1 of 14 celltrion, inc., exhibit 1063 · 2017. 5. 10. · reprinted from j . . if.ji . biol. (...

Reprinted from J . . If.JI. Biol. (1985) 186. 651- 663

Domain Association in Immunoglobulin Molecules

The Packing of Variable Domains

Cyrus Chothia, Jiri Novotny, Robert Bruccoleri and Martin Karplus

3

1 of 14 Celltrion, Inc., 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 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 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 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 sidechain 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 genera l model for t he 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:!:?- :?836/85/23065 1- 13 S03.00/0 651

(i.e. class T) and minor (i.e. class 11 ) histocompatibilit.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

© 1985 ..\c.,tdemie Press Inc. (London) Ltd.


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 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 t he 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 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 fourstranded P-sheets of the two domain types a1·e homologous: t he five- or fourstranded 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 VL-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 among. the constant and variable domains (Novotny & Franek, 1975; BeaJe & Feinstein, 1976: '.'Jovotny Pf al., 1977).

Different antibody molecules in t he same 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; and 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 the domains from the conserved frame.work residues and the hypervariable 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-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 crystallographic 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>< in t.\'Pi<«l l immunoglobulin domains. \ ' t>rti«t->< represent. the position of C'cx atoms: t hos,.. in /J ·:<ht·t>ts art- linked b~· ribbons: and those between strand:< hy lines. (a) The \ 'L domain of KOL: tilt' P-shet-t involved in \ 'L- \'H rnntat·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). ·


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 Protein t,vpes (A) (~o) (kJ) (A) Reference

Fab KOL .I.I, )'lII 1·9 human

26 -3010 Marquart el al. (1980)

Fab :\EW J.I , yII :?·O human

19 - 2592 0·21 Saul et al. (1978)

Fab MCPC: 603 " · yl 2·7 :?4 - 3703 0·26 Segal el al. (1974) 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 cycles of constrained energy minimiz.ation with the 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 rootmean-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 crystallographi c· 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 using the methods of Lee & Richards ( 1971) and 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; the 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 prest-nt. The P-strands and P·sht-et>< ,,, .. re defined h_,. 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 (Paul ing 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 explicit ly from the 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 the 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 characteristics 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).


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 domaindomain contacts also have ,·an der \\'aals ' interactions 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 -YH 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 Hydrophobi(· Polar Total

KOL \ ' L domain 11:!1 fi!'i~ 17i9 ;;so 311 891 KOL \'H domain 1:!16 700 1926 615 250 865 \'1 .- \'H in KOL 2337 1358 3705 1195 561 1756 ~ E\\. \'I, domain 1233 7H 19i7 529 :l~i 916 ~ P,\\' VH domain I 186 801 1985 506 386 892 \'L \ ' H in NEW :!419 1545 3962 1035 773 1808 :\H'P(' 603 VL domain 1082 689 1771 676 299 975 :\l<!P(' 603 \ ' H domain 1156 760 1916 619 324 943 \ 't,... \·H in MCPC 603 2238 1449 3687 l:!fli'i Ii:.!:! 1918 \ ' J,_ \·H ~,·erag~ 2331 1714 3785 1 HI.~ 652 18:.!i


Pac/.."ing of l mmunoglobulin Variable Domains

J s ~~L::912 J1 l ::~ I WI06 l TIS

96 Rl43 9 1 Y39 3 2 H24 I LS4 I 064 I FSS

9 7 :::::::9 0 33

I F96 I AO I N24 98F95 8906::::34 K30 I FllO I 09 I A9

....C.:99··· ··· ··ea 35

100 T7 I I l I Gl4 Y48 Y41 1.. 45

101~32 •·•···· y~~=:::T~~~- --_4!6 t~b 10

1

2 ••.•..• 8

1

6 3

1

7 •••••. . 4 5-........_ p69

44P67

103 a s ··· ···· 3 a 0 29 I P7o .•.••.. 0 38

J j j o~ c~U 104·····--94 39··· · ···~2/ P6I

I I I I

VL

(a)

