Reprinted from J . . If.JI. Biol. ( 1985) 186. 65 1- 663
`
`3
`
`Domain Association in Immunoglobulin Molecules
`The Packing of Variable Domains
`
`Cyrus Chothia, Jiri Novotny, Robert Bruccoleri
`and Martin Karplus
`
`1 of 14
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`

`

`J . ;)Jul. Biol. (1985) 186, 65 1- 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 I ngold Laboratories
`Department of Chemistry, University College London
`20 Gordon Street, London WOl H OAJ, England
`
`Jiri Novotny, Robert Bruccoleri
`
`Molecular &: Cellular Laboratory
`Massachusetts General Hospital
`and
`
`Harvard Medical School, Boston MA 021J.J. U.S.A.
`
`and
`Martin Karplus
`
`Department of Chemistry
`Haruard University , Cambridge MA 02138, U.S.A.
`
`(Received 17 July 1984, and in revised form 19July1985)
`
`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 1800A 2 of
`protein surface is buried between the domains. Approximately three quarters of this
`interfat·t> is formed by the packing of the \' L and \'H /3-sheets in the consen·ed
`' 'framework ' ' and one quarter from contac·t.s between the hypel'\·ariable regions. 'fhe
`/3-sheets that form the int<>rface han• edge strands that are strongly t.\\'isted (coiled) by
`/J-bulges. As a result, the edge strands fo ld back over t heir own /3-sheet at two diagonally
`opposite corners. When the VL and VH domains pack together. residues from t.hese edgr
`strands form the central part of the interface and give what we call a three-layer packing;
`i.e. t here is a third layer composed of side-chains inserted between th!'. two backbone side(cid:173)
`chain layt>r:s that. are usually in contact. This three-layer packing is different fro m
`previously described /3-sheet packings. The 12 residues that forJ'!l the central part of the
`three observed VI.r-VH packings are absolutely or very strongly conserved in all
`immunoglooulin sequences. This strongly suggests that the structure described here is a
`general model for t he association of VL and \'H domains and that the th ree - la~·pr packing
`plays a central role in forming t.he antibody combining ,-ite.
`
`1. Introduction
`
`Immunoglobulins art' the best-studied examples
`of a large and ancient fam ily of proteins, which also
`includes /3-microglobulins. Thy- 1 antigens, major
`
`(i.e. class T) and minor (i.e. class 11 ) histo(cid:173)
`compatibilit.y antigens and cell surface receptors.
`Fun<'tionall\' . all these struC'tures a re involved in
`cell recognfrion processes (Jensenius & Williams,
`1982). either actively as vehicles endowed with
`
`IHl:!:?- :?836/85/23065 1- 13 S03.00/0
`
`651
`
`© 1985 ..\c.,tdemie Press Inc. (London) Ltd.
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`2 of 14
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`

