Journal of
`OLECULAR
`BIOLOGY
`
`Editors in Chief
`J. C. KENDREW S. BRENNER
`
`olume 186
`
`Number 3
`
`5 Decem.ber 1985
`
`San Diego
`Orlando
`London
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`
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`
`JMOBAK 186 (3) 483- 677
`ISSN 0022- 2836
`
`PFIZER EX. 1063
`Page 1
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`

`

`Journal of Molecular Biology
`
`Editors-in-Chief
`J . C. Kendrew, 7 All Saints Passage. Cambridge CB2 3LS. England
`f:l . Brenner , M.R.C. La bora tory of Molecula r Biology . Uni versity P ostgrad uate Medi cal School
`Hills Road. Cambridge CB2 2QH , England
`
`nen es :
`
`}
`
`Gene stru cture
`Gene mod ifi cation
`Gene ex pression
`Gene regulation
`
`( 'ells:
`
`Ce ll development
`Cell fun ct ion
`
`Organellt> structures }
`Macromolec ula r
`assemblies
`
`Molen d e8:
`
`Macromolec ula r
`stru cture
`
`Physical chemistry
`
`L etters to the
`Editor:
`
`General
`Pre limina ry X-ray data
`
`Editors
`S. B ren ner (address a bove) .
`P. Chambon , Laboratoire de Genet iq ue Moleculaire des E ucaryotes du
`CN RS. Institu t de Chimie Biologique. F aculte de Medi cine, II Rue
`Humann . 67085 Strasbourg Cedex . F rance.
`J.I.J. Gottesman , Laboratory of Molecular Biology . National Cancer
`Instit ute , Nationa l Institu tes of Hea lt h, Bethesda, Mel 20205,
`U.S.A.
`I. Herskowitz, Depa rtment of Biochemistry a nd Biophysics, School of
`Medi cine. Uni versity of California . San Francisco, C'A 941 43. U.S.A.
`B. Mach , Depa rtement d~ Mi cro biologie , C.M.ll .. 9 a \'. de Champel, CH-
`1211 Geneve 4. Swi tzerland .
`A'. Matsubam , Instit ute for Molecula r a nd Cellular Biology. Osaka
`Cni versity , Ya mada -oka , Sui ta. Osaka 565, J apan .
`H. E . Hu xley . M. R.C' . Labora tory of Molecula r Biology , Un iversity
`Postgrad uate Med ical Schoo l. Hills Road , Ca mbridge CB2 2QH,
`England .
`A . Kl ug. M.R. C'. Laboratory of Molecula r Biology. University
`Postgradu ate Med ical Schoo l. Hills Road. Cambridge CB2 2QH.
`Engla nd .
`R. H1tber . Ma x-Pi anck-Tnstit ut fii r Biochemie. 8033 Mart insried bei
`Miin chen. Germ any .
`J . C. K endrew (address ~bove) .
`G . A . Gilbert , De pa rt ment of Biochemistrv. Universitv of Birmingham.
`P .O. Box 363 , Birmingham B l5 2TT~ England .
`·
`S . Brenner (address abo\'e) .
`R . Hu.ber (add ress abo,·e) .
`J . C. K endrew (address a bove) .
`
`{
`{
`{
`
`Associate Editors
`( '. R . Cant01· , Depart ment of Human Genetics and Development. College of P hysicians Hurgeons of Columbia
`Uni versity , 70 1 West 168 Street, Room 1602. New York , NY 10032, U.S .A.
`1'. Lu zzati , Cent re de Genetique Molecul aire. Cent re National de Ia Recherche Scient ifi que. 9 1 Gif-sur-Yvette . France.
`J . H. M iller. Depa rt ment of Biology, U niversity of California , 405 Hilgard Avenue. Los Angeles. CA 90024 . U.H.A .
`M . F. M oody , School of Pha rm acy , Uni versity of London. 29/39 Brunswi ck Sq uare. London WC I N I AX.
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`PFIZER EX. 1063
`Page 2
`
`