( c }

I PSO I 100 111 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 10 3W71 93Ao::::35TO I W73 I AO I E9

,.,,<.10 4 ... ··-·92 36

105014 I I I I ~~5 91 ;~L::37~~ 47::~

I06"'--107::::::.9bv33 Ja~~::::l wes

I I I -.........4 5 t:2! 10 8 a9········3 048 I l. 88

·••···•· 9020 I I I 0 32 44 ~~0 109 ···--··aa ···- ·- · / R96

··· · ·-· 40 4'3

I I I I

VH

(b)

655

Figure 2. P-Sheet residues that form the \"l,-\'H intt>rfarl' 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) The 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. 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

st rands of the P·sheet.


656

150

100

q) 60 -; ! GI 0 i:I 0

.D .14 ()

&I - 50

-100

- 150

20

150

100

q) 50 ... ·e ... GI i:I 0 .D )4 0

0

"' al -60

- 100

-150

65

25

fl3

r·. Chothia. J. Novotny . R. Bruccoleri and M. Karplus

30 35

90

40

Sequence nUDlber

{o)

L3

95

Sequence DUDlber

(bl

45

100

50 55

{J4

105

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.


150

100

CZ> 50 -;: 'it .. ti d 0

0

~ (J ., iQ -50

-100

-150 Hl {J5

30 35

150

100

CZ> 50 ..., .. :s ti 0 d 0 .0 .lll (J .,

Q:l -50

-100

-150

90

Packing of Immunoglobu.lin l'rtriablr Dcnnains

40

95 100

45

Sequence nU1Ilber

( c)

105

Sequence nUDlber

(d)

Fig. 3.

50

110

657

55 60

115 120

greater d ifferences. Conservation of /]-bulge <·onformatio ns is especially striking and implies that they are important a rchitectura lly, as previously suggested by Richa rdson ( 1981 ). The correspondence in the /]-sheets is made en·n more

e1·ident h.\· t he difference in behavior of t.he h_q1errnriahle loops. The overall simila ri ty of P-shel!l geometries is confi rmed h.1· a least.-squares fit of their atomic co-ordinates. Fits of t he mainc·hain <lloms oft.he three VL /J-sheet.s to each other


658 ('. Ghothia. J. Novotny. R. Bruccoleri and M. Karplus

Pro 100

109 88 40

43

VL VH VL-VH

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. 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

\' Lt KOL 0·76 0·55 0·88 1·11 0·94 XE\\" 0·82 0·96 1·05 0·97 MC'P(' 0·70 1·00 0·84

\'Ht KOL 0·87 0·65 ;\ P,\\" 0·87 l\l ('PC'

B. Fit" of bot.11 fl -sheet regions of the r /.- 1· H inlttfareB§

KOL NEW MC.PC

KOL 0·87 0·70 NEW 0·87 ;\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.


Packing of Immunoglobulin Variable Domains 659

VL

VH

(a) (b)

(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.


660 <'. Chotlt ia. J . Novotny. R. Bruccoleri and M. Karplus

Table 4 R1»<id11e.• burifd in

Residue at this position An"t'"sible surface in area. of residue (A 2 )

Domain Residue ~o KOL N~;\r MC:PC KOL :>;~\\' MC:PC

\ ' L 34 Asn Ly~ Ala 2 39 0 36 Tyr Tyr Tyr I) 0 I 3B (:In O ln Gin :! 7 17 .1-J P ro Pro Pro 8 5 5 46 Leu Leu Leu 17 35 s 87 Tyr Tyr Tyr 9 I II 89 Ala Gin Gin 0 I 0 91 Trp Tyr Asp 3 12 0 96 Tyr Arg {A>u 6 5 3 98 Phe Phe Phe 10 9 2

VH 35 T\T 'l'hr Gin 0 :! ti 37 v·al V11I Val 0 3 I 39 Gin Gin Gin 8 :!O :!I 45 Li'u Leu Leu 10 6 3 47 Trp Trp Trp II 6 -I 91 Phe Tyr Tyr II 8 JI 93 Ala Ala Ala 0 0 I 95 A~p Asn Asn Cl 0 5

JOO Pro lie Phe 0 32 0 103 Trp Trp Trp :!7 :!I! :!ti

t Data taken from Kabat t i al. (1983).

