Reprinted from J . . If.JI. Biol. (1985) 186. 651-663
`
`3
`
`Domain Association in Immunoglobulin Molecules
`The Packing of Variable Domains
`
`Cyrus Chothia, Jiri Novotny, Robert Bruccoleri
`and Martin Karplus
`
`1 of 14
`
`BI Exhibit 1063
`
`

`

`J. ;)Jul. 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 WOlH 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 1800A2 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 fold back over their 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. there is a third layer composed of side-chains inserted between th!'. two backbone side­
`chain layt>r:s that. are usually in contact. This three-layer packing is different from
`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 the association of VL and \'H domains and that the three-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 family of proteins, which also
`includes /3-microglobulins. Thy- 1 antigens, major
`
`IHl:!:?-:?836/85/230651-13 S03.00/0
`
`651
`
`(i.e. class T) and minor (i.e. class 11) histo­
`compatibilit.y antigens and cell surface receptors.
`Fun<'tionall\'. all these struC'tures are involved in
`cell recognfrion processes (Jensenius & Williams,
`1982). either actively as vehicles endowed with
`© 1985 ..\c.,tdemie Press Inc. (London) Ltd.
`
`2 of 14
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`BI Exhibit 1063
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`

`652
`
`('. Ghothia. J. No-votny, R. Bruccoleri nnd M. Karplus
`
`this paper. In particular, the nature of the interface
`between V L and VH domains is examined by
`C'omparing tht· Fab fragments of KOL, NEW and
`l\lt!P(' 603 myeloma proteins whose X-ray
`structures are known. The relative contributions to
`the buried surface between the domains from the
`frame.work residues and the hyper­
`conserved
`variable regions are determined. Attention
`is
`focused on the unique packing oft he interfa<·es and
`the reasons for this packing are examined.
`
`2. Materials and Methods
`{a) Fab fragment co·ordinal""
`Cartesian co-ordinates 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
`graphic residuals (R factors) for the 3 Fab fragments. To
`facilitate 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.,
`
`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 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, the homology among primary structures
`of irnmunoglobulin, P-microglobulin, Thy-! 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 el al ..
`1973; Orr Pl al., 1979: Feinstein, 1979; Cohen el al.,
`1980, 198la; 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 the chains
`of
`of domains made up
`composed
`being
`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 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
`stranded P-sheets of the two domain types a1·e
`homologous: the five- or four- stranded P-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 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 VL-VH, CL-CHI
`and CH3-CH3. Edmundson et al. (1975) were the
`rotational
`to note the phenomenon of
`first
`allomerism between the variable and constant
`domain dimers, that is. whereas t.he e-c dimers
`interact ria a close packing of their four-strand
`sheets. the V-\' dimers pack "inside out", with the
`face-to-face. The
`five-st.randed sheets oriented
`reversal of domain-domain interaction is reflected
`in the amino acid sequence homology between. and
`constant and variable domains
`the
`among.
`(Novotny & Franek, 1975; BeaJe & Feinstein, 1976:
`(b)
`'.'Jovotny Pf al., 1977).
`Figure 1. The P·:<lwt>t>< in t.\'Pi<«ll immunoglobulin
`same
`the
`in
`Different antibody molecules
`domains. \'t>rti«t->< represent. the position of C'cx atoms:
`organism bind different antigenic structures. The
`t hos,.. in /J·:<ht·t>ts art- linked b�· ribbons: and those
`variation in ,;pec·ific·it y
`between strand:< hy lines. (a) The \'L domain of KOL: tilt'
`is produced by ,;e,·eral
`in \'L-\'H rnntat·t:< is closer to the
`involved
`P-shet-t
`mechanisms: mutations, deletions and insertions in
`,.it'W!'r (unbroken line). (b) The :<<111H·
`\'L domain rotated
`the binding regions of the VL and VH domains; and
`90°. i\ntp th<1t the int.erfa<,e·forming
`by <1p111:oxi111att-ly
`the association of different light and heavy chains.
`P-slwt>t is strongl.'· t\\'i:<tt'd at diagonallv opposite corners
`Aspeds of the second mechanism are analyzed in
`(drawing hy .-\. ;\(. l,t-sk).
`
`(o)
`
`·
`
`3 of 14
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`Paclcing of 1 mmunoglobulin Variable Domains
`
`653
`
`Table I
`Summary of X-ray crystallographic data
`
`X-ray data
`Minimized
`Land H
`chain Resolution R factor Energy r.m.s. shift
`t,vpes (A)
`(�o)
`(A)
`(kJ)
`
`Reference
`
`.I.I, )'lII
`
`J.I, yII
`"· yl
`
`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.
`
`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 the 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 the 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
`
`were used in the present stud,1·:
`
`VH domain dimers. The structures were subjeC't.ed to 100
`cycles of constrained energy minimiz.ation with the
`program CHARMM 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 (-0·50 kcal/atom) with an average root­
`mean-square co-ordinate different from the 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
`essentiall.1· identical
`results were obtained from the 2 types of co-ordinates
`sets.
`
`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-strands are twistt-d
`(C'hothia,
`1973).
`The
`out-of-planarit.y
`angle
`(180°-160°} == 20 can he obtained explicitly from the
`w (see. e . g. Chou el al .. 1982). Wt- define the local
`backbone torsion angles <p. i/I and
`values of thi- principal
`9 = (-2-) (180-lrll.
`
`backbone twist for 2 consecutive residues as:
`
`(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
`using the methods of Lee & Richards ( 1971) and
`Richmond & Richards (1978). The latter program was
`obtained from Yale University. The water probe radius
`lrl
`used was l ·4 A and the section interval along the Z axis
`was 0·05 A; the atom van der \\'aals' radii used were :! A
`where r is the torsion angle CP-<.'a-(''11.-C'P and lrl denotes
`for all the (Pxtended) tetrahedral carbon atoms, l ·85 A
`its magnitude. When glycine residues that lack cp atoms
`for all the planar (.�p2 hybridized) carbons. l ·4 A and
`are encountered, the torsion angle 9 is measured with
`I ·6 A for carbonyl and hydrox�·J oxygens. respectively.
`rt-:>pect to t.he C'P atom following the gly«ine. Thus.
`1 ·5 A for a carbonyl OH group. 2·0 A for all the
`glycine residues contribute to the local backbone twist
`(extended) tetrahedral nitrogen atoms. l ·5 A. l ·7 A and
`indirl·rtl�'. by being induded in the virtual bond Ca-Ca
`l ·8 A for s-p2-hybridized nitrogen atoms carrying no
`that spans from the residue pre<'eding the gl.n·i1w to that
`2·0 A for
`hydrogen, I and 2 h�'drogen atoms. respecti,·el.'··
`which follows it.
`a sulfhydr,vl group and l ·85 A for a divalent sulfur atom
`Backbone twist profiles (plots of 9 as a funttion of the
`with no h�·drogens.
`amino acid residue) ,;er\'e to C'haraC'tf·rize polypeptide
`chain conformations. Certain conformational character­
`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. It 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
`
`(c) P-Strands a11d P-sheets
`Protein struC'tures were analyzed 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
`prest-nt. The P-strands and P·sht-et>< ,,, .. re defined h_,.
`intn-strand ba<"kbone (C' = 0 . . . H-N) hydrogen-
`their
`
`(T = 3606/11) in a c-orresponcling wa.v to that de><«ril, .. d for
`
`b b)· Chou Pf rd. ( 1982).
`
`4 of 14
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`