`652
`
`('. Ghothia. J. No-votny, R. Bruccoleri nnd M. K arplus
`
`recognition specificity (antigen-combing antibodies)
`or passively as surface structures that a re being
`recognized (histocompatibility antigens). Only t he
`immunoglobulin tertiary structures are known to
`date (Schiffer el al., 1983; Epp el al., l 974: Saul et
`al., 1978; Segal et al., 1974; Marquart et al., 1980;
`Deisenhofer, 1981: Phizackerley et al., 1979).
`However, t he homology among primary structures
`of irnmunoglobulin, P-microglobulin , Thy- ! antigen,
`some of t he histocompatibility antigen domains,
`T-cell receptor P chain a nd
`the transepithelial
`·'secretory component" has been interpreted as
`evidence for a common fold (Cunningham el al ..
`1973; Orr Pl al., 1979: Feinstein, 1979; Cohen el al.,
`1980, 198l a; Novotny & Auffray, 1984: Yangai et
`al .. 1984; Hedrick et al., 1984; Mostov et al., 1984).
`r\ typical antibody molecule (IgG I) consists of
`two pairs of light chains (M, 25,000) and two pairs
`of heavy chains ( ii/, 50,000), each of t he chains
`composed of domains made up of
`being
`approximately 100 amino acid
`residues. The
`domains are autonomous folding units; it has been
`demonstrated experimentally (Hochman et al.,
`1973: Goto & H amaguchi , 1982) that a polypeptide
`chain segment corresponding to a single domain can
`be refolded
`independently of t he rest of t he
`polypeptide chain . All the immunoglobulin domains
`a re formed by two P-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 P-sheets of the two domain types a1·e
`homologous: t he five- or four- stranded P-sheet of
`t he variable domains derives from the three-strand
`sheet of the constant domains by the addition, at
`one side, of a two-stranded P-hairpin or a single
`P-strand, respectively.
`In a complete im~unoglobulin molecule, domains
`that correspond to different polypeptide chains
`associate to form domain dimers V L-VH , CL-CH I
`and CH3-CH3. Edmundson et al. (1975) were the
`first
`to note
`the phenomenon of rotational
`allomerism between
`the variable and constant
`domain dimers, t hat is. whereas t.he e-c dimers
`interact ria a close packing of their four-strand
`sheets. the V-\' dimers pack "inside out" , with t he
`five-st.randed sheets oriented
`face-to-face. The
`reversal of domain-domain interaction is reflected
`in the amino acid sequence homology between. and
`the constant and variable domains
`among.
`(Novotny & Franek, 1975; BeaJe & Feinstein, 1976:
`'.'Jovotny Pf al., 1977).
`t he same
`in
`Different antibody molecules
`organism bind different antigenic structures. The
`variation in
`,;pec·ific·it y
`is produced by
`,;e,·eral
`mechanisms: mutations, deletions and insertions in
`the binding regions of the VL and VH domains; a nd
`the association of different light and heavy chains.
`Aspeds of the second mechan ism a re analyzed in
`
`this paper. In particular, the nature of the interface
`between V L and VH domains is examined by
`C'omparing t ht· Fab fragments of KOL, NEW and
`l\lt!P(' 603 myeloma proteins whose X-ray
`structures a re known. The relative contributions to
`t he buried surface between t he domains from the
`conserved fra me.work
`residues and
`the hyper(cid:173)
`variable
`regions are determined. Attention
`is
`focused on the unique packing of t he interfa<·es and
`the reasons for this packing are examined .
`
`2. Materials and Methods
`
`{a) Fab fragment co·ordinal""
`Cartesian co-ordi nates for Fab fragments KOL. XEW
`and MCPC 603 were obtained from the Brookhaven Data
`Bank {Bernstein el al.. 1977). Table I lists the domain
`classification. the nominal resolutions and the crystallo(cid:173)
`graphic residuals (R factors) for the 3 Fab fragments. To
`fac ilitate comparisons of the 3 structures. their residue
`numbering was changed from that used in the original
`descriptions to that used by Kabat el rt!. (1983). Thus. in
`this paper residues that are structurally homologous have
`the same sequence number.
`To obtain consist.ent sets of atomic co-ordinates. the
`original co-ordinates were dissected into indi,·idual \'I.,
`
`(o)
`
`( b )
`Figure 1. The P·:<lwt>t><
`t.\'Pi<«l l immunoglobulin
`in
`domains. \ ' t>rti«t->< represent. the position of C'cx atoms:
`linked b~· ribbons: and those
`t hos,..
`in
`/J·:<ht·t>ts art-
`between strand:< hy lines. (a) The \ ' L domain of KOL: tilt'
`P-shet-t involved in
`\ 'L- \'H rn ntat·t:< is closer t o t he
`,.it'W!'r (unbroken line). (b) The :<<1 11 H· \'L domain rotated
`by <1p111:ox i111att-ly 90°. i\ntp th<1t the int.erfa<,e·forming
`P-slwt>t i s strongl.'· t\\'i:<tt'd at diagonallv opposite corners
`(drawing hy .-\ . ;\( . l,t-sk).
`·
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`Paclcing of 1 mmunoglobulin Variable Domains
`
`653
`
`Table I
`Summary of X-ray crystallographic data
`
`Land H
`chain
`t,vpes
`
`.I.I, )'lII
`
`J.I, yII
`
`" · yl
`
`X-ray data
`
`Minimized
`
`Resolution R factor Energy r.m.s. shift
`(A)
`(A)
`(kJ)
`(~o)
`
`Reference
`
`1·9
`
`:?·O
`
`2·7
`
`26
`
`19
`
`:?4
`
`-3010
`
`Marquart el al. (1980)
`
`- 2592
`
`0·21
`