`

`J. Mol. Biol. (1985) 186, 651 - 663
`
`Domain Association in Immunoglobulin Molecules
`The Packing of Variable Domains
`
`Cyrus Chothia
`
`M RC Laboratory of Molecular Biology
`Hills Road, Cambridge CB2 2QH
`
`and
`Christopher Ingold Laboratories
`Department of Chemistry, University College London
`20 Gordon Street, London WC1H OAJ, England
`
`Jiri N ovotny, Robert Bruccoleri
`
`Molecular & Cellular Laboratory
`Massachusetts General Hospital
`
`and
`Harvard Medical School, Boston MA 02114, U.S.A .
`
`and
`Martin Karplus
`
`Department of Chemistry
`Harvard University, Cambridge MA 02138, U.S.A.
`
`(Received 17 July 1984, and in revised form 19 July 1985)
`
`We have analyzed the structure of the interface between VL and VH domains in three
`immunoglobulin fragments: Fab KOL, Fab NEW and Fab MCPC 603. About 1800 A 2 of
`protein surface is buried between the domains. Approximately three quarters of this
`interface is form ed by the packing of the VL and VH fj-sheets in the conserved
`" fram ework " and one quarter from contacts between the hypervariable regions. The
`fj-sheets that form the interface have edge strands that are strongly twisted (coiled) by
`P-bulges. As a result, the edge strands fold back over their own fj-sheet at two diagonally
`opposite corners. When the VL and VH domains pack together, residues from these edge
`strands form the central part of the interface and give what we call a three-layer packing;
`i.e. there is a third layer composed of side-chains inserted between tht: two backbone side(cid:173)
`chain layers that are usually in contact. This three-layer packing is different from
`previously described fj-sheet packings. The 12 residues that form the central part of the
`three observed VL-VH packings are absolutely or very strongly conserved in all
`immunoglobulin sequences. This strongly suggests that the structure described here is a
`genera l model for the association of VL and VH domains and that the three- layer packing
`plays a central role in forming the antibody combining site.
`
`1. Introduction
`
`Immunoglobulins are the best-studied examples
`of a large and ancient family of proteins, which also
`includes fj-microglobulins. Thy-1 antigens, major
`
`(i .e. class II) histo(cid:173)
`(i.e. class I) and minor
`compatibility antigens and cell surface receptors.
`Functionally, all these structures are involved in
`cell recognition processes (Jensenius & Williams,
`1982), either actively as vehicles endowed with
`
`0022- 2836/85/230651-13 03 .00/0
`
`651
`
`© 1985 Academic Press Inc. (London) Ltd.
`
`PFIZER EX. 1063
`Page 3
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`

`

`652
`
`C. Chothia, J. Novotny , R . Bruccoleri and M . Karplus
`
`recognition specificity (antigen-combing antibodies)
`or passively as surface structures that are being
`recognized (histocompatibility antigens). Only the
`immunoglobulin tertiary structures are known to
`date (Schiffer et al. , 1983; Epp et al. , 1974; Saul et
`al., 1978; Segal et al., 1974; Marquart et al., 1980;
`Deisenhofer, 1981; Phizackerley et al., 1979).
`However, the homology among primary structures
`of immunoglobulin, /1-microglobulin , Thy-1 antigen,
`some of the histocompatibility antigen domains,
`T-cell receptor p chain and the transepithelial
`"secretory component" has been interpreted as
`evidence for a common fold (Cunningham et al. ,
`1973; Orr et al. , 1979; Feinstein , 1979; Cohen et al. ,
`1980, 1981a; Novotny & Auffray, 1984; Yangai et
`al., 1984; Hedrick et al., 1984; Mostov et al., 1984).
`A typical antibody molecule (IgG 1) consists of
`two pairs of light chains (Mr 25,000) and two pairs
`of heavy chains (Mr 50,000), each of the chains
`being
`composed of domains made up of
`approximately 100 amino acid
`residues. The