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 spacefilling 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 graphica l inspection of their packing and by the fits of the coordinates 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 int erfaC'e::; can be described in tRrms of a t.hree·laver structure: an inner layer consisting of large si.dechains 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

V Ir- I' H i ntflfflre.<

Principal re~idues found No. of sequenc·es known that u t this positiont

inc:lude I his positiunt (itl.-n tit y 11nd number of <·a"<'•)

362 Alall7. Asn92. His51. Ser37 318 Tyr243. Phe40. Val28 302 Gln279 :!:31$ Prol90. Phe:!9. \ 'all4 235 Leul!'>i . Oly3:! . Pro19. Ya113 227 T,·rl till. Phe65 :!17 G.ln I :!x. Ala35 :!11 Trp59. Tyr31 . N-r:!7 199 Trp-16. T~·r31. J:!6. R20 :!06 Phe203

:!17 C:ln53. Asn-1:! . !:'t·r34. Lys:!:! 200 \ 'a1178. lle lll 183 0 111176 163 Leu160 157 Trpl51 159 Tvrl:!8. Phe30 161 Aial -16 131 Asp53. c:l~· 18 113 Phe76. Met I I. Leu6 1:!5 Trp 11 8

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 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',\' a re 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 tha t 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 fou nd in ot.her sequenn·:< (Table 4) . [nspection of t he thrt·(' structures shows


Packing of lmmunoglobulin Var-iable Domains 661

I 96 0·23W

I 0·16 y

98 0·99 F

I 99 l·OOG

I 101 l·OOG

I

I 1000·73F

O·IO L

103 0·94W

I 104 0·98 G

I 106 0·98 G I

I 91 (>-28 w

I 0·25 y

89 0·600

I 0·16A

87 0·99 F/Y I

I 95 0·40 D

I 0·14G

93 0·91 A

l 91 0·99 Y/F

I

I 34 0·32 A

I 0·25N

36 0·90Y/F

I 38 0·92 a I

I

35 0·23E

I 0·19N

37 0·89V

I 0·10 l

39 0·960 I

VL

416 0·67 L

I 0·14 G

44 0·80 p I 0·12 F

VH

I 47 0·93 w

l 45 0·98 L I

Figure 6. The conservation of residues that form \ ' J,

\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 <·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 VL

and 104-106 in VH is absolutely conserved in \"L and VH sequences (Fig. 6). Thus the pattern of

conserved residues in \'L and \.t-1 sequences

suggests that the three-layer parking found for the structures studied here is a genera.I model for \ ' lr

VH associations.

(e) 7'/iri'e·frtyl'I· pocki11y n.~ r1 I/I'll' P-sheet jlf1d·i11y class

The packing of P-sheets in proteins has been

analyzed in some detail (Chothia et "'" 1977: Chothia & Janin. 1981, 1982: Cohen et al .. 198 lb). Observed parkings h1wc· been divided into t \\'O

classes: the aligned and the orthogonal. The three

lay<' 1' packings described here for the \" L-nl

32

x=+IBA -

SS

x= oA-el

76

X=OA

x= +IBA

Figure 7. An example of the aligned class of P-sheet

parkings. This Figure shows the parking of 2 fl-sheets within 11 VL domain. (a) Shows arrangement of the

P-sheets: c 'o: atoms in one sheet are indicated by open <·in·lt•s and thmw in t.he other sheet h,· filled <' ir<'les.

&ctions cut through a space.filling modei of the packing <1t .r = O ,\ and .r = 18 A are shown in (b) and (c). In (b)

and (c·) the strands of the sheet are approximately

perpPndirular to the page. Note how the strands of one

P-sheet make d irt><·t contact with the strands in t.he other sheet. Residues from the edge strand:> do not form ii

middle layl'r as they do in the 3-layer packings illustrated

in Fii.:s .t and 5. Adapted from C'hothia & Janin (1981).


