Rrprint1•d from J . .l/ol. Biol. (1987) 196. !lOl-!)17
`
`38
`
`Canonical Structures for the Hypervariable Regions
`of Immunoglobulins
`
`Cyrus Chothia and Arthur M. Lesk
`
`1 of 18
`
`BI Exhibit 1062
`
`

`

`J. Jin/ Biol. (1987) 196. 90I-91i
`
`Canonical Structures for the Hypervariable Regions
`of Immunoglobulins
`Cyrus Chothia 1 •2 and Arthur M. Lesk 1.3t
`1 11/ RC La-Ooratory of Molecular Biology
`Hills Road. Cambridge CB2 2QH
`England
`
`2Ghristopher h1gold Laboratory
`l "niversity College Lo1ulon
`20 Gordon St-reet
`London WCJl-f OAJ, England
`3 EM BL Biocom pa ting Programme
`llfeyerhofstr. 1, Postfach 1022.09
`D-6.900 Heidelberg
`Federal Republic of Germany
`
`(Received 13 November 1986, and in rn•ised form �.1 April 1987)
`
`\\'e have analysed the atomic structures of Fab and \"L fragmrnt-s of immunoglobulins to
`determine the relationship between th�i1· amino aC'id sequences and t.h<' three·dimeni;ional
`structures of their antigen binding .:it-t-s. \\'e identif�· the relatively few residues that,
`through their packing, hydrogen bonding or the abilit�· to assume unusual </J. if! or w
`conformations, arc primarily responsible
`for the main-chain conformations of the
`hypervariable regions. These residues are found to oc·<'nr at sitt'>1 within the hypervariable
`regions and in the conserved /J-sheet. framework.
`Examination of the sequences of immunoglobulins of unknown structur<' shows that
`many have hypervariable regions I hat are similar in siZ(' to one of the known ;;t ruc·l un·:< and
`contain identical residues at the sitrs responsible for the observed c·onformation. 'f'his
`implies that these hyperva.riable regions ha.ve «onformations close t,o those in the known
`structures. For five of the hypervariable regions, the l'f'J>erloire of conformations appears to
`be limited to a relath·e]y :-:mall number of discn·t.e st rurtural da;:;;es. \\'c· call the c·ommonly
`ON·nrring ma.in-chain conformations of the hypervariab)e regions "1•anonical st.ructurc•s".
`is being test eel and refined
`The accuracy of the analysis
`l1y the predid i1111 of
`immunoglobulin struc·tures prior to their experimental determination.
`
`1. Introduction
`The specificity of immunoglobulins is detnrnined
`by th" sequenl't' and size of the hypt-n·ar1able
`regions in the variable domai11s. These regions
`produce a surface complementary to that of the
`antigen. The subject of this paper is the relation
`lit>lwt·1·n the amino acid sequerwrs of antibodies and
`the strul'ture of their binding ;;itt>>1. The results we
`:wts of
`report are related to
`two prf'vious
`ohsrrvations.
`
`�ean\'tk· Ha<·kensack Campu3, Teanf'1·k. :\.J 07666.
`t Also associated with Fairleigh Dickinson l'nivf'rxit.v.
`lXA.
`
`fir:-1 set com·ernl< the sequenct>s of the
`The
`hypervariable regions.
`l\aliat and his colleagul's
`(Kahat ti nl .. 1977: Kabat, 1978) rompared lh1·
`s('qnrn1:es oft lw h.vpernviable re�ions then known
`and found t.hat. at 1 3 sitex in the light. thain:-.
`and at >lf'\'E'll positions in t ht> ht'a".'' l"11ain,.;. the
`rt·sidue!-l are consen-rrl.
`1'h1·y argnecl th1\t the
`resid11C's at Hwst' i;it1·s art' invoked in I he st.ructure.
`rat hl'r than t hl' spl.'1:ificity. of tht> hyp1·rrnrialtlt·
`regions. 'I'lw.v suggested that lht'.'!' n>.,id111·s ban· a
`fo.:Pcl position in antibndic•:-: and that thi:-: 1·01dd hr
`used in tht· modrl building of combining sites to
`limit I he conformal ions and positions of 1 h1· silt':-.
`whose residut>' var·it·d. Padla.n (1979) also 1·xamitwd
`the sl'querwes of the hypervariable n·gion of light
`901
`
`2 of 18
`
`BI Exhibit 1062
`
`