`654
`
`C. f'hothia. J. Novotny, R. Bruccoleri and M. Karp/11.,
`
`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
`
`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.
`
`·t
`
`(b) Conformation of interface P-sheets
`The deviation of the conformations of the
`/J-sheets that form 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
`average 9 = + 20°. right-handed a helices to lines of
`P·sheets correspond to horizontal lines with an
`
`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
`
`3. Results
`(a} Dom fl i 11-r/0111f/ i11 N111/rtrl .-11 1/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
`nd \'JI was taken
`surface on the association of \'L
`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
`domain contacts also have ,·an der \\'aals' inter­
`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
`the
`structure
`of
`the
`unassociated domains.
`Of the total area buried between the \'L-YH
`dimers about one quarter comes from residues in
`the h>·pervariable regions and about thrcT quarters
`in P-sheets. Figure � shows t.he
`from residues
`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.
`
`'a
`
`Table 2
`Accessible surfaces and those lost on 1·L-l'H association (.·F)
`
`isolated surface
`
`Contact surfa<'e
`
`Hydrophobic Polar Total
`
`Domain pair
`
`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.�
`
`311
`250
`561
`:l�i
`386
`773
`299
`324
`Ii:.!:!
`
`652
`
`891
`865
`1756
`916
`892
`1808
`975
`943
`1918
`18:.!i
`
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`