662 C. Clwthia, J. Novotny, R. Bruccoleri and M. Karplus

interface do not fit into either of these classes. Orthogonal P-sheet packings are quite different: in that class the main chain directions of the packed sheets are inclined at approximat e]~· 90° and one or more strands pass from one P-sheet to the next with little or no interruption (Chothia & Janin, 1982).

The a ligned packings do ha\•e some similarities to three-layer packings in that both involve P-sheets that are essentially independent. The packing of residues at the interface, however, is ,-er.r different. In aligned packings the P-sheets pack face-to-face with some of the side-chains of each strand making direct C'ontact with strand~ on the opposite sheet. Examples of such packings are found within each of the \'L and VH domains (Fig. 7). Aligned P-sheet packings can be described as two-layer structures with the side-chains of the two layers packed t.ogether. In:>t-rtion between tlw two sheets of the side-chain from a residue of an edge strand is uncommon and where it does occur is only found at the margins of tlw interface. In the th1·ee-layer packings described here the residues from the edge strands form a complete la~·er at the tent.er of the interface (Fig. 5).

The different residue packing in the t.wo classes results in different geometry. In aligned packings the angle bPtween the mean chain directions of the packed P-sheets is about - 30° ( -20° to - 50°) and arises from the twist of lhe individual fl-sheets. The angle of the three-layer packings described here is -50°. This angle arises from the bulkier size of the residues that form the middle laye1-. as well as from the concave curvature of th~ interface-forming fl-sheets (cf. Fig. 1). In aligned packings the P-sheets are about 10 A apart. In three-la.'·er packings the eentral regions of each sheet are 1-1 . .\ apart: the larger value arises from the additional central layer. This central layer extends through the whole Jengt.h of the interface. from the " bottom" up to the " top" where the binding :-:itf' is located. and participates in forming the floor of the antigen combining ca,·it . .'-. For example. some oft.he aromatic third -layer side-chains (Phe98 in the \'L domains) were shown to be indispensable for the antigenic :<pet·ifitity (Azuma ff/ al., 1984), even though the.\' are on).'· marginally exposed to solvent:. (Kovotny et al .. 1983).

The general shape of the aligned fl-sheet. paC'king is that of a twisted prism (Fig. 7: ('hot hia & Janin , ( 1981 ). If we ignore the regular parts of the edge strands, three-la.'·er paC'king:< have the general shape of a twisted hyperboloid with ellipti«al crn:<,.;sections (Figs -I and 5: :'\ovotn.\· Pf al. , 1983, 1984-).

Important for the three-layer packings described here are the aromatic- side-chains that form the center of the contact. They pack with their sideC'hains approximately perpendicular to each other as typified in benzene crystals (('ox Pl al .. 1958; \\\«knff. 1969; Nockolds ef al .. 1975; Thomas et al .. 1982; Williams, 1980: Burley & Petsko, 1985; ~m·otn~· & Haber, 1985). This contrasts with the residues most commonl.v found at the interface of aligned packings. They are aliphatic residues like

Val, Ile and Leu that approximate the close packing expected for hard spheres.

4. Conclusion

T mmunoglobulins are composed of sets of domain dimers. Single domains are sandwiches composed of two P-sheet backbone layers with the hydrophobic side-chains in between. For the VL-VH domain dimers we have found that they cannot be described simply as the packing together of two of these sandwich structures. Instead, the \'L-VH interface has a three-layer structure with a set of primarily aromatic side-chains interposed between the sandwich structure making up each of the domains. The three-layer packing is faci litated by highly twisted edge strands that bend at places where P-bulges occur. This mode of P-sheet packing is different from those described previously and produces a sheet- sheet interface that is signifi cantly bulkier than typical "aligned" sheetrsheet interfaces (Chothia & Janin , 1981). The interfaces between VL and VH in Fab KOL, Fab NEW and Fab ~ICPC 603 have very similar structures of this three-layer type. The pattern of residue conservation found in the sequences of other immunoglobulins strongly suggests the same structure occurs generally in VL-VH association. This is in accord with the presence of P-bulges at homologous postions in the edge P-strands. and their highly conserved conformation. The three· layer P·sheet packing thus plays a central .-ole in forming the antibody combining site.