`

`902
`
`C. Chothia nm/ A. M. Lesk
`
`variable domain of tl1e light chain (Vdt they list
`some 200 complete and 400 partial sequences; for
`the variable domain of the heavy chain (VH) they
`list about 130 complete and 200 partial sequences
`In this paper we use the residue numbering of
`Kabat et al. ( 1983), except in the few instance�
`structural
`superposition of certain
`where the
`hypervariable regions gives an alignment different.
`from that suggested by the sequence comparisons.
`In Table l we list the immunoglobulins of known
`structure
`for which atomic co-ordinates
`are
`available from t,he Protein Data Bank (Bernstein et
`al., 1977), and give the references to the crystallo­
`graphic analyses. Amzel & Poljak (1979), Marquart
`& Deisenhofer ( 1982) and Davies & Metzger (1983)
`have written reviews of the molecular structure of
`immunoglobulins.
`The \'L and VH domains have homologous
`references, Ree Table l ). Each
`structures
`(for
`contains two large fl-pleated sheets that pack face
`to face with their main chains about 10 A apart
`(l A = O· I nm) and inclined at an angle of -30°
`(Fig. I). The P-sheets of each domain are linked by
`a conserved disulphide bridge. The antibody
`binding site is formed by the six hyperl'ariable
`regions; three in VL and three in \'H. These regions
`link strands of the P-sheets. Two link strands that
`are in different {J-sheets. The other four are hair-pin
`turns: peptides that link two adjacent strands in
`the same P-sheet (Fig. 2). Sibanda & Thornton
`(1985) and Efimov (1986) have described how the
`conformations of small and medium-sized hair-pin
`turns depend primarily on the length and sequence
`of the turn. Thornton et al. (1985) pointed out that
`the sequence-conformation rules for hair-pin turns
`can be used for modelling antibody combining sites.
`The results of these authors and our own
`unpublished work on the conformations of hair-pin
`turns, are summarized in Table 2.
`
`chains. He found that residues that are part of the
`h.YJ>l"'rvariable regions. and that are buried within
`the domains in the known structures. are conserved.
`The residues he found conserved in V,i sequenl"e�
`\\"ere different to those conserved in VK �t>quen<"t'S.
`The i<t>1·ond ,:;t:>t· of observations concerns the
`conformation of the hypervariable regions. The
`results of tht· structure analysis of Fab and Bence·
`Jones prot\>ins (Saul l't al .. 1978; Segal ''/ al., 197+:
`�larquart et al .. 1980; Suh et n.l., 1986; !-ichiffer et al ..
`1973: Epp et al., 1975; Fehlhammer et al .. 1975:
`Colman el rd .. 1977; Furey el al., 1983) show that in
`several cases hypervariable regions of the same size,
`but with different sequences, have the same main­
`chain conformation (Pa<llan & Davies, 1975;
`Fehlhamnwr et al .• 1975; Padlan et al., 1977;
`Padlan, 1977b; Colman et al .. 1977; de la Paz et a.I ..
`1986). Details of these observations are given
`below.
`In this paper. from an analysis of the immuno­
`globulins of known atomic stru<:ture we determine
`the limits of the P-sheet framework common t.o the
`known st.ructures (see section 3 below). We then
`identify the relative!.'· few residues that, through
`packing, hydrogen bonding or the ability to assume
`unusual </>. I/! or w conformations, are primarily
`responsible
`for
`the main-chain conformations
`observed in the hypervariable regions (see sections 4
`to 9, below). These residues are found to occur at
`sites within the hypervariable regions and in the
`conserved 8-sheet framework. Some correspond to
`residues identified by Kabat r:t al. (1977) and by
`Padlan (Padlan et al .. 1977; Padlan. 1979) as being
`important for determining l,he conformation of
`hypervariable regions.
`immuno­
`Examination of t.he sequences of
`glohulins of unkno\\"n structure shows that in manv
`('asE's the �t>I of residues responsible for one of th.e
`ohserved hypt>rni.riablf' conformations is present.
`This suggests that most of the hypen·ariable
`regions in immunoglobulins han? one of a small
`3. The Conserved �Sheet Framework
`disl"rete set of main-chain conformations that wf'
`imm unoglobulin
`<'all ··canoniC'al st rudme>s '' . Sequence variations at
`Comparisons
`of
`the first
`structures determined showed that. t.he fram1>work
`the silt's not responsible for the ('()nformation of a
`«anonical stru<'lure will modulate the
`partic·ular
`surfa(·t· 1 hat it. prest>nls to an antigen.
`regions of different moleC'ules are very similar
`t A bbrf'viatiom:: used: \'1. and \'H, variahle
`Pri0>· tu thi� anal)·sis, alt.empts to model thf'
`regions of
`thE' immunoglobulin light and hea''.'' c'hains.
`<"Ombining sites of antihodit>>' of unknown strm·ture
`COR,
`ha\'t> bt·t>n ha>w<I on tlw assumption that hyper­
`re�pl"'l'!in•l
`.Y: r.m.:< .. root·mean-:>quare;
`1·omplementarity·df.'tf.'rmining region.
`,·ariable n·gions of the same si:1a· have similar
`ba!·kbone �I rutturt>s (sf."e st>(·tion I:.!. ht'ln\\"). :\,; \\"€'
`show bf:'low. and a� has bt>t·n n·;ilir,ed in part be.fore.
`(·t>rtain insta.ru·es. :\lodelling
`this is t.r11t> only in
`based on the ,.;<·ts of rl'�idtws idf'ntified here a:<
`respon::;ihle
`for th\· olisl'rn·d ('Onformations of
`
`
`hyp1·/'\'arfablc regions ll'ould be 1·xµel·kd to giYl'
`mort• acn1rat<· rPsults.
`
`2. Immunoglobulin Sequences and Structures
`Kahal et 11/. (1983) ha,·p publish!:'<! a mllrr·tion of
`the known
`immunoglobulin seque>H't>.�. For the
`
`11
`l TTI
`Ill
`
`Table 1
`I 1111111111oglob11/i11 mriable domain,s of k1101c11
`a.tomic structure
`('hain
`T�·pe
`l'r(llt>i11
`L
`H
`l'alo'XE\nl ,.\I
`�'ah )J('fl('fiO:i "
`�·11h Kill.
`,!J
`�'ab .Jr;:l!I
`"
`\'L }{)<;}
`"
`'"- RHJ<:
`J.l
`
`Refertnce
`Saol el 11/. (1978)
`p/ rt/. (1974)
`�t'j!lll
`Marquart el al. (198\ll
`Soh el al. (J!lSli)
`Epp el,,/, (1975)
`Fort')' el al. ( Hl83)
`
`3 of 18
`
`BI Exhibit 1062
`
`

