His.
`
`Table l. Antibody residues involved in contact with lysozyme. Sequence
`positions are numbered as in Kabat eta/. (1) except for V H CDR3, where the
`numbers of Kabat et a/ .. (1) arc given in parentheses.
`
`Antibody residues
`
`Lysozyme residues in contact
`
`Light chain
`CDR1
`
`FR2
`CDR2
`CDR3
`
`Heavy chain
`FR1
`CDR1
`
`CDR2
`
`CDR3
`
`750
`
`His 30
`Tyr 32
`Tyr49
`Tyr 50
`Phe 91
`Trp 92
`Ser 93
`
`Leu 129
`Leu 25, Gin 121, lie 124
`Gly22
`Asp 18, Asn 19, Leu 25
`Gin 121
`Gin 121, Ile 124
`Gin 121
`
`Thr 30
`Gly 31
`Tyr 32
`Trp 52
`Gly 53
`Asp 54
`Arg99 (96)
`Asp 100 (97)
`Tyr 101 (98)
`Arg 102 (99)
`
`Lys 116, Gly 117
`Lys 116, Gly 117
`Lys 116, Gly 11 7
`Gly 117, Thr ll8, Asp 119 .
`Gly 117
`Gly 117
`Arg 21, Gly 22, Tyr 23
`Gly 22, Tyr 23, Ser 24, Asn 27
`Thr ll8, Asp 119, Val120, Gin 121
`Asn 19, Gly 22
`
`A computer graphics analysis indicates that a His residue could be
`placed in the interface, in the space occupied by Gin 121, with small
`displacements of the contacting antibody side chains, maintaining
`the H bonds made by Gln 121. Conformational energy calculations
`(30) confirm this possibility, the total energy being little changed on
`substitution of His for Gln 121. The buried hydrogen bond is
`maintained with good geometry, and only very small shifts of neigh(cid:173)
`to accommodate
`the mutation.
`boring groups are necessary
`This seems to rule out steric hindrance in explaining the absence of
`complex formation when His occurs at position 121. Other possible
`explanations for the effect of this anlino acid substitution include the
`following. (i) His 121 could be charged, and consequently unstable
`in the hydrophobic pocket occupied by Gin 121; (ii) its side chain
`may have a different orientation from that of Gln, fornling, for
`example, a salt bridge with Asp 119; and (iii) substitution ofHis for
`Gln at position 121 may induce a local change of conformation in
`the polypeptide backbone making the antigenic determinant unrec(cid:173)
`ognizable by the antibody. Not enough information is available to
`decide on the relative importance of these factors. Nevertheless, the
`
`Table 2. Lysozyme residues in contact with antibody.
`
`Lysozyme
`residues
`
`Antibody residues
`in contact (No.)
`
`Lysozyme
`residues
`
`Antibody residues
`in contact (No.)
`
`Asp 18
`Asn 19
`Arg 21
`Gly22
`Tyr 23
`Scr 24
`Leu 25
`Asn 27
`
`1 L chain
`2H, L
`1H
`4 H(3), L
`2H
`1 H
`1 L
`1H
`
`Lys 116
`Gly 117
`Thr ll8
`Asp 119
`Val120
`Gin 121
`Ile 124
`Leu 129
`
`3H
`6H
`2H
`2H
`1H
`5 H (1), L(4)
`2L
`1L
`
`Table 3. Hydrogen bonded interactions between antibody and lysozyme.
`Sequence positions are numbered as in Kabat et a/. ( 1) except for V H CD R3
`where the numbers of Kabat eta/. (1) are given in parentheses.
`
`Antibody residue
`
`Lysozyme residue
`
`Light chain
`
`Heavy chain
`
`Ne2 His 30
`OTJ Tyr 50
`0
`Phe 91
`
`Oy1 Thr30
`N
`Gly 31
`N
`Gly 53
`NTJ1 Arg 99 (96)
`0&1 Asp 100 (97)
`0&2 Asp 100 (97)
`0T] Tyr 101 (98)
`OTJ Tyr 101 (98)
`OT] Tyr 101 (98)
`
`• Denotes the closest interactions (distances s 2.5 A).
`
`Leu 129
`0
`0&1 Asp 18
`Ne2 Gln 121*
`
`Lys 116*
`0
`Lys 116
`0
`Gly 117*
`0
`Gly 22
`0
`N&2 Asn 27
`0-y Ser 24 *
`Val120
`N
`Gln 121
`N
`0&1 Asp 119
`
`SCIENCE, VOL. Z33
`
`BIOEPIS EX. 1059
`Page 7
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`

`

`Fig. 5. Stereo view of the environment of Gin 121
`with (above) atoms drawn with their van der Waals
`radii, showing the close packing of the three antibody
`aromatic rings around the antigen side chain. The
`dotted line indicates the hydrogen bond from Ne2 of
`Gin to the main chain carbonyl oxygen of V L Phe 9 L
`
`CDRIL
`
`fact that the His residue is not induced to fit into the interface
`position occupied by Gln 121 is in agreement with a "lock and key''
`model (see below) of complex formation between conformationally
`stable antigen and antibody structures.
`Japanese quail lysozyme has an Asn at position 121 and the
`additional differences Asn 19 ~ Lys and Arg 21 ~ Gln in the
`antigen-antibody interface. Asn 121 would be unable to form
`hydrogen bonds as strong as those of Gln 121. In addition,
`replacement of Asn 19 by Lys causes the loss of weak interactions
`between Ool of Asn 19 and N of VH Arg 102. The positively
`charged Lys 19 side chain would be repelled by VH Arg 102 and
`would probably remain outside the interface, further reducing
`packing efficiency. Only main chain atoms of Arg 21 make contact
`with Fab and the side chain is external; therefore changes at this
`position in the antigen surface are probably not detrimental to
`complex formation.
`The equilibrium affinity constant of Fab DL3 binding of hen
`lysozyme is 4.5 x 107M - 1
`• Other monoclonal antibodies to lyso(cid:173)
`zyme that we (25) and others (31) have obtained and characterized
`show similar affinity constants for the homologous lysozyme anti(cid:173)
`gen. Moreover, the determined equilibrium constants of protein
`antigens with their specific antibodies range from I05M - 1 to
`l0 10M - 1 (32). Thus, DL3 is a typical antibody of the monoclonal
`response in BALB/c mice and one of an about average affinity
`constant in immune responses to protein antigens in general.
`Comparison of evolutionarily related proteins has been used to
`identifY antigenic sites in proteins such as lysozyme (5, 25, 33). The
`detection of antigenic determinants by these fine specificity studies
`is biased toward the recognition of evolutionarily variable residues,
`such as Gln 121 by antibody DL3. As our r