`domains are autonomous folding units; it has been
`demonstrated experimentally
`(Hochman et al. ,
`1973; Goto & Hamaguchi, 1982) that a polypeptide
`chain segment corresponding to a single domain can
`be
`refolded
`independently of the rest of the
`polypeptide chain . All the immunoglobulin domains
`are formed by two /1-sheets packed face-to-face and
`.covalently connected together by a disulfide bridge.
`The topology of the N-terminal , variable domains
`in both the light and heavy chains differs from that
`of the C-proximal constant domains. While the two
`variable domain sheets consist of five and four
`strands, respectively, the constant domain sheets
`are three- and four-stranded (Fig. 1). The four(cid:173)
`stranded /1-sheets of the two domain types are
`homologous; the five- or four- stranded /1-sheet of
`the variable domains derives from the three-strand
`sheet of the constant domains by the addition , at
`one side, of a two-stranded /1-hairpin or a single
`/1-strand, respectively.
`In a complete immunoglobulin molecule, domains
`that correspond to different polypeptide chains
`associate to form domain dimers VL- VH , CL- CH1
`and CH3-CH3. Edmundson et al. (1975) were the
`first
`to note
`the phenomenon of
`rotational
`allomerism between
`the variable and constant
`domain dimers, that is, whereas the C-C dimers
`interact via a close packing of their four-strand
`sheets, the V- V dimers pack " inside out", with the
`five-stranded
`sheets oriented
`face-to-face. The
`reversal of domain- domain interaction is reflected
`in the amino acid sequence homology between , and
`among,
`the constant and variable domains
`(Novotny & Franek, 1975; Beale & Feinstein , 1976;
`Novotny et al. , 1977).
`Different antibody molecules
`same
`the
`in
`organism bind different antigenic structures. The
`variation in specificity
`is produced by several
`mechanisms: mutations, deletions and insertions in
`the binding regions of the VL and VH domains; and
`the association of different light and heavy chains.
`Aspects of the second mechanism are analyzed in
`
`this paper. In particular, the nature of the interfi
`between VL and VH domains is examined ~
`comparing the Fab fragments of KOL, NEW a~
`MCPC 603 myeloma proteins whose X-ra
`structures are known. The relative contributions~
`the buried surface between the dom ains from the
`conserved framewbrk
`residues and
`t he hyPe
`regions are determined. Attention ~­
`variable
`focused on the unique packing of th e interfaces an:
`the reasons for this packing are examined.
`
`2. Materials and Methods
`
`(a) Fab fmgrnent co-ordinates
`
`Cartesian co-ordinates for Fab fragm ents K OL , NEW
`and MCPC 603 were obtained from the Brookhaven Data
`Bank (Bernstein et al., 1977) . Table I lists t he domain
`classification , the nominal resolutions and t he crystallo(cid:173)
`graphic residuals (R factors) for the 3 F ab fragments. To
`facilitate comparisons of the 3 structures, t heir residue
`numbering was changed from that used in t he original
`descriptions to that used by Kabat et al. (1983). Thus, in
`this paper residues that are structurally homologous have
`the same sequence number.
`To obtain consistent sets of atomic co-ordinates, the
`original co-ordinates were dissected into individual VL-
`
`(a)
`
`I'
`(b)
`·
`.
`lobu tn
`.
`.
`Ftgure 1. The P-sheets 111
`typiCa l tmmunog ms:
`domains. Vertices represent the position of Ox a~~ose
`linked by ribbor~s; a nd L' the
`those
`in P-sheets are
`between strands by lines. (a) The VL domam of KO ·the
`P-sheet involved in VL-VH contacts is clos~r tot ted
`
`viewer (unbroken line). (b) The same VL domam t ;in!!
`
`by approximately 90°. Note tha t the interface- or nel'll
`P-sheet is strongly twisted at diagonally opposrte cor
`(drawing by A. M. Lesk) .
`
`PFIZER EX. 1063
`Page 4
`
`