`Saul et al. (1978)
`
`- 3703
`
`0·26
`
`Segal el al. (1974)
`
`Protein
`
`Fab KOL
`human
`Fab :\EW
`human
`Fab MCPC: 603
`mouse
`
`The energy given for Fab KOL is that of the unminimized crystallographic data.
`
`VH domain dimers. The structures were subjeC't.ed to 100
`the
`cycles of constrained energy minimiz.ation with
`program CH ARMM version 16 using the adopted-basis
`Newton-Raphson procedure (Brooks et al .. 1983) with
`constraints of 41 ·8k.J (lOkcal) present on all the atoms
`(Bruccoleri & Karplus. unpublished results). Typically,
`the constrained minimization converged from original
`positive values of potential energy to values of about
`- 2· 1 kJ/atom (-0·50kcal/atom) with an average root(cid:173)
`mean-square co-ordinate different from t he original X-ray
`structure of 0·3 A (see Table 1). The results indicate that
`the C'rystallographic structures were satisfactory and that
`acceptable values of potential energy can be achieved by
`small adjustments of the co-ordinates. Thus, both energy
`minized structures and the crystallographic· co-ordinates
`were used in the present stud,1·: essentiall.1·
`identical
`results were obtained from the 2 types of co-ordinates
`sets.
`
`(b) Computation of solveul-al·ce.s.~ible surfaces
`and f011fart ar,,a.~
`Solvent-accessible surfaces (Lee & Richards. 1971) were
`computed with programs written by A. :\'f. Lesk using the
`method of Shrake & Rupley (1973) and by T . Richmond
`the methods of Lee & Richards ( 1971) and
`using
`Richmond & Richards (1978). The latter program was
`obtained from Yale Univers ity. The water probe radius
`used was l ·4 A a nd the section interval along the Z axis
`was 0·05 A; t he atom van der \\'aals' radii used were :! A
`for all the (Pxtended) tetrahedral carbon atoms, l ·85 A
`for all the planar (.~p2 hybridized) carbons. l ·4 A and
`I ·6 A for carbonyl and hydrox~·J oxygens. respectively .
`1 ·5 A for a carbonyl OH group. 2·0 A for all
`t he
`(extended) tetrahedral nitrogen atoms. l ·5 A. l ·7 A and
`l ·8 A for s-p2-hybridized nitrogen atoms carrying no
`hydrogen , I a nd 2 h~'drogen atoms. respecti,·el.'·· 2·0 A for
`a sulfhydr,vl group and l ·85 A for a divalent sulfur atom
`with no h~·drogens.
`
`(c) P-Strands a11d P-sheets
`Protein struC'tures were a nalyzed using the C'HARM'.\1
`program (Brooks et al .. 1983) in the so-called explicit
`hydrogen atom representation: aliphatic hydrogens were
`combined together with their heavy atoms into"extended
`atoms" whereas hydrogens bound to polar atoms. ~nd
`possibly
`involved in hydrogen bonds were expli(·1tly
`,,, .. re defined h_,.
`prest-nt. The P-strands and P·sht-et><
`their intn-strand ba<"kbone (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 kJ/ bond or
`less) were taken to be parts of t he P-sheets (cf. Fig. 3 of
`Novotny el al .. 1983). This method of defining P-strand
`boundaries gives results essentially identical to those
`obtained by visual inspection of crystallographic models,
`although it tends to be somewhat more restrictive (the 2
`methods sometimes differ in inclusion of t he JI\- or
`C-terminal P-strand residues). Ambiguities arise in cases
`of edge P-strands that start and end with irregular
`conformations (P-bulges); such cases are discussed in
`more detail below.
`
`(d) P-Strand conformation
`
`In a typical extended polypeptidt- chain segment. the
`dihedral angle between the 2 consecutive side-chains is
`not 180° as in the ideal P-sheet (Pauling et al., 1951) but
`closer to - 160°; that is, the P-st rands are twistt-d
`(C'hothia,
`1973).
`The
`out-of-planari t.y
`angle
`(180°-160°} == 20 can he obtained explicitly from t he
`values of thi- principal backbone torsion angles <p. i/I and
`w (see. e.g. Chou el al .. 1982). Wt- define the local
`backbone twist for 2 consecutive residues as:
`
`9 = (-2-) (180 - lrll.
`
`lrl
`where r is the torsion angle CP-<.'a- (''11.-C'P and lrl denotes
`its magnitude. When glycine residues that lack cp atoms
`are encountered, the torsion angle 9 is measured with
`rt-:>pect to t.he C'P atom following the gly«ine. Thus.
`glycine residues contribute to the local backbone twist
`ind irl·rtl~'. by being induded in the virtual bond Ca-Ca
`that spans from t he residue pre<'eding the gl.n·i1w to that
`which follows it.
`Backbone twist profiles (plots of 9 as a funt t ion of t he
`amino acid residue) ,;er\'e to C'haraC'tf·rize polypeptide
`chain conformations. Certain conformational character(cid:173)
`istics of polypeptides are more clearly seen using 9 n1lues
`instead of the <pijl ,·alues for indi,·idual residues. Jn our
`plots, the value of the torsion angle C'a-CP-C'rt.-C'P is
`assigned to the second (C') residue. The angle 9 is related
`to "the amount of twist per 2 residues", defined a:> f> by
`Chou et al. (1982): in fac-t. 9 = !b. I t thus follows that 9
`ran be obtained from th<> helical parameters 11 (number of
`residues per t.urn). h (the rist' per residut-) and T
`(T = 3606 / 11) in a c-orresponcling wa.v to that de><«ril, .. d for
`b b)· Chou Pf rd. ( 1982).
`
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`