`

`The Structure of Hypervariable Regions
`
`L2
`f
`
`903
`
`N
`
`c
`
`N
`
`Figure 2. A drawing of the arrangement. of the
`hypervariable regions in immunoglobulin binding site:;.
`The squares indicate the position of residues at the ends
`of the /J-sheet strands in the framework regions.
`
`residues
`framework of 79
`common /J-sheet
`(Fig. 3(b)). For different pairs of VH domains the
`r.m.s. difference in the position of the main-chain
`atoms is between 0·64 and l ·-1.:l A.
`The combined P-sheet framework consists of \"L
`residues-!. to 6, 9 to 13, 19 to 25. 33 to -1-9, 53 to 55,
`61 to 76, 84 to 90, 97 to 107 and VH residues 3 to I:?,
`17 to 25, 33 to 52, 56 to 60 , 68 to S:!. 88 to 95 and
`102 to 112. A fit of the main-chain atoms of these
`156 residues in the four known Fab structures gives
`r.m.s. differences in atomic positions of main-chain
`atoms of:
`
`KOL
`:-:EWM
`�kP('603
`
`� F,\\"i\I
`
`l·39A
`
`McPC603
`
`,J539
`
`1·15 A
`1·47 A
`
`1·14 A
`1·37 A
`t·03 A
`
`The major determinants of the tertia,..r stn1ttun·
`of the framework are the residues buried within and
`between the domains. We calculated the ac:«essible
`surface area (Lee & Richards, 1971 ) of each residue
`jn the Fab and VL structures. In Table .i we list the
`residues commonly buried within the \'L and \'H
`domains and in the interface between them. These
`are essentially the same as those identified by
`Padlan (1977a) as buried within the then known
`structures and conserved
`in the then known
`sequences. Examination of the :'WO to 700 \'1..
`sequences and 130 to 300 VH sequences in the
`Tables of Kabat et al. (1983) shows that in nearly
`a.II the sequences listed there the residues at these
`positions are identical with, or very similar to. \host'
`in the known structures.
`There are two positions in the \'._ :·wqut-nces at
`which the nature of the conserved residues depends
`on the chain class. Tn VA sequences, the residues at
`positions 7 l and 90 are usually Ala and l:ier/ Ala,
`respectively; in V� sequences the correllponding
`residues are usually Tyr/Phe and Gln/Asn. These
`residues make conta<'I with t lw hypervariable loops
`and play a role in determining the conformation of
`
`Figure 1. The structure of an immunoglobulin V
`domain. The drawing is of KOL Vv Strands of .8-sheet are
`represented by ribbons. The three hypervariable regions
`are labelled LI, L2 and L3. L2 and L3 are hairpin loops
`that link adjacent P-sheet strands. Ll links two strands
`that are part of different .8-sheets. The VH domains and
`their hypervariable regions. H l, H2 and H3, have
`homologous structures. The domain is viewed from the P­
`sheet that forms the Vt. -VH interface. The arrangement of
`the 6 hypervariable regions that form the antibody
`binding site is shown in Figure 2.
`
`(Padlan & Davies, 1975). The structural similarities
`of the frameworks of the variable domains were
`seen as arising from the tendency of residues that
`form the interiors of the domains to be conserved,
`and from the conservation of the total volume of
`the interior residues (Padlan,
`l977a, 1979). In
`atldition, the residues that form the central region
`of the interface between Vi.. and VH domains were
`observed to be strongly conserved (Poljak et al.,
`1975; Padlan, l977b) and to pack with very similar
`geometries (Chothia et al., 1985).
`In this section we define and describe the exact
`�xt.ent of the structurally similar framework regions
`in the known Fab and VL structures. This was
`de�rmined by optimally superposing the main­
`chain atoms of the known structures (Table 1) and
`calculating the differences in position of at-Oms in
`homologous residuest.
`In Figure 3(a) we give a plan of the /J-sheet
`�ramework that, on the basis of the superpositions,
`is �ommon to all six VL structures. It contains 69
`res1.dues. The r.m.s. difference in the position of the
`main-chain atoms of these residues is small for all
`pairs of VL domains; the values vary between 0·50
`and 1·61 A(Table3A). The four VH domains share a
`t For these and other calculations we used a program
`system written by one of us (see Lesk, 1986).
`
`4 of 18
`
`BI Exhibit 1062
`
`