`

`Packing of Immunoglobulin Variable Domains
`
`653
`
`Table 1
`Summary of X -ray crystallographic data
`
`Land H
`chain
`types
`
`.!.I , yiii
`
`.!.I, yii
`
`K , yi
`
`Protein
`
`Fab KOL
`human
`Fab NEW
`human
`Fab MCPC 603
`mouse
`
`X-ray data
`
`Minimized
`
`Resolution R factor Energy
`(A)
`(kJ)
`(% )
`
`r.m.s. shift
`(A)
`
`Reference
`
`1·9
`
`2·0
`
`2·7
`
`26
`
`19
`
`24
`
`-3010
`
`Marquart et al. (1980)
`
`-2592
`
`0·21
`
`Saul et al. (1978)
`
`-3703
`
`0·26
`
`Segal et al. (1974)
`
`The energy given for Fab KOL is that of the unminimized crystallographic data.
`
`· dimers. The structures were subjected to 100
`the
`of constrained energy minimization with
`CHARMM version 16 using the adopted-basis
`son procedure (Brooks et al., 1983) with
`of41·8kJ (lOkcal) present on all t he atoms
`& Karplus, unpublished results). Typically,
`·ned minimization converged from original
`values of potential energy to values of about
`( -0·50 kcalfatom) with an average root(cid:173)
`co-ordinate different from the original X-ray
`of 0·3 A (see Table 1 ). The results indicate that
`llfDwo• •v~•"' l'-'"' c structures were satisfactory and that
`of potential energy can be achieved by
`ustments of the co-ordinates. Thus, both energy
`structures and the crystallographic co-ordinates
`used in the present study; essentially
`identical
`were obtained from the 2 types of co-ordinates
`
`(b) Computation of solvent-accessible sU?jaces
`and contact areas
`surfaces (Lee & Richards, 1971) were
`with programs written by A. M. Lesk using the
`of Shrake & Rupley (1973) and by T . Richmond
`the methods of Lee & Richards (1971) and
`& Richards (1978). The latter program was
`from Yale University . The water probe radius
`1·4 A and the section interval along the Z axis
`A; the atom van der Waals' radii used were 2 A
`t he (extended) tetrahedral carbon atoms, 1·85 A
`the planar (sp2 hybridized) carbons, 1·4 A and
`for carbonyl and hydroxyl oxygens, respectively ,
`for a carbony l OH group, 2·0 A for all
`t he
`tetrahedral nitrogen atoms, 1·5 A, 1·7 A and
`for sp2-hybridized nitrogen atoms carrying no
`1 and 2 hydrogen atoms, respectively , 2·0 A for
`group and 1·85 A for a divalent sulfur atom
`hydrogens.
`
`(c) {3-Strands and {3-sheets
`structures were analyzed using the CHARMM
`(Brooks et al. , 1983) in the so-called explicit
`atom representation: aliphatic hydrogens were
`together with their heavy atoms into"extended
`whereas hydrogens bound to polar atoms and
`involved
`in hydrogen bonds were explicitly
`. The {3-strands and {3-sheets were defined by
`Inter-strand backbone (C = 0 ... H -N) hydrogen-
`
`bonding pattern. A hydrogen bond list was generated in
`CHARMM for all the polypeptide chain segments under
`consideration and amino acids with hydrogen bonds of
`nearly optimal geometry (energy of -4·18 kJfbond or
`less) were taken to be parts of the {3-sheets (cf. Fig. 3 of
`Novotny et al. , 1983). This method of defining {3-strand
`boundaries gives results essentially identical to those
`obtained by visual inspection of crystallographic models,
`although it tends to be somewhat more restri ctive (the 2
`methods sometimes differ in
`in clusion of the N- or
`C-terminal {3-strand residues). Ambiguities arise in cases
`of edge {3-strands that start and end with irregular
`conformations ({3-bulges); such cases are discussed in
`more detail below.
`
`(d) {3-Strand conf ormation
`
`In a typical extended polypeptide chain segment, the
`dihedral angle between the 2 consecutive side-chains is
`not 180° as in the ideal {3-sheet (Pa uling et al., 1951) but
`closer to -160°;
`that is, the {3-strands are twisted
`The
`out-of-planarity
`angle
`(Choth ia ,
`1973).
`(180° -160°) = 20° can be obtained explicitly from the
`values of the principal backbone torsion angles cp, 1/1 and
`w (see, e.g. Chou et al. , 1982). We define the local
`backbone twist for 2 consecutive residues as:
`s = ( -~) (180-l-rl) ,
`where T is the torsion angle C{J-CIX- C' IX- C' f3 and 1-rl denotes
`its magnitude. When glycine residues that lack C{J atoms
`are encountered, the torsion angle S is measured with
`respect to the C' f3 atom following the glycine. Thus,
`glycine residues contribute to t he local backbone twist
`indirectly, by being in cluded in t he virtual bond CIX-CIX
`that spans from the residue preceding the glycine to that
`which follows it .
`Backbone twist profiles (plots of S as a function of the
`amino acid residue) serve to characterize polypeptide
`chain conformations. Certain conformational character(cid:173)
`istics of polypeptides are more clearly seen using S values
`instead of the cpljl values for individual residues. In our
`plots, the value of the torsion angle C1X-C{3-C'1X- C' f3 is
`assigned to t he second (C') residue. The angle S is related
`to " the amount of twist per 2 residues " , defined as b by
`Chou et al. ( 1982); in fact, S = tb. It thus follows t hat S
`can be obtained from the helical parameters n (number of
`residues per turn) , h (the rise per residue) and T
`(T = 360°/n) in a correspond ing way to t hat described for
`b by Cho u et al. (1982).
`
`PFIZER EX. 1063
`Page 5
`
`