`654
`
`C. f'hothia. J. Novotny, R. Bruccoleri and M. Karp/11.,
`
`3. R esults
`(a} Dom fl i 11-r/0111f/ i11 N111/rtrl .-111/flf<'·'
`\\'e identified the residues that form the interfat'e
`between \ .L and \ ' H b.'· calrulation of the soh·ent ·
`accessible surface of the domains, first in isolation
`and second when associated. Anv residue t.hat Inst
`surface on the association of \' L 'and \'JI was taken
`as part of the int.erface between them. Wt' also
`<let.ermined which residues form van der \\'aals '
`contacts across the interface (distance C'utoff ~·I ..\).
`The lists of residues obt.ained by the two methods
`were ,·ery similar. Thus, exc·ept for a few marginal
`cases. the residues that lose surface in domain(cid:173)
`domain contacts also have ,·an der \\'aals' inter(cid:173)
`actions between the domains, indicating that t hf'
`\"1.,-\'H interface is tightly packed.
`The total surface areas of the separated \' L and
`VH domains and that buried on the a><sMiation 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 atoms
`are similar to those found in other <.'ase:> (Chothia &
`Janin. 1975) . For the bovine pancreatic tr.'·psin
`inhibitor and trypsin it is known that t.he structure
`of
`the
`isolated proteins does not
`changP
`significantly on association. In most cases, as for
`t.he VL and \'H domains considered here. there are
`no data concerning
`t he
`structure of
`the
`unassociated domains.
`Of the total area buried between the \ ' L -Y H
`dimers about one quarter comes from residues in
`the h>·pervariable regions and about thrcT quarters
`from
`residues
`in P-sheets. Figure ~ shows
`t.he
`residues that form the interfaces and the areas that.
`are buried for the three \'H- \' L packings. Two
`important, features are e\·ident. in this Figun·. First.
`homologous residues form the interface in the three
`structure:;. Second, the pattern formed by the
`contact residues is most unusual. The contacts of
`residues on the edge strands of the P·sheets are
`more extensive than those of residues on the innt-r
`strands. This is the opposite of the bPhavior found
`in previously described ]3-sheet p1t<·kings, where it
`is the central strands that have tht- largest contact.
`
`For example. for pac·king of P-sheet,s i11 the same
`domain , the region of maxima.I cont.att generallv
`runs diagonally across l ht- sheets at 45° with respec·t
`to the P-strands (Cohen et al., 1981 b; Chothia &
`Janin, 1981). The point is clearly illustrated in the
`('a backbone plot in Figure 2(c): here, for each of
`tht- Ca atoms a C'ircle is displayed, the area of which
`is proportional to the total contact area made by
`the residue with the other sheet. As we descrih~
`below. the unusual packing is a direC't. consequence
`of the distortions present in this type of P-sheet.
`
`(b) Conformation of interface P-sheets
`The deviation of the conformations of the
`/J-sheets that for m the interface between \ 'L and
`\'H from the idealized flat structure (i .e. lwisting,
`coiling and bending) can be characterized by the
`variations in the twist angle 9 (see Materials and
`Methods). On such twist profiles, regular twisted
`P·sheets correspond to horizontal lines with an
`average 9 = + 20°. right-handed a helices to lines of
`9 = - 110° and tight reverse turns as triplets of
`points of approximately the same magnitude and
`alternating sign. The insertion of an additional
`residue in an edge strand of a /J-sheet, so that two
`edge residues face one another on an inner strand,
`forms what has been called a /J·bulge (Richardson el
`al., 1978). Such insertions can have a ,·ariety of
`tonformational effects depending upon the exaC'I
`<Pl/I ,·alues of the inserted residue and those of its
`neighbors. Usually a sharp bend or local coiling is
`produced in the edge strand: this gives rise to a
`single- or double-point peak or trough in the 9
`values.
`In Figure 3 we show the 9 values for the \'L-\'H
`interface segments (/J-strands with the adjacent
`hypervariable loops) in KOL, NE\\' and MCPC 603.
`Two
`important features of these
`/J-sheets are
`p,·ident from
`the Figure. First.. most of the
`individual values of 9, and the patterns formed b.'·
`the variations in 9 angles, are very similar in the
`different sheets, partit·ularly in the inner P·strands
`(/J 1, /13. P5 and P8 of Fig. 3) and in the P-bulges: the
`edge P-strands {P'2. P4. P6 and P9 of Fig. 3) have
`
`Table 2
`Accessible surfaces and those lost on 1·L-l'H association (.·F)
`
`isolated surface
`
`Contact surfa<'e
`
`Domain pair
`
`Hydrophobic Polar Total
`
`KOL \' L domain
`KOL \'H domain
`\'1 .-\'H in KOL
`~ E\\. \'I, domain
`~ P,\\' VH domain
`\'L \' H in NEW
`:\H'P(' 603 VL domain
`:\l<!P(' 603 \ ' H domain
`\'t,...\·H in MCPC 603
`\ ' J,_\·H ~,·erag~
`
`11:!1
`1:!16
`2337
`1233
`I 186
`:!419
`1082
`1156
`2238
`2331
`
`fi!'i~
`700
`1358
`7H
`801
`1545
`689
`760
`1449
`1714
`
`17i9
`1926
`3705
`19i7
`1985
`3962
`1771
`1916
`3687
`3785
`
`Hydrophobi(· Polar Total
`;;so
`615
`1195
`529
`506
`1035
`676
`619
`l:!fli'i
`1 HI.~
`
`891
`865
`1756
`916
`892
`1808
`975
`943
`1918
`18:.!i
`
`311
`250
`561
`:l~i
`386
`773
`299
`324
`Ii:.!:!
`652
`
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`