`

`904
`
`f'. Chothia and A. M. Lesk
`
`�tru\"lur't'
`
`Sequence'
`
`I :! 3 .j
`:-. c:. <:- x
`
`:! -3 I
`1:::-1
`
`x. (:. x. x
`
`X-X- (:. X
`x.x.x.x
`
`X-X·X·G
`
`Table 2
`('1111f11r111ritio11 of hair-pin 11lrnS
`Conformationb
`(.)
`iP•) 1/12 </12. t/13
`+55 +35 +85 _5d
`�.
`or
`+65 -12.'i -105 +10'
`+711 -115 -90 0'
`+50 +45 +85 -20•
`+60 +20 +85 +:?;;'
`iP2 t/12 </13 t/13 </14 t/14
`I/II
`iPl
`-50 -35 -95 -10 +145 + l51i
`-135 + 175
`iP3 t/13 tP4 t/14 </15 1/15
`</12 t/12
`-95 -50 -105 0 +85 -160 3/3
`-75 -10
`+65 -50 -130 -� -90 +130
`+;;o +55
`
`Frequency'
`
`6/6
`
`6/7
`7/8
`41-I
`4/4
`
`1/1(3/3)
`
`13/15
`
`3/3
`'!/:!
`1/1
`
`and X-G·X·X·
`
`I 2 3 .j 5•
`3 :?/ "-.i
`x x x x (;
`I I
`1-- --
`x x x x x
`___ .,
`/3'-.
`,P:? 1/12
`2 3 4 5•
`.j
`:!
`</13 t/13 </14 1/14
`l /I
`(;
`x X·X X· X -60 -:!.';
`-90 0 +85 +JO
`D
`·�--5
`:! 3 .j 5 6' iP2 t/I:?
`:!- .j
`iP3 1/13 </14 1/14 tP5 1/15
`t:
`I I
`x x x.x x. x -65 -30
`-65 -45 -95 -5 +70 +35
`:! 5
`x
`I I
`1:::6
`The data in thi• TahlP arP. from Rn nnpuhlish�d anlllyois of proteins whose atomic atrut•t ur .. has been
`determined at a resolution of 2 A or higher. The conformations dl"S<·ribed here for the 2-residue X-X·
`X-1: turn and the 3-residue turns are new. The other conformations have �n described by Sibanda &.
`Thornton (1!111.;1 and by Efimo,· (1986). \\"e list only conformations found more than once.
`• X indic·,11�� no residu.- ..-�tri .. tiuu exct'pt that certain sites cannot ha,·p Pro. M this �idue requires
`a 4> v11lue of - -w and cannot form a hydrogen bond to its main-chain nitrogen.
`• Residu�s whose </I.I/I valuPS are not given havt' a P conformation.
`' �·requencies are given as 111/n,. wlwrt- 112 is the number of cases where we found the structure in
`column I with the ..equence in column 2 and n1 the number of these cases that have the conformation
`in t'Olumn 3. r.., .... pl for the frequenc·iE>• in bra<'ket.'!. data is given only for non-homologous proteins.
`d.•.r Tht·>i· nrc• typP I'. 11' and Ill' turn�.
`1 Different t'Onformations are found for the single �ases of X-D-G-X-X and X-C·X·G-X.
`h Differl!nt conformations are found for the single cases of X-X-X-X -X. X.(;.(:.);.X
`(;.The:!\·"""" of X-X-X-\.\. have different <·onformations.
`' Different l'onformations are found for the:! cases of X·G·X-X-X-X.
`34, 50 to 56 and 89 to 97 in \"Land 31 to 35. 50 �o
`these loops. Thi:< is discussed in sec-t ion:< 5 and 7,
`65 and 95 to 102 in \"H· This point is discussed m
`lit' low.
`The conservation of
`lhf> framework :<I ru<'l ure
`sec-t ion 11, below.
`t-xtends In the residues immediately adjan·nt lo the
`hypervariable region:<. lf thr l'"""l'n·t'cl franH'works
`4. Conformation of the L1
`of a pair of molecules are
`superposed, the
`differences in t h1· fJt>:<it ion:< of 1 lws<' t«·:<idm's is in
`Hypervariable Regions
`mn:<t c·1\s1·:< le:<s than 1 .l. and in all but one case l!'ss
`In t.he known \ · structures thC' conformations of
`I he LI regions, re�idues 26 to' 32, are char�cterist�c
`<·ontrnst. residut's in tht•
`than 1·8 . .\ (Tablt>5). Jn
`hyperva.riablc region adjaecnt to the eonsern�d
`of the <-lass of tlw light. chain. Jn V, domat�s t�e�r
`conform a I ion is helical and in t he \". domains it IS
`fram(·\\·ork <·an differ in posi ti on !.>· 3 .. \ or more.
`The six loops. whose main-c·hain conformation�
`extended (Padlan et al .. 1977; Padlan. 1977b; de la
`,·ary and whi<·h an· part of the antil11ul>· c·ombinin).(
`Paz et rtl .. 1986). These conformational differences
`:<it ... are formed l1y r1·.;i1hu·-. :!Ii to :l:! .. )0 to .i:! and
`are the result of sequence differences in b�t h the LI
`region and the framework (Lesk & Chot h1a, 1982).
`!ll to !Iii in \"L <lomainll, and 26 to:{:!. 53 to 1)5 and
`96 to 101 in lh f' \'H domains LI, L:!. L3. HI. H:? and
`l imi t:< <1n· ,.:nnwwha t
`H3,
`respeC'tin·l,L Tht>il'
`,._
`(a) VA do111ai11.<
`difft-rt-nt from
`tho:-1·
`11f
`I he <'omplt>mt-ntarit
`ciE>t er minin� reJZions defitwd li.y Kil hat ,,, rd. ( J !}8°3)
`Fig ure + shows the c-onformation of the LI
`
`011 the hasts of sequc•nc·<- \';Hinbility: re sid1tt's :!..t to
`rrgions of the \"., domains. The LI regions in RHE
`
`5 of 18
`
`BI Exhibit 1062
`
`