`

`654
`
`C. Chothia, J. Novotny, R . Bruccoleri and M. Karplus
`
`3. Results
`
`(a) Domain-domain contact surfaces
`We identified the residues t hat form t he interface
`between VL and VH by calculation of t he solvent(cid:173)
`accessible surface of t he domains, first in isolation
`and second when associated . Any residue that lost
`surface on t he association of VL a nd VH was taken
`as part of t he interface between t hem. We also
`determined which residues form van der Waals'
`contacts across the interface (distance cutoff 4·1 A).
`The lists of residues obtained by the t wo methods
`were very similar. Thus, except for a few margina l
`cases, t he residues that lose surface in domain(cid:173)
`domain contacts also have van der Waals' inter(cid:173)
`actions between the domains, indicating that the
`VL-VH interface is tightly packed.
`The total surface areas of the separated VL and
`VH domains and t hat buried on t he association is
`shown in Table 2. The values for the buried surface
`area (between 1700 and 1900 A 2
`) and the fraction of
`the buried surface that is composed of polar ato ms
`are similar to those found in other cases (Chot hia &
`J anin , 1975). For the bovine pancreatic trypsin
`inhibitor and t rypsin it is known that t he structure
`of
`t he
`isolated
`proteins does
`not
`cha nge
`significantly on association . In most cases, as for
`the VL and VH domains considered here, there a re
`no
`data
`concerning
`the
`structure of
`the
`unassociated domains.
`Of t he total area buried between t he VL-VH
`dimers about one quarter comes from residues in
`t he hypervariable regions and about three quarters
`/3-sheets. Figure 2 shows
`the
`from
`residues
`in
`residues that form the interfaces and t he areas t hat
`are buried for the three VH- VL packings. Two
`important features are evident in t his Figure. First,
`homologous residues form the interface in the three
`structures. Second, t he pattern formed by
`t he
`contact residues is most unusual. The contacts of
`residues on the edge strands of the /3-sheets are
`more extensive than t hose of residues on the inner
`strands. This is the opposite of the behavior found
`in previously described ]1-sheet packings, where it
`is the central strands that have the largest contact.
`
`For example, for packing of /3-sheets in t he
`domain , the region of maximal contact
`runs diagonally across the sheets at 45°
`to the /3-strands (Cohen et al. , 1981 b;
`J anin , 1981). The point is clearly illustrated in
`Ca backbone plot; in Figure 2(c) ; here, for each
`t heCa ato ms a circle is displayed , the area of
`is proportional to t he total contact area made
`t he residue with the other sheet. As we
`below, the unusual packing is a direct
`of the distortions present in t his type of /3-sheet.
`
`(b) Conf ormation of interface /3-sheets
`The deviation of
`t he conformations of
`/3-sheets that form the interface between VL
`VH from t he idealized flat structure (i.e.
`coiling and bending) can be characterized by
`variations in the twist angle 9 (see Materials
`Methods). On such twist profiles, regular
`/3-sheets correspond to horizontal lines with
`average 9 = + 20°, right-handed a helices to lines
`9 = -110° and tight reverse turns as triplets ci
`points of approximately t he same magnitude IIlii
`a lternating sign . The insertion of an additional
`residue in an edge strand of a /3-sheet, so that two
`edge residues face one another on an inner strand,
`forms what has been called a /3-bulge (Richardson d
`al. , 1978). Such insertions can have a variety of
`conformational effects depending upon the exact
`cptjl values of the inserted residue and those of i
`neighbors. Usually a sharp bend or local coiling it
`produced in the edge strand ; this gives rise to I
`single- or double- point peak or trough in the 8
`values.
`In Figure 3 we show the 9 values for t he VL-VB
`interface segments (/3-strands with the adjacent
`hypervariable loops) in KOL, NEW and MCPC 603.
`/3-sheets are
`important features of these
`Two
`the Figure. First, most of the
`evident
`from
`individual values of 9, and the patterns formed by
`t he variations in 9 angles, are very similar in the
`different sheets, particularly in the inner /3-strands
`(/31 , /33, /35 and /38 of Fig. 3) and in the /3-bulges; the
`edge /3-strands (/32, /34, /36 and f39 of F ig. 3) have
`
`Table 2
`Accessible surfaces and those lost on V L--V H association ( A2
`
`)
`
`Isolated surface
`
`Contact surface
`
`Domain pair
`
`Hydrophobic Polar Total
`
`Hydrophobic Polar Total
`
`KOL VL domain
`KOL VH domain
`VL-VH in KOL
`NEW VL domain
`NEW VH domain
`VL-VH in NEW
`MCPC 603 VL domain
`MCPC 603 VH domain
`VL-VH in MCPC 603
`VL-VH average
`
`1121
`1216
`2337
`1233
`1186
`2419
`1082
`1156
`2238
`233 1
`
`658
`700
`1358
`744
`80 1
`1545
`689
`760
`1449
`171 4
`
`1779
`1926
`3705
`1977
`1985
`3962
`1771
`1916
`3687
`3785
`
`580
`615
`1195
`529
`506
`1035
`676
`619
`1295
`1195
`
`311
`250
`561
`387
`386
`773
`299
`324
`623
`652
`
`891
`865
`1756
`91 6
`892
`1808
`975
`943
`1918
`1827
`
`PFIZER EX. 1063
`Page 6
`
`