`Pac/.."ing of l mmunoglobulin Variable Domains
`
`655
`
`I
`I
`I
`I
`I N20
`I Yl2
`I AO
`I E9
`I AO
`I
`I
`I
`47::~
`91 ;~L::37~~
`Ja~~::::l wes
`I06"'--107 ::::::.9bv33
`I
`-.........4 5 t:2!
`I l. 88
`I 0 32
`44 ~~0
`R96
`/
`···- ·- ·
`4'3
`40
`I
`I
`
`J s ~~L::912
`J1
`l TIS
`I WI06
`l ::~
`I FSS
`I 0 64
`I LS4
`I N24
`I AO
`I F96
`I A9
`I FllO
`I 09
`....C.:99··· ··· ··ea
`I
`I
`l
`I Gl4
`101~32 •·•···· y~~=:::T~~~---_4!6t~b
`I P 7o
`a s ··· ···· 3 a 0 29
`0 38
`.•.••..
`c~U
`j
`j o~
`J
`39··· · ··· ~2/ P6I
`104·····--94
`I
`I
`I
`I
`
` •••••. . 4 5-........_
`69
`44P67
`
`p
`
`17
`
` ••.•..• 8
`
`
`
`3
`
`16
`
`12
`
`10
`
`103
`
`96 Rl43
`
`9 1 Y39
`
`3 2 H24
`
`9 7 :::::::9 0
`
`33
`
`98F95
`
`8906 ::::34 K30
`
`35
`
`100 T7
`
`Y48
`
`Y41
`
`1.. 45
`
`100 111
`
`02 7
`101 052
`
`96
`
`DO
`9 5 N31 •.. • 3 3
`
`102 .... .•. 9 4
`
`34
`
`I PSO
`I F62
`I 018
`l W75
`I W73
`
`10 3W71
`
`93Ao:::: 35TO
`
`,.,,<.10 4 ... ··-·92
`
`36
`
`105014
`
`I ~~5
`
`a9········3 048
`·••···•· 9020
`
`10 8
`
`I
`I
`I
`I
`109 ···--··aa
`··· · ·-·
`I
`I
`
`VL
`
`(a )
`
`VH
`
`(b)
`
`( c }
`Fig ure 2 . P-S heet residues that for m the \"l,-\'H intt>rfa rl' in the Fabs KOL. :\ F,\\' and :llC'P(' 603. Residue numbers
`are those of Kabat Pl al. (1983). (a) \ 'L interface-forming P·shert: (b) \ "H interfac·t>-forming P·sheet. Brokrn lines
`indicate hydrogen bonds. At each position where a residue forms part of th!' interfaC'e. wi- gi\•e the residue i<h·ntit.'· in
`KOL. NEW and MCPC 603. and the accessible surface of the residue that is buried in the \'L-\"H interface. :\ott· the
`P· bulges in the edge strands at positions 4:3. 4-~ and 100. 101 in \'I. and 44. 4'1 and 105. 106 in the \ 'H . (c) T he P-sheet
`from KOL VH domain. Residues making contacts to thE> \"T, domain across the domain-domain int.erfat·e are circled.
`The main -chain atoms are displayed. T he circles associated with each (.'(!( atom ha,·e an tllt'a proportional to the
`111·1·1·ssiblt- surface area lost when tht> \ ' L- \ "H dimer forms. Note the large an·a" assoc-iated with residues in the edge
`st rands of t he P·sheet.
`
`6 of 14
`
`Celltrion, Inc., Exhibit 1063
`
`