`

`The Str11c/11re of II Y/ll'l'l"flfiah/1' Regions
`
`905
`
`76
`
`61
`
`19
`
`18
`
`82
`
`68
`
`----·
`
`C-----
`
`6
`
`4
`
`-----c
`
`9
`
`53
`
`55
`
`C-----
`-----
`
`7
`
`3
`
`w----­
`�::::
`
`H2
`
`60
`
`84
`
`, ,
`
`, .. '"'
`13 '
`
`',_
`
`107
`
`8
`
`12
`
`,,''
`,,,
`' ' -,
`......
`
`112
`Figure 3. Plane of the tl·sheet framework that is conserved in the \"L and \'H domains of the immunoglobulins of
`mown atomic structure.
`
`and KOL contain nine residues designated 26 to 30.
`30a, 30b, 31 to 32; �E\\'�1 has one additional
`residue. The LI regions in RHE and KOL have the
`3ame conformation: their main-chain atoms have a
`r.m.s. difference in position of 0·28 A. Superposition
`of the LI region of �EWM with those of KOL and
`RHE shows that the additional residue is inserted
`between residues 30b and 31 and has little effect on
`the conformation of the rest of the region:
`superpositions of the main-chain a.toms of 26 to 30b
`and 31 to 32 in NEWM to 26 to 32 in KOL and
`RHE give r.m.s. differences in position of 0·96 A
`and 1·�5 A. Thus, the sequence alignment for the Vl
`Ll regions of KOL, RHE and NEWM implied by
`the structural superposition is:
`
`30 :Joa 30b
`
`:111,. 31
`:17
`:!(;
`:1:!
`28 29
`Position
`A�n
`Sn Ala Thr Asp Ile <:iv �r
`RHE
`:-;.,r
`Thr Ser &>r Asn lie c(,· S.-r
`KOL
`lie Thr
`:>IE\\'�I �r S.-r Ser A•n lie (;I�· Ala l:ly Asn His
`Tn all three structures, residues :?Ii to 29 form a
`t�·pe J turn with a hydrogen bond between the
`carbonyl of 26 and the amide of :?!1. Residues 27 to
`30b form an irregular helix (Fig. 4). This helix :-.it:<
`across the top of the /J-sheei core. The side-cha.in of
`residue 30 penetrates deep into the core occupying a
`cavity between residues 25. 33 and 71. The major
`determinant of the conformation of LI
`in the
`observed structures is the packing of residues :!5.
`30, 33 and 71. V;. RHE, KOL and NEWM. have the
`
`6 of 18
`
`BI Exhibit 1062
`
`