`

`Packing of I mmunoglobulin Variable Domains
`
`655
`
`31
`
`I
`I TIS
`I FSS
`
`32 H24
`
`I PSO
`100111
`I F62
`I 018
`
`027
`101DS2
`
`I
`96
`I
`I
`DO
`95N31 ____ 33
`I
`I N20
`
`95 L76 ::::92
`
`I s23
`I ::~
`I LS4
`
`96 Rl43
`
`I WI06
`I 064
`
`91 Y39
`
`34
`102 _______ 94
`I Yl2
`I W7S
`I AO
`103W71
`93Ao:::: 35TO
`I W73
`I AO
`I E9
`I
`I
`I
`was
`j~~~r===y~~ T::~
`
`10~4
`02s
`I
`106:
`
`F32
`
`V6
`
`104 ______ _ 92
`
`36
`
`"4s t11~s~
`I G30
`44GI3
`R96
`
`107:::_-_-_:90
`
`38:::::::46
`
`I
`1018
`
`1 ________ 1 048
`819 ________ 319g~~
`
`40 ______ "43 /
`I
`I
`
`109::::::::-88
`I
`I
`
`VH
`
`(b)
`
`I L4S
`
`4
`
`t;b
`
`16
`
`44 =~
`I ::;
`
`43A63
`P61
`
`-------
`37 - _____ _45~
`
`8
`
`:::::::3
`
`15
`
`18
`
`/
`39 _______ "42
`I
`I
`
`1
`104 _______ 84
`I
`I
`
`VL
`
`(a)
`
`33
`97 ::::::: 90
`I AO
`I N24
`I F96
`98 F9S
`89 06 :::: 34 K30
`I 09
`I A9
`I FIIO
`....-:::::99--- -----88
`I Y48
`
`35
`I Y41
`
`100~(4
`
`A32
`
`11
`-------
`..........
`102 ___ __ -- 86
`
`sr~~==::316~!~
`I
`I
` gii
`
`I
`
`10
`
`3
`
`( c )
`2. P-Sheet residues that form the VL-VH in terface in the Fabs KOL, NEW and MCPC 603. Residue numbers
`of Kabat et al. (1983). (a) VL interface-forming P-sheet; (b) VH interface-forming P-sheet. Broken lines
`hydrogen bonds. At each position where a residue forms part of the interface, we give the residue identity in
`NEW and MCPC 603, and the accessible surface of the residue that is buried in the VL-VH interface. Note the
`in the edge strands at positions 43, 44 and 100, 101 in VL and 44, 45 and 105, 106 in the VH. (c) The P-sheet
`VH domain. Residues making contacts to the VL domain across the domain-domain interface are circled .
`----·u-,,. .. ,,,·n atoms are displayed. The circles associated with each Cct atom have an area proportional to the
`surface area lost when the VL-VH dimer forms . Note the large areas associated with residues in the edge
`of the P-sheet.
`
`PFIZER EX. 1063
`Page 7
`
`

`

`656
`
`C. Chothia, J. Novotny, R. Bruccoleri and M. Karplus
`
`CZ>
`
`.... .,
`·~ ....
`
`Ql
`1=1
`0 ..a
`..!If
`()
`aS
`Ill
`
`150
`
`100
`
`50
`
`0
`
`-50
`
`-100
`
`-150
`
`20
`
`150
`
`100
`
`50
`
`0
`
`-50
`
`- 100
`
`CZ>
`
`.... .,
`·~ ....
`
`Ql
`1=1
`0 ..a
`..!If
`()
`aS
`Ill
`
`Sequence nuxnber
`
`(a}
`
`85
`
`90
`
`95
`
`100
`
`105
`
`Sequence nuxnber
`
`( b}
`Figure 3. The backbone twist (3) profiles of VL--VH interface-forming segments. The segments shown include:;
`hypervariable loops (Ll , L2, L3 , Hl , H2 and H3) and the {J-strands. The {J-strands are indicated by bars at the bot ce
`of the plots and labeled {J I through {J9 according to Novotny et al. (1983) . {J-B ulges are denoted by open boxes. Seq~~: 2
`numbers correspond to the Kabat et al. (1983) numbering system and are the same as in Fig. 2. (a) and (b)
`ill·
`interface-form ing segments of the VL domain ; (c) and (d) the 2 interface-forming seg ments of the VH doma
`(0) KOL; (0) NEW; (L",.} MCPC 603.
`
`PFIZER EX. 1063
`Page 8
`
`

`

`Packing of Immunoglobulin Variable Domains
`
`657
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`Sequence number
`(c)
`
`90
`
`95
`
`100
`
`105
`
`110
`
`115
`
`120
`
`Sequence number
`
`(d)
`
`Fig. 3.
`
`/3-bulge
`of
`differences. Conservation
`·••vnn.•u. .. rm " is especially striking and implies th at
`are important architecturally , as previously
`(1981).
`The
`Richardson
`by
`pondence in t he /3-sheets is made even more
`
`t he
`in behavior of
`t he difference
`evident by
`hyperva ri a ble
`loops. The overall
`similarity of
`/3-sheet geo metries is confirmed by a least-squ ares
`fi t of t heir ato mic co-ordinates. Fits of t he main(cid:173)
`chain ato ms of t he t hree VL /3-sheets to each other
`
`PFIZER EX. 1063
`Page 9
`
`