`

`Pac/.."ing of lmmunoglobulin Variable Domains
`
`655
`
`Js ��L::912
`J1
`l ::�
`I WI06 l TIS
`96 Rl43 91 Y39 32 H24
`I 064 I FSS
`I LS4
`97 :::::::90 33
`I F96
`I AO I N24
`98F95 8906::::34 K30
`I A9
`I FllO I 09
`....C.:99··· ·····ea 35
`100T7
`Gl4
`I
`I
`l
`Y48 Y41 1..45
`101�32
`I
`•·•···· y��=:::T���---_4!6t�b
`816
`317 •••••.. 45-........_ p69
`1012 ••.•..•
`44P67
`103 as·······3a029
`I P7o
`.•.••.. 038
`j o�
`c�U
`j
`J
`39·······�2/ P6I
`104·····--94
`I
`I
`I
`I
`
`I PSO I
`100111 96
`I F62 I
`027 DO I
`101 052 95 N31 •..• 33
`I 018 I N20 I
`
`102 .....•. 94
`34
`l W75 I AO I Yl2
`103W71 93Ao::::35TO
`I E9
`AO
`I W73 I
`,.,,<.104 ... ··-·92
`36
`105014
`I
`I
`I ��5
`I
`91;�L::37�� 47::�
`I06"'--107::::::.9bv33 Ja��::::l wes
`I
`I
`-.........45 t:2!
`I
`I l.88
`3 048
`a9········
`108
`·••···•· 9020
`44��0
`I 032
`I
`···-·-· / R96
`I
`109···--··aa
`4'3
`·····-· 40
`I
`I
`I
`I
`
`VL
`
`(a)
`
`VH
`
`(b)
`
`( c }
`are those of Kabat Pl al. (1983). (a) \'L interface-forming P·shert: (b) \"H interfac·t>-forming P·sheet. Brokrn lines
`Figure 2. P-Sheet residues that form the \"l,-\'H intt>rfarl' in the Fabs KOL. :\ F,\\' and :llC'P(' 603. Residue numbers
`· in
`indicate hydrogen bonds. At each position where a residue forms part of th!' interfaC'e. wi- gi\•e the residue i<h·ntit.'
`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) The P-sheet
`from KOL VH domain. Residues making contacts to thE> \"T, domain across the domain-domain int.erfat·e
`strands of the P·sheet.
`
`are circled.
`The main-chain atoms are displayed. The 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
`
`6 of 14
`
`BI Exhibit 1063
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`

`

`656
`
`r·. Chothia. J. Novotny. R. Bruccoleri and M. Karplus
`
`150
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`100
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`60
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`
`20
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`25
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`30
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`Sequence nUDlber
`{o)
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`105
`
`Sequence DUDlber
`(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. The P-strands are indicated by bars at the bottom
`
`oftht- 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
`
`BI Exhibit 1063
`
`

`

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

`

`658
`
`('. Ghothia. J. Novotny. R. Bruccoleri and M. Karplus
`
`Pro
`100
`
`109
`
`88
`
`40
`
`43
`
`VL-VH
`VH
`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}.
`
`(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.
`sheets can be achieved as a 2-fold symmetry
`l n fact, the least-squares superposition of the two
`
`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
`
`1·11
`0·88
`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'
`
`of the r /.-1· H inlttfareB§
`B. Fit" of bot.11 fl-sheet regions
`
`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
`atoms
`following
`least-squares fits of
`their co-ordinates.
`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
`
`BI Exhibit 1063
`
`