`

`906
`
`( '. Chothia and A. M. Lesk
`
`Table 3
`/JijJ,.tt·11r•'·' in i1111111111oylril111li11 frflllll'trork
`.. 11rnrl11 rP.� ( . .t)
`For l'•""' of \" domains '"� give. the r:m.s. difference in. thl'
`atomic ('1Qsitio11' of frame\\·ork mam chain atoms aftpr optimal
`:-.Ultt'f1Ht"'lti11f\
`:\ l't. tfomn;uli
`Framework residues al'I' 4 tu 6, !I tu 13, 19 to:?:.. 33 lo 4!l. 53 to
`:;:;. 61 tu 7ti. 114 to !10 and 97 to I07.
`)\t"Prno:i
`J!;39
`KOL ;l;E\01 REI
`1·41
`1·61
`1·46
`1·47
`()·74
`1·36
`l·:.!3
`l ·13
`1-1:.
`1 :!-1
`1·28
`1·53
`()·77
`0·50
`0·76
`
`RHE
`l\llL
`:'\E\01
`REI
`)(('('( 'Oll:l
`B. J 'u d1m111hl"'
`Framework residues art> 310 l:!. 17 to :!5. 33 to 52, 56 to 60. 68
`to X:!. i;s to 95 and Ill:! lo I I:!
`:'\F.\DI M('PC'603
`IHj,.j
`H:!
`1·27
`
`KOL
`:'\E\Dl
`)lt'l't ·1�1:1
`
`.J539
`
`0·89
`1·29
`0·89
`
`for which the sequences of the Ll regi ons are
`known. The 21 st-quences in subgroups I, II, V and
`\. f ha ,.e L I regions that are the same length as
`those found in RHE, KOL or NEWl\1. Of these, 18
`conserve the residues responsible for the observed
`conformations:
`
`Residue
`position
`25
`30
`33
`71
`2!!
`
`Residue in
`KOL/RHE/NEWM
`<:1,,·
`lit
`Val
`Ala
`Asp/Asn
`
`R�iduesin
`18 \', sequences
`I>!( ;r,.
`17 V�l. l llt
`17 \"al. I lie
`18Ala
`11 Asp. 6 Asn, I Ser
`
`The ronservation of these residues implies that
`these 18 L I regions have a conformation that is the
`same as that in RH E, KOL or NEWM.
`Subgroups TII and I\' have 13 sequences for
`which the LI regions are known (Kabat et al.,
`1983). These regions are shorter than those in RHE
`and KOL and in the other V, subgroups. They also
`have a quite different pattern of conserved residues.
`Kabat f:I al. ( 1983) listed 29 mouse \·.domains for
`which the sequence of the Ll region is known.
`These L l regi ons are the same size as that in
`X F:\ DI. They also have a pattern of residue
`<:on:sen·alion similar to, but not identical with, that
`in KOl./XEWM: Ser at position 25, Val at 30, Ala
`at 33 and Ala at 71. This suggests that the fold of
`
`,..amt' residues at these sitE>s: C:ld5. Ile30, Val33 and
`·
`Ala71. (Another LI residue,
`Asp29 or Asn29. is
`buried hy the conliwts it makes with L3.)
`Kabat el al. (1983) listed 33 human \'1 domains
`Table 4
`Rl'.sirlue.< ro11111uml,11 b11rin/ 11•itlti11
`
`\"L domains
`
`Residues in
`known
`strudures
`
`Position
`4
`Ii
`1!\
`:!:I
`:!I
`:!."}
`:$:!
`:1.;
`37
`47
`411
`ti:?
`64
`71
`73
`;;)
`-<'
`10
`86
`'<><
`!)tJ
`!17
`!l!I
`1111
`Ill:!
`10�
`
`Position
`"
`6
`18
`20
`.,.�
`:!of
`:1-t
`3G
`38
`48
`411
`51
`69
`n1
`XO
`>t?
`86
`88
`lNI
`II:.!
`10�
`106
`107
`10!)
`
`l'L and l'n domains
`r., domains
`Residue� in
`A.k . ..\.0
`A.S.A.'
`known
`cA1l
`(,\2)
`stru�ture�
`14
`L
`6
`L.M
`16
`Q
`Q.f:
`I:!
`,.
`L
`:!I
`II
`I.�I
`I
`L
`0
`ti
`('
`('
`0
`1:.A.X
`s
`13
`S.\'.T.A
`4
`�l.Y
`3
`\".L
`\\"
`\\"
`0
`ll
`13
`30
`Q
`s
`R
`L.l. \\"
`I
`I,\·
`I
`2-t
`0
`A.G
`�-
`l.\".S
`4
`11
`l.\'.M
`t; A
`13
`13
`.\.F.Y
`2
`0
`L.F
`L.F
`0
`0
`L
`ti
`0
`M.L
`I.\'
`2
`D
`�
`D
`11
`3
`A.!'
`fl
`A(;
`y
`0
`\"
`('
`ti
`0
`l'
`A S.(J.:'\
`I
`II
`1;
`\".T.1:
`(;
`18
`19
`c;
`T.S
`17
`3
`2
`,.
`c:
`II
`T
`I
`L.\·
`:!
`. • )li·an ac���s;;ible .�urfaC'e. area (A.X .• \.) of lht> ro«idu .. s i11 th� 1''1ib �truC'tures �EWM, MCPC603.
`1,01, and .J.1. U and 111 the\ L 'tru<·tures REI and HHE.
`
`7 of 18
`
`BI Exhibit 1062
`
`