`

`656
`
`r·. Chothia. J. Novotny . R. Bruccoleri and M. Karplus
`
`q)
`
`-;
`!
`
`GI
`i:I
`0
`.D
`.14
`()
`&I
`
`150
`
`100
`
`60
`
`0
`
`- 50
`
`-100
`
`- 150
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`Sequence nUDlber
`{o)
`
`150
`
`100
`
`50
`
`0
`
`-60
`
`- 100
`
`-150
`
`fl3
`
`65
`
`90
`
`L3
`
`95
`
`{J4
`
`100
`
`105
`
`Sequence DUDlber
`
`q)
`
`...
`·e ...
`
`GI
`i:I
`0
`.D
`)4
`0
`
`"' al
`
`(bl
`Figure 3. The ba<'kbone twist (9) profiles of VL-VH interface-forming segments. The segments shown include t.ht"
`hypervariable loops (LI . L2. L3. HI , H2 and H3) and the P-strands. T he P-strands are indicated by bars at the bottom
`ofth t- plot:-i and lahelt'd Pl throuf:!h P911.<'<"ording to :'\m"<•t11.\· el 11/. (1983). P·Bulgei; 111· .. denoted liy open liuxt>". ~t'((ltt'll<'t'
`nurnhers correspond to the Kabat Pl al. ( 1983) numbering system and arc the same as in Fig. 2. (a) and (b} The 2
`intt•1fat·(··formi11g ""'J.!llWnt:< of the \'L domain; (c) and (d) tlw 2 interfat,.·fonning segments of tht" \'H domain.
`(0) KOL; (0) :'\l·:W. (b.l :\IC'P<' 60:3.
`
`7 of 14
`
`Celltrion, Inc., Exhibit 1063
`
`