We thank John <.'rei;»well for Figure drawings. and National Institutes of Health and the Royal ~otiety for support.

References

Azuma.. T., Igras. V .. Reilly, E. B. & Eisen. H. (1984). Proc .. Vrit . Acad. Sci .. U.S.A. 81. 6139-6143.

Beale. D. & Feinstein. A. (1976). Q'Uart . Rev. Bio-phys. 9. 135- 180.

Bernstein. F . (' .. Koetzle. T. F .. Williams, G. J.B., :'lleyE-r. E . F .. Brice, ~I. D .. Rodgers. J. R ., Kennard. 0 ., Shimanouchi, T . & Tasumi, M. (1977). J. Mo/.. Biol. 112. 535-542.

Brooks, B .. Bruccoleri. R. E., Olafson, B. D., States, D. J .. :-iwaminathan, S .. & Karplus, M. (1983). J. ('11mput. ( 'h P11t. 4. 187- 217.

Burley.:-;. & Petsko. G. (1985). 8cie11re. 229. 23- 28. l'hothia. ('. (197:3). J . .\fol. Biol. 15. 295-302. ('hothiit. (' . &. .Ja.nin. J . (1975). Nature (London), 256.

705-708. l'hothia. f' . & .Janin. J . (198 1). Proc . .Vat. AMd. Sci ..

l".S . ..t . 78, .Jl4(H.150. C 'hothia, ( '. & Janin. J. ( 1982). Biochemistry. 21, 3955-

3965. C'hothia. <: .. Levitt. :\I. & Richardson. D. (1977). Proc.

.\.(Jf. Amd. Sri .. (l.S.A . 14 , .J13Q-4134. C'hou. K. (' .. Pottle. M .. Kernethv. G., l 1t>da. Y. &

Scheraga. H. (1982). ,J. Mol. Bi~l. 162, 89-112. Cnht-n. F . C., Rtt-rnberg. M. J . E . & Taylor. W.R. (1980).

.\"fJ/UrP. (London). 285. 378- 382.


Packing of Immunoglobulin I 'ariable Domains 663

Cohen. F. r .. Novotny. J .. Sternberg. M. J .E., Campbell. D. G. & Williams. A. F . (1981a). Biocht111. J. 195. 31-40.

Cohen. F. (' .. Sternberg. M. J . E. & Taylor. W. R. (198Ib). J . Mol. Biol. 14'1. 253- 272.

C'ox. E. C., Cruickshank. D. W. J . & Smith . J . A. S. (1958). Proc. Roy. Soc. ser. A 247, 1- 21.

Cunningham, B. A .. Wang. J . J .. Berggard. I. & Peterson. P . .-\ . (1973). Biochemi.,lr!I. 12. -181 1-4816.

Deisenhofer. J . (1981). Biorliemi81tJJ. 20. n 61-2370. Edmundson . .-\. B .. Ely, K . R. .. Abola. E. E .. Srhiffer. lll.

& Panagiotopoulos. N . (1975). Biod1e111i.)try. 14. 3953-3961.

Epp. 0 .. Colman. P.. Fehlhammer. H., Bode. W .. Schiffer. M. & H uber. R. ( 1974). Eur. J. Biochem. 45. 513-5:?.J..

Feinstein, A. (1979) . . Va11m (London). 181 , 230. Goto, Y. & Hama.guchi, K. (1982). J. Mol. Biol. 156, 911-

926. Hedrick, S. lll.. Nielsen. E. A .. Ka,•aler. J .. Cohen. 0 . 1.

& Davis. ) f. )1. (1984) . .\'ature (London). 308. J.l5-149.

Hochman, J .. Inbar. D. & Givol. D. (1973). Biorhm11~1ry. 12. 1130-11 35.

Jensenius. J . C'. & Williams. A. F. (1982). Salim

(London). 300, 583-588. Kabat. E. A .. \\'u, T. T .. Bilofsky. H .. Reid-Milner. ~I . &

Perry, H . (1983) Sequences of Proteins of ! 11111111110·

1-0gical htteresl. Publir Health :-\t-n·it·e. l\ .I.H .. Washington. D.C.