`

`Packing of Immunoglobulin Variable Domains
`
`659
`
`VL
`
`VH
`(b)
`
`(a)
`
`(d)
`(c)
`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'di1111�.
`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
`
`BI 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)
`
`Principal re�idues found
`
`u t this positiont
`(itl.-ntity 11nd number of <·a"<'•)
`
`Alall7. Asn92. His51. Ser37
`Tyr243. Phe40. Val28
`Gln279
`Prol90. Phe:!9. \'all4
`T,·rl till. Phe65
`Leul!'>i. Oly3:!. Pro19. Ya113
`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. llelll
`0111176
`Leu160
`Trpl51
`Tvrl:!8. Phe30
`Aial-16
`Asp53. c:l�· 18
`Phe76. Met I I. Leu6
`Trp 118
`
`362
`318
`302
`:!:31$
`235
`227
`:!17
`:!11
`199
`:!06
`:!17
`200
`183
`163
`157
`159
`161
`131
`113
`1:!5
`
`No. of sequenc·es known that
`
`Domain Residue
`inc:lude I his positiunt
`�o KOL N�;\r MC:PC KOL :>;�\\' MC:PC
`I)
`0
`2
`I
`17
`5
`s
`II
`0
`0
`3
`2
`ti
`I
`:!I
`3
`
`\'L
`
`VH
`
`34
`36
`.1-J
`3B
`
`Ala
`Asn
`Ly�
`Tyr
`Tyr Tyr
`Gin
`(:In Oln
`Pro
`Pro
`Pro
`Leu
`46
`Leu
`Leu
`87
`Tyr
`Tyr
`Tyr
`Gin
`Ala Gin
`89
`Trp Tyr Asp
`91
`Arg
`Tyr
`96
`{A>u
`Phe Phe Phe
`98
`Gin
`T\T
`'l'hr
`35
`v·al
`Val
`V11I
`37
`Gin
`Gin
`Gin
`39
`45 Li'u Leu Leu
`Trp Trp Trp
`47
`91
`Phe Tyr Tyr
`Ala
`Ala
`93
`Ala
`Asn
`A�p Asn
`95
`Phe
`JOO Pro
`lie
`Trp
`103
`Trp
`Trp
`
`39
`0
`7
`5
`35
`I
`I
`12
`5
`9
`:!
`3
`:!O
`6
`6
`8
`0
`0
`32
`:!I!
`
`:!
`8
`17
`9
`0
`3
`6
`10
`0
`0
`8
`10
`II
`II
`0
`Cl
`0
`
`:!7
`
`-I
`5
`
`JI
`I
`
`0
`:!ti
`
`t Data taken from Kabat ti al. (1983).
`
`these six residues form the center of the interfac·e.
`They are in contact. wiLli ea.uh other in pairs and
`make a herringbone pattern.
`.
`ll
`Details of how residues pack at the \·1, \
`interfaee can be seen in sections cut through space­
`filling models. Figure 5 shows sections of the KOL
`\.L-\.H 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 /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
`ordinates of the main-c·hain atoms forming the \' 1.­
`\.H interfaces described above (Table 3).
`The packing of the /J-sheets at the three n�-\·H
`interfaC'e::; can be described in tRrms of a t.hree·laver
`.
`de­
`structure: an inner layer consisting of large si
`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
`
`that form the \'1,-VH interface were conserved in
`the other immunoglobulin sequences then known.
`They predicted that the 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 their 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
`in V L.
`absolutel.Y. or ,·ery st.rongly. conserved:
`residues 36. 38. H. 87 and 98: in \·H, residues 37,
`.t5, �i. 91, 93 and 103. ,.\,. shown in Figure 6,
`39 .
`these 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 three structures studied have
`a range of residues at these positions that is fairl.\·
`representative of those found in ot.her sequenn·:<
`(Table 4). [nspection of the thrt·(' structures shows
`
`11 of 14
`
`BI Exhibit 1063
`
`

`

`Packing of lmmunoglobulin Var-iable Domains
`
`661
`
`VL
`
`x = +IB A -
`
`x = o A -
`
`416
`
` 0·67 L
`
`I 0·14 G
`
`44 0·80 p
`I 0·12 F
`
`VH
`
`I
`
`91 (>-28 w
`I 0·25 y
`I 0·16A
`
`89 0·600
`
`87 0·99 F/Y
`I
`
`I
`34 0·32 A
`
`I 0·25N
`I
`
`36 0·90Y/F
`
`38 0·92 a
`I
`
`I
`95 0·40 D
`
`32
`
`SS
`
`el 76
`
`X = O A
`
`I
`
`35 0·23E
`
`37 0·89V
`
`39 0·96 0
`
`I
`
`47 0·93 w
`l
`
`I
`96 0·23W
`
`I 0·16 y
`
`98 0·99 F
`I
`99
`I
`
`l·OO G
`
`l·OOG
`
`101
`I
`
`I
`100 0·73 F
`O·IO L
`
`103 0·94W
`I
`104 0·98 G
`I
`
`106 0·98 G
`I
`
`I 0 · 1 4 G
`l
`
`93 0·91 A
`
`91 0·99 Y/F
`
`I
`
`I 0 · 1 9 N
`I 0·10 l
`I
`45 0·98 L
`I
`\IH interfaces. On a plan of the VL and \'H P-sheets \\'t'
`Figure 6. The conservation of residues that form \' J,­
`show the principal residues found at sites buried in the
`interface and at sites involved in the formation of tht'
`sequences that contain the given residue, for example.
`P-bulges. At each site we note the proportion of known
`0·99 of known \'I, sequi>n«>s ha,·e Phe at position 98 (see
`Table 4). The one-letter <·odi> for amino acids is used.
`
`that the different residues are accommodated by
`small conformational changes in the ends of the
`P-strands and somewhat larger changes in the
`hypervariable regions. The Gly-X-Gl.'· sequenr<'
`that produces the P-bulge at residues 99-101 in V L
`and 104-106 in V H is absolutely conserved in \"L
`and VH sequences (Fig. 6). Thus the pattern of
`conserved residues
`in \'L and \.t-1 sequences