`

`658
`
`C. Chothia, J. Novotny , R . Bruccoleri and M. Karplu
`
`Pro
`100
`
`84 39
`
`VL
`
`109
`
`88
`
`40
`
`43
`
`VH
`
`VL-VH
`
`F igure 4. The key residues in the edge strands involved in VL-VH packings (Fab KOL). Note how in (a) Pro«,
`Ty r96 and Phe98 in VL and in (b) Leu45 , ProlOO and Trpl03 in VH fold over the central strands of their /3-sheet and
`so in (c) form the core of the VL-VH packing (see also the position of these residues in Fig. 5).
`
`(30 residues) , and of the three VH /3-sheets to each
`other
`(32
`residues)
`give
`root-mean-square
`differences in atomic positions of between 0·73 and
`1·23 A (see Table 3) . If a few peripheral residues are
`removed from the fits the r.m.s.t differences are
`redu ced to 0·55 to 0·87 A. Table 3 also reports the
`results of least-squares fits of the VL /3-sheets to the
`VH /3-sheets. The r.m .s. differences are only a little
`greater than for the fits of the VL or VH /3-sheets to
`each other, 0·70 to 1·11 A. Thus, the six regions of
`/3-sheet that form the VL- VH interface in KOL,
`NEW and MCPC 603 have very similar structures.
`In fact, the least-squares superposition of the two
`sheets can be achieved as a 2-fold symmetry
`operation, i.e. rotation around an axis passing
`through the centroid of the interface.
`The second feature of the /3-sheets illustrated in
`Figure 3 is the different amounts of twist found in
`the edge and inner strands. The two central strands
`in both VL and VH have ~ values in the range that
`indicate a degree of twist commonly found
`in
`/3-sheets. The average ~ value tends to be the same
`for both the inner and the edge strands, but the
`twist of the edge strands is dominated by /3-bulges
`(Figs 2 and 3) with characteristic ~ values ± 70. Its
`effect is to fold the ends of the edge strands over
`central strands. This occurs at two diagonally
`opposite corners of the /3-sheets. Side-chains of
`residues 44 (Pro) , 96 (Tyr, Arg, Leu) and 98 (Pro) in
`VL and 45 (Leu) , 100 (Pro, Ile, Phe) and 103 (Trp)
`cover residues in the inner strands (Fig. 4(a) and
`(b)). The other parts of the edge strand residues,
`45-46 and 101- 104 in VL, 46--48 and 106--109 in
`VH, lie next to the inner strand in the normal
`manner.
`
`t Abbreviation used: r.m .s., root-mean -sq uare.
`
`(c) Packing of the /3-sheets at the V ~ V H interface
`As noted above, the strong twists that occur in
`the edge strands of VL and VH means t hat residues
`at two diagonally opposite corners fold over the
`/3-sheets: 44, 96 and 98 in VL (Fig. 4(a)), and 45,
`100 and 103 in VH (Fig. 4(b)) . Figure 4(c) show
`that when the VL and VH domains pack together
`
`Table 3
`The fit of /3-sheets forming V ~ V H interfaces
`A. Fits of individual {J-sheets
`
`VLt
`
`VHt
`
`KOL NEW MCPC
`
`KOL NEW MCPC
`
`0·76
`
`0·55
`0·82
`
`0·88
`0·96
`0·70
`
`l · ll
`1·05
`1·00
`0·87
`
`0·94
`0·97
`0·84
`0·65
`0·87
`
`VLt KOL
`NEW
`MCPC
`VHtKOL
`NEW
`MCPC
`
`B. Fits of both {J-sheet regions of the V L-V fl interfaces§
`
`KOL NEW MCPC
`
`0·87
`
`0·70
`0·87
`
`KOL
`NEW
`MCPC
`The Table give r.m.s. differences in position of the mai~ cht::
`atoms
`following
`least-sq uares
`fits of
`their co-ordma
`·
`ces
`Differences are given in A.
`t VL residues u ed to determine fits and r .m.s. differen
`ces
`33- 39, 43- 47 , 84- 90 and 98- 104.
`t VH residues used to determine fits and r.m.s. differen
`f VL
`33- 40, 44- 48, 88- 94 and 102- 109.
`§Residues used in fits 33- 39, 43-47 , 84-90 and 98--104 °
`and 34-40, 44-48, 88-94 and 103- 109 of VH.
`
`PFIZER EX. 1063
`Page 10
`
`