Lee. B. & Richards. F . lll. (1971). J . Mol. Biol. SS . 379-400.

Marquart, M .. Deisenhofer. J . & Huber, R. (1980). J . .llol Biol. 141. 369-391 .

i\fostov. K. E .. Friedlander. lit. & Blobel. G. ( 1984). Nattire (London) , 308. 37-43.

Nockolds. ('. E .. Kretsinger, R. H .. Coffee, C' J . & Bradshaw, R. A. (1975). Proc. Sot . Acad. Sci .. U.S .A. 69. 581-584.

Novotny. J . & .-\uffray. ('. (1984) . .V11r/. Acid Res. 12. 243-255.

Xo\'otn.\·. J . & Franek. F . (1975) . . \'at1m (lo11do11 ). 258. 641 - 043.

Novotn ~· . J . & Haber. E. (1985). Proc. Nat. Acad. Sci .. U.S.A . 82. 4592-4596.

Novotny. J .. Vitek, A. & Franek, F. ( 1977). J. ,\fol. Biol. 113. 711 -718.

Novotny, J .. Bruccoleri. R.. X1:well. J., ~I urph~· . D., Haber, E. & Karplus. )[. (1983). J. Biol. ('hem . 258. 14433- 14437.

Xovotn}•. J .. Bruccoleri. R. & Newell. J . (1984). J . .\fol. Biol. 177, 567-5i3.

Orr. H. T ., Lancet, D .. Robb. R. J .. Lopez de Castro. J. & Strominger, J . L. ( 1979). Natiire ( LondM). 282. 266-270.

Pauling. L .. C'orey. R. E. & Branson. H. R. (1951). Proc. Nat . A rad. Sri .. U.S .A . 37. 205-211.

Phizackerley. R. P .. \\'ishner. B. C .. Br.rnnt. S. H .. Amzel. L. M., Lopez de Castro. J . A. & Poljak. R. J . (1979). J/ol . lmm1mol. 16. 841- 850.

Poljak. R. J .. Amzel. L. M., Chen. B. L .. Phizarkerley. R. P. & Saul, F. (1975). /1111111111oge11elit11. 2. 393- 394.

Richmond. T. J. & Richards, F . M. (1978). J . • llo/. Biol . 119. 537- 555.

Richardson. J . S .. Getzoff. E. D. & Ri<'hardson. D. ('. (1978). Proc . .Vol . ..lead. Sri .. CS.A . 75, 2574-2578.

Richardson. J . :-\. (1981). Adm11. Protein Chem. 34. 167-339.

Saul, F. A .. Amzel. L. lll. &: Poljak. R. J . (1978). J. Biol.

Chem. 2S3. 585-597. Segal. D .. Padlan. E. A., Cohen. G. H .. Rudikoff. :-\ ..

Potter. lll. & Davies, D. (1974). Proc . .\'nt . ...!rod. 81'i .. l'.S.A . 71. 4298-4302.

Schiffer. M., Girling, R. L .. Ely. K. R. & Edmundson. A. B. (1973). Biochemistry. 12. 1620-1631.

Shrake, H. & Rupley, J. A. (1973). J . .llol. Biol. 79. 351-371.

Thomas. K. A. Smith. G. 1\1., Thomas, T. B. & Feldmann. R. J . (1982). Proc. Sat . ..lead. Sci .. l '.S .A . 79. 4843-4847.

Williams, D. E. (1980). Acta ('rystallogr. sect . ..I , 36. 715-723.

\\'y('koff. R. \\' . G. (1969). Jn Crystal Slruc/11res. 2nd edit.. vol.6. part l , pp. 1-2. Wiley. Xt-w York.

Yanagi. \'. , Yoshikai. Y .. Leggett. K .. Clark. KP . Aleksander. I. & Mak, T. \\' (1984). .\'alure (London). 308. 1.J.5- 149.

Edited by R. Huber


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