`

`CZ>
`
`-;:
`
`'it ..
`~ (J .,
`
`ti
`d
`0
`
`iQ
`
`Packing of Immunoglobu.lin l'rtriablr Dcnnains
`
`657
`
`150
`
`100
`
`50
`
`0
`
`-50
`
`-100
`
`-150
`
`Hl
`
`{J5
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`Sequence nU1Ilber
`( c)
`
`150
`
`100
`
`50
`
`0
`
`-50
`
`-100
`
`-150
`
`CZ>
`
`..., ..
`:s
`
`ti
`d
`0
`.0
`.lll
`
`(J .,
`
`Q:l
`
`90
`
`95
`
`100
`
`105
`
`110
`
`115
`
`120
`
`Sequence nUDlber
`
`(d)
`
`Fig. 3.
`
`/]-bulge
`of
`greater differences. Conser vation
`<·onformatio ns is especially striking and implies that
`they are important a rchitectura lly, as previously
`( 1981 ).
`by
`Richa rdson
`The
`suggested
`correspondence in the /]-sheets is made en·n more
`
`in behavior of t.he
`t he difference
`e1·ident h.\·
`h_q1errnriahle
`loops. The overall simila rity of
`P-shel!l geometries is confi rmed h.1· a least.-squares
`fit of their atomic co-ordinates. Fits of t he main(cid:173)
`c·hain <lloms oft.he three VL /J-sheet.s to each other
`
`8 of 14
`
`Celltrion, Inc., Exhibit 1063
`
`

`

`658
`
`('. Ghothia. J. Novotny. R. Bruccoleri and M. Karplus
`
`Pro
`100
`
`109
`
`88
`
`40
`
`43
`
`VL
`Figure 4. The key residues in the edge strands involved in VL-VH packings (Fab KOL). Note ho~ in (a) Pro44.
`Tyr96 and Phe98 in \'Land in (b) Leu45, ProlOO and Trp103 in YH fold over the central strands of their P-sheets and
`so in (c) form the core of the VL-\'H packing (see also the position of these residues in Fig. 5}.
`
`VL-VH
`
`VH
`
`(30 residues). and of the three VH P·sheets to each
`other
`(32
`residues)
`give
`root-mea.n-squa.re
`differences in atomic positions of between 0·73 and
`I ·23 A (see Table 3). If a few peripheral residues are
`removed from the fits the r.m.s.t differences are
`reduced to 0·55 to 0·87 A. Table 3 also reports the
`results of least-squares fits of the VL P-sheets to the
`\"H /3-sheets. The r.m.s. differences are only a little
`greater than for the fits of the VL or VH P-sheets to
`each other, 0·70 to HI A. Thus, the six regions of
`P-sheet that form the VL-VH interface in KOL,
`NEW and MCPC 603 have very similar structures.
`ln 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 3 values in the range that
`indicate a degree of twist commonly found in
`P-sheets. The average 3 value tends to be the same
`for both the inner and the edge strands, but the
`twist of t lw edge strands is dominated by /3-bulges
`(Figs 2 and 3) with characteristic S 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 P-sheets. Side-chains of
`residues H (Pro). 96 (Tyr, Arg, Leu) and 98 (Pro) in
`\.Land .J.5 (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, 4~8 and 106- 109 in
`VH. lie next to the inner strand in the normal
`manner.
`
`t Abbreviation used: r.m.s., root-mean-square.
`
`(c) Packing of the P-sheets at the T'f.r-l 'H i11te1face
`As noted above, the strong twists that occur in
`the edge strands of VL and VH means that residues
`at two diagonally opposite corners fold over the
`/3-sheets: 44, 96 and 98 in VL {Fig. 4(a)), and -15.
`100 and 103 in VH (Fig. 4(b)). Figure -l(c) shows
`that when the VL and VH domains pack together
`
`Table 3
`The fit of /3-sheets forming V l.r- r H interfaces
`A . Fit.y of i11dit'id11al {J-sheets
`
`VLt
`
`VHt
`
`KOL NEW M('P("
`
`KOL NEW MCPC
`
`0·76
`
`0·55
`0·82
`
`0·88 1·11
`0·96 1·05
`0·70 1·00
`0·87
`
`0·94
`0·97
`0·84
`0·65
`0·87
`
`\' Lt KOL
`XE\\"
`MC'P('
`\'Ht KOL
`;\ P,\\"
`l\l('PC'
`
`B. Fit" of bot.11 fl-sheet regions of the r /.- 1· H inlttfareB§
`
`KOL NEW MC.PC
`
`0·87
`
`0·70
`0·87
`
`KOL
`NEW
`;\1(' J>('
`
`The Table !!ll' P,, r.m.s. differences in position of the main chain
`following
`least-squares
`fits of
`their co-ordinates.
`atoms
`Differences arl' given in A.
`t \"L residues used to determine fits and r.m.~ . differen~es
`33- 39. 4:3- -47. s.J- 90 and 98- 104.
`t \" H residues used to determine fits and r. m .~. differences
`33-40, H-48. 88- 94 and 102-109.
`§Residues used in fits 33- 39. 43-47. 84-90 and 9S-104 of \'L
`and 3HO. 44-48. 88-94 and 103-109 of VH.
`
`9 of 14
`
`Celltrion, Inc., Exhibit 1063
`
`