`

`Packing of Immunoglobulin Variable Domains
`
`659
`
`VL
`
`VH
`
`(b)
`
`(a)
`
`VH
`
`VL
`
`(c)
`
`(d)
`
`5. Residue packing at the KOL VL-VH interface .. This Figure shows superimposed serial sections cut through
`ling model of the interface. VH residues are shown by broken lines and VL residues by continuous lines. The
`ax is that relates VL to VH is perpendicular to the page. Each part of the Figure shows 4 sections,
`by I A, superimposed . (a) ections 0 to 3 A; (b) sections 4 to 7 A; (c) sections 8 to ll A; and (d) Sections 12 to
`
`PFIZER EX. 1063
`Page 11
`
`

`

`660
`
`C. Chothia, J. Novotny , R. Bruccoleri and M. Karplus
`
`Table 4
`V L-V H interfaces
`Residues buried in
`
`Residue at this position
`in
`
`Accessible surface
`area of residue (A 2
`)
`
`Domain Residue
`No . KOL NEW MCPC
`
`KOL NEW MCPC
`
`No. of sequences known that
`include this positiont
`
`Principal residues found
`at this posit iont
`(identity and num ber of cases)
`
`VL
`
`VH
`
`34
`36
`38
`44
`46
`87
`89
`91
`96
`98
`35
`37
`39
`45
`47
`91
`93
`95
`100
`103
`
`Asn
`Tyr
`Gin
`Pro
`Leu
`Tyr
`Ala
`Trp
`Ty r
`Phe
`Tyr
`Val
`Gin
`Leu
`Trp
`Phe
`Ala
`Asp
`Pro
`Trp
`
`Lys
`Tyr
`Gin
`Pro
`Leu
`Tyr
`Gin
`Tyr
`Arg
`Phe
`Thr
`Val
`Gin
`Leu
`Trp
`Tyr
`Ala
`Asn
`Ile
`Trp
`
`Ala
`Tyr
`Gin
`Pro
`Leu
`Tyr
`Gin
`Asp
`Leu
`Phe
`Gin
`Val
`Gin
`Leu
`Trp
`Tyr
`Ala
`Asn
`Phe
`Trp
`
`2
`0
`2
`8
`17
`9
`0
`3
`6
`10
`0
`0
`8
`10
`II
`0
`0
`0
`0
`27
`
`39
`0
`7
`5
`35
`I
`I
`12
`5
`9
`2
`3
`20
`6
`6
`8
`0
`0
`32
`28
`
`0
`I
`17
`5
`8
`II
`0
`0
`3
`2
`0
`I
`21
`3
`4
`II
`I
`5
`0
`26
`
`t Data taken from Kabat et al. ( 1983).
`
`362
`318
`302
`238
`235
`227
`217
`211
`199
`206
`217
`200
`183
`163
`157
`159
`161
`131
`113
`125
`
`Ala117 , Asn92, His51 , Ser37
`Tyr243, Phe40, Val28
`Gln279
`Prol90, Phe29, Val14
`Leul57 , Gly32 , Prol9 , Vall3
`Tyrl60, Phe65
`Glnl28, Ala35
`Trp59, Tyr31 , Ser27
`Trp46 , Tyr31 , 126, R20
`Phe203
`Gln53 , Asn42, Ser34, Lys22
`Val178, Ile 19
`Gln 176
`Leul60
`Trpl51
`Tyrl28 , Phe30
`Ala146
`Asp53 , Gly 18
`Phe76, Met I I , Leu6
`Trp 11 8
`
`these six residues form the center of the interface.
`They are in contact with each other in pairs and
`make a herringbone pattern.
`Details of how residues pack at the VL- VH
`interface can be seen in sections cut through space(cid:173)
`filling models. Figure 5 shows sections of the KOL
`VL-VH interface. The central role played by the
`three pairs of edge residues, Tyr96 and Trpl03 , and
`Leu45 and Pro44 are seen in parts (b) , (c) and (d) of
`the Figure. The inner strands of the P-sheets, 32- 39
`and 84- 92 in VL and 33-40 and 88- 95 in VH , only
`make interdomain contacts at one end of the
`interface where the side-chains of Gln38 and Gln39
`hydrogen bond
`to each other (Fig. 5(a)) . The
`structures of the VL-VH interfaces in NEW and
`MCPC 603 are very similar to that of KOL
`illustrated here. This is demonstrated by graphical
`inspection of their packing and by the fits of the co(cid:173)
`ordinates of the main-chain atoms forming the VL(cid:173)
`VH interfaces described above (Table 3).
`The packing of t