`

`the mouse \'� Ll regions is a distorted version of
`that found in tht> known human structures.
`
`:!fj
`
`Residue
`J539
`REI
`MCPC-1m:1
`
`3la. 3Jb
`
`:-\l"I'
`
`f:ly
`
`(b) 1·. domains
`In Figure ,-, we illust rat-e the 1·onformation of the
`LI regions in the three known \.K structures: J539.
`REI and \l('PC603. In J539 L1 has six residuE>s. in
`RET it has seven and in MCPC603 13. The LI
`region of J539 has an t>xtended l'Onformation. In
`REI,
`residues 26 to 28 have an extended
`conformation and 29 to 3� form a distorted type IT
`turn. The six additional residues in MCPC'l>ll:3 all
`occur in the region of this turn (Fig. 5). Tn the t.hree
`;;tnu·turt':> the main chain of residues :W to 29 and
`:3:! haw the same conformation. A fit of the main·
`('hain atoms of these residues in J539. REI and
`�ICPC603 give:; r.m.s. differences in position of 0-4 7
`to I ·03 A. The sequenc-e alignment implied b�· the
`�t rul"lural superposition is:
`:Jo
`27
`28
`29
`31
`�(.'!'
`\'al
`Ser
`:-\c-r
`Ser
`[le
`Ly•
`Set Olu Asp
`lie
`Ser Ulu Ser Leu Leu Asn
`In J539, REI and MCP(.'603. residues 26 to :rn
`extend across the top of /J-sheet framework with
`one, 29. buried within it. The main contacts of 29
`are with residues 2. 25. 33 and 71. The penetration
`of residue 29 into the interiOI' of the framework is
`not as great as that of residue 30 in the Y1 domains,
`and the deep c-avity that exists in \"1 domains is
`filled in V" domains hy tht> large side-chain of the
`i·esirlue at position 71. In J539. REI and :\lCPC603,
`the residues involved in the packing of L l (2. ::!fl.
`:!\!. 33 a.nd 71) are very similar: Ile. Ala/Ser. \·al/
`Ile/teu, Leu and Tyr/Phe, r·espeet.i\·t>ly.
`The six residues 30 to 30f in :'ltCPC603 form a
`hair-pin loop that extends a wa>· from the domain
`(Fig. 5) and does not have a well-ordered conforma·
`tion (fiegal "' al., 197�)-
`Kabat et al. (1977) noted that residues at c-ertain
`position!' in the Ll regions of the V" ,.;equt>rH·f'>< then
`known were conserved, and suggested that they
`havt' a structural role. The structural role of
`residues at positions 25. 29 and 33 is confirmed by
`the above analysis of the \"" structures and t,he
`pattern of residue conservation in t.he muc:h larger
`,,, al.
`number of sequences known now. Kabat
`(1983) listed 65 human and llH- mou�e \'" sequenc·e><
`for which the residues between posit.ions 2 and 33
`are known. For about half of these. the residue at
`position 71 is also known. Thes\-' data ><how that
`there are 59 human and l-l-8 mouse i<equt>nt·e:< that
`ha,·t.' residues ,-en· similar to thosl' in the known
`'
`stnwtures at the
`site:-; involved in the packing of
`LI:
`Poi;ition
`2
`�;}
`29
`33
`71
`
`. J.�39/REl/�lf 'l'f ·1;u:i
`
`Ile
`Ala Ser
`Val Ile Leu
`Leu
`Tyr Phe
`
`Human\" •
`;,7 lie. I Met. 1 \'al
`!'d Ala, 7 Ser
`ao Ile. ti \'al. x Leu
`;;; Leu. t \"al
`:.'X Phe. I Tyr
`
`The 8tr11rf11re of Hypen•oriable Heyions
`
`907
`The numher of residue,:; in the LI region m these
`
`:<(·quen1T1' ,-aries:
`
`Hesi<lut- size of LI
`�umber of human r.
`:-;umber of mouse \"•
`
`7
`fi
`:lx
`I I 40
`
`9 JU
`
`8
`14
`
`1:3
`
`11
`
`It
`:!
`I
`�
`:�:! 31i 30
`
`The conservation of r<'sidut>>< at the positions buried
`between LI and tlw framework implies thal in the
`large majority of\"" domains rt>sidur:-: :W l o 29 han·
`a conformation clos€" to that found in the known
`struct.ures and tha.t the remaining residues. if small
`in number, form a turn or, if large. <.I hair-pin loop.
`
`5. Conformation of the L2
`Hypervariable Regions
`The L2 regions have the same conformation in
`the known strndurt>s (Padlan Pl al., 1977; Padla.n.
`:Jl e
`31d
`31f
`3:? Ser
`
`31c·
`
`Tyr
`.. �\:-.:11 Glu Lys Asn Phe
`1977/J: de la Paz ,,, rd .• 1986) expect for .'.\E\nl.
`\1·here it i:< deleted. v\'p find that the similaritil'" in
`the L2 structures arise from the conformat.ional
`requiremE-nts of 1:1 three-residue turn and
`tht·
`c-onservation of the framework residues <lgainst
`which L2 pack:<.
`The know structures L2 consists of three n·,.;irlue,.;.
`50 to n2:
`
`ResiduP
`:;o
`51
`
`!):!
`
`RHE
`'l\·r
`.\:<II
`
`Asp
`
`KOi.
`
`Arg
`.\�1·
`Ala
`
`Rf<:!
`
`�H 'PC603
`
`.Ja:m
`Ulu
`lie
`Ser
`
`<a"
`Olu
`� ... r
`S�r
`Al;.
`.\la
`These three rt>sidue·s link two adjal"ent ,.;!.rands in
`the framework P·><lwl'I. Residues 49 and f>:J an ..
`h�·drogen bonded to each othel' ,.;o that the L2
`region is a three-residue hair-pin turn (Fig. 6).
`
`51 / ""'
`50
`52
`I
`I
`49= = =53
`The c'onformations of L2 in tht- fi,-f' stru<'turt-:-< art'
`ver,v similar: r.m.�. differenc:t.'i< in position of their
`main·thain atom!> are lwt·wt>t>n 0· 1 and 0-97 A. 1'ht>
`only difference among the conformations i" in the
`orientaticm of the peptide bt-twet'n residues i'ill and
`51. In W'PC603 this difference is associated with
`the Gly re:-;ichw at po,.;ition 50. The >1ide-<:hain>< of L:2
`all point towards the surfac·e. The main-chain pac·k,.;
`
`1:34 lie. 14 \"al
`1(14 .\la. 4 �r
`Ml Leu .. ii \"al. :38 llt-
`$14 Lo·u. 44 �It-I, i \"al. 3 llt­
`!H Plw. �Ii 1\r
`
`8 of 18
`
`BI Exhibit 1062
`
`