`

`Packing of Immunoglobulin Variable Domains
`
`659
`
`VL
`
`VH
`
`(b)
`
`(a)
`
`(c)
`
`(d)
`Figure 5. Residue packing at the KOL VL-VH interfac·e .. This Figure shows superimposed serial sertions ('Ill t.hrough
`a space·filling model of the interface. VH residues are shown by bro.ken lines and VL residues by continuous Jines. The
`pseudo 2-fold axis that relates \' L to \.H is perpendicular to t.he page. Earh part of the Figure shows 4 ~t'd i 1111~ .
`separated by I A. superimposed. (a) Sections 0 to 3 A: (b) sertions 4 to 7 A: (c) sections 8 to 11 ..\: and (d) Rertions I:! to
`ISA.
`
`10 of 14
`
`Celltrion, Inc., Exhibit 1063
`
`

`

`660
`
`<'. Chotltia. J . Novotny. R. Bruccoleri and M. Karplus
`
`Table 4
`V Ir- I' H i ntflfflre.<
`R1»<id11e.• burifd in
`
`Residue at this position
`in
`
`An"t'"sible surface
`area. of residue (A 2
`)
`
`Domain Residue
`~o KOL N~;\r MC:PC
`
`KOL :>;~\\' MC:PC
`
`No. of sequenc·es known that
`inc:lude I his positiunt
`
`Principal re~idues found
`u t this positiont
`(itl.-n tit y 11nd number of <·a"<'•)
`
`\ ' L
`
`VH
`
`34
`36
`3B
`.1-J
`46
`87
`89
`91
`96
`98
`35
`37
`39
`45
`47
`91
`93
`95
`JOO
`103
`
`Asn
`Ly~
`Tyr
`Tyr
`(:In
`O ln
`P ro
`Pro
`Leu
`Leu
`Tyr
`Tyr
`Ala
`Gin
`Trp
`Tyr
`Tyr Arg
`Phe
`Phe
`'l'hr
`T\T
`v·al
`V11I
`Gin
`Gin
`Li'u
`Leu
`Trp
`Trp
`Phe
`Tyr
`Ala
`Ala
`A~p Asn
`Pro
`lie
`Trp
`Tr p
`
`Ala
`Tyr
`Gin
`Pro
`Leu
`Tyr
`Gin
`Asp
`{A>u
`Phe
`Gin
`Val
`Gin
`Leu
`Trp
`Tyr
`Ala
`Asn
`Phe
`Trp
`
`2
`I)
`:!
`8
`17
`9
`0
`3
`6
`10
`0
`0
`8
`10
`II
`II
`0
`Cl
`0
`:!7
`
`39
`0
`7
`5
`35
`I
`I
`12
`5
`9
`:!
`3
`:!O
`6
`6
`8
`0
`0
`32
`:!I!
`
`0
`I
`17
`5
`s
`II
`0
`0
`3
`2
`ti
`I
`:!I
`3
`-I
`JI
`I
`5
`0
`:!ti
`
`t Data taken from Kabat t i al. (1983).
`
`362
`318
`302
`:!:31$
`235
`227
`:!17
`:!11
`199
`:!06
`:!17
`200
`183
`163
`157
`159
`161
`131
`113
`1:!5
`
`Alall7. Asn92. His51. Ser37
`Tyr243. Phe40. Val28
`Gln279
`Prol90. Phe:!9. \'all4
`Leul!'>i. Oly3:!. Pro19. Ya113
`T,·rl till. Phe65
`G.ln I :!x. Ala35
`Trp59. Tyr31 . N-r:!7
`Trp-16. T~·r31. J:!6. R20
`Phe203
`C:ln53. Asn-1:!. !:'t·r34. Lys:!:!
`\ 'a1178. lle lll
`0 111176
`Leu160
`Trpl51
`Tvrl:!8. Phe30
`Aial-16
`Asp53. c:l~· 18
`Phe76. Met I I. Leu6
`Trp 11 8
`
`these six residues form the center of t he interfac·e.
`They are in contact. wiLli ea.uh other in pairs and
`make a herringbone pattern.
`Details of how residues pack at the \· 1, \.ll
`interfaee can be seen in sections cut through space(cid:173)
`filling models. Figure 5 shows sections of the KOL
`\.L-\.H interface. The central role played by the
`t hree pairs of edge residues. Ty r96 and Trpl03. and
`Leu45 and Pro44 are seen in parts (b), (c) and (d) of
`t he Figure. The inner strands of t he /J·sheets. 32- 39
`and 8.t- 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-\'H interfaces in XE\\' and
`~1CPC 603 are very similar to that of KOL
`illustrated here. This is demonstrated b.Y graphical
`inspection of their packing and by the fits of the co(cid:173)
`ordinates of the main -c·hain atoms forming the\' 1.(cid:173)
`\ .H interfaces described above (Table 3).
`The packing of the /J-sheets at the three n~-\· H
`int erfaC'e::; can be described in tRrms of a t.hree·laver
`structure: an inner layer consisting of large si.de(cid:173)
`chains from strongly twisted ends of the edge
`strands; and two outer layers formed hy th(' main
`and side-chains of the inner /J-strands and the
`middle part of the edge strands (Figs .t and 5).
`
`(d ) Tlirn'-hr!J'' prirkiny "·~ o general model for
`I' L- 1' H (l.~sorinfirm.~
`Ten ~·ears ago Poljak et "'· ( 1975) examined their
`Fab XE\\' structure and noted that th(' residues
`
`t hat form the \'1,-VH interface were conserved in
`the other immunoglobulin sequences t hen known.
`They predicted t hat t he mode of association of
`other \·L- \ 'H dimers would be the same as that
`found in Fab NEW. The structures and many
`sequences determined since then. and the work
`reported here, confirm t heir prediction.
`The three structures studied here include a widt>
`range of immunoglobulins: human ). and y to mouse
`" and y (Table I). In KOL. :-J E\\' and :\IC'PC 603
`residues at about ten posit.ions in VL and in VH are
`buried in the interface between the domains. The
`amino acid sequences of many other immuno·
`globulins have been determined and a. tabulation
`published by Kabat et al. (1983). We examined the
`tables of
`\ "L and \. H sequences to find what
`residues occur at positions homologous to the 20
`buried in the \.L-,.H interfaces studied here. The
`results of t.his ::;111Tt',\' are given in Table .t and
`Figure 6.
`At I:! of the :!O positions residue identity is
`absolutel.Y. or ,·ery st.rongly . conserved: in V L.
`residues 36. 38. H . 87 and 98: in \·H , residues 37,
`39 . .t5, ~i . 91 , 93 and 103. ,.\,. shown in Figure 6,
`t hese residues form the whole of the central and
`lower regions of the interface. The eight positions
`that have some variation in residue identity are all
`in the upper part of tlw intRrfaces where the.'· are
`adjacent to and partially buried h~· the hyper·
`variable regions. The t hree structures studied have
`a range of residues at these positions t hat is fairl.\·
`representative of those fou nd in ot.her sequenn·:<
`(Table 4) . [nspection of t he thrt·(' structures shows
`
`11 of 14
`
`Celltrion, Inc., Exhibit 1063
`
`

`

`Packing of lmmunoglobulin Var-iable Domains
`
`661
`
`VL
`
`x=+IBA -
`
`32
`
`I
`91
`
`(>-28 w
`
`89 0·600
`
`I 0·25 y
`I 0·16A
`
`87 0·99 F/Y
`I
`
`I
`95 0·40 D
`
`93 0·91 A
`
`I 0·14G
`l 91 0·99 Y/F
`
`I
`
`I
`96 0·23W
`0·16 y
`
`I
`I
`I 101
`
`98 0·99 F
`
`99
`
`l·OOG
`
`l·OOG
`
`I
`
`I
`1000·73F
`O·IO L
`
`103 0·94W
`
`104 0·98 G
`
`I
`I 106 0·98 G
`
`I
`
`39 0·960
`I
`I
`Figure 6. The conservation of residues that form \ ' J,(cid:173)
`\IH interfaces. On a plan of the VL and \ ' H P-sheets \\'t'
`show the principal residues found at sites buried in the
`interface and at sites involved in the formation of tht'
`P-bulges. At each site we note the proportion of known
`sequences that contain the given residue, for example.
`0·99 of known \'I, sequi>n«>s ha,·e Phe at position 98 (see
`Table 4). The one-letter