`

`908
`
`26
`
`I'. ( '/lfJlhin nnd A . • 1/. /,Psk
`
`32
`
`Hypervariable
`region
`LI
`L2
`1,,3
`H I
`H:!
`H:l
`
`Table 5
`DifferenrP.� in the positions of the framework rP.,idiua
`'"�jfm• 11 t to the hypervariable regions i 11
`i 111 m·u.noglobul in structures
`Differences in
`A<lja<·1>11t framework
`re<iduc·•
`position (.\}
`fl·:!+I
`0·3--0·5
`0·8-1·0
`
`:?!;
`-1!1
`!)()
`:,?;,
`52
`95
`
`33
`53
`97
`33
`56
`ltl�
`
`O<H).S
`I�� J.4
`�8-1-2
`0.3-1·�
`l·�-1·7
`II·� J.i
`
`fl·a 1·2
`
`0·8-:H
`0·5-1·2
`
`KOL LI
`Figure 4. The C'onformation of the �I regi
`of \'A
`on
`.
`.
`framt-work struC'ture: st'\' seC'tion �.
`l\:OL. The side-chain of lle30 is buried w1thm the
`
`against the consPrved framework residues llf'4 i and
`Gly6+/Ala6.t (Fig. 6).
`Kabat "' al. (I 983) gi,·<· t ht· se4uenn•)< of the L:!
`regions of I 74 \'L domains. l n all cases t lw.\' are
`three residues in length. Of the l 7-i. 122 do not
`residue at position :)I). The residllPl< at position 48
`and 64 are almost ah:<oh1tf>ly conserved as I le an<l
`Gh-. These size and sequenN' identities imply that
`:tl.:Oost all L:? regions ha,•e a conformation close to
`that found in the known structures.
`
`contain (:ly and 49 have. like :\ICPC603, a Cly
`
`6. Conformation of the L3
`Hypervariable Regions
`The L3 region, residues 91 to 96. forms the link
`11c·lwf>c·n two adjac:ent :<trands of P-sheet. Our
`anah·;;i,. of the strurt ure), and sequen<'e" known for
`this · region suggests that the large majority of •
`chains have a common conformation that is quite
`different from the conformations found in J. chains.
`
`(a) l'A domains
`The L3 region of \'; � E\DI has six rt'><idues and
`those of KOL and R.11 E ha.1·c· t'ight. :-i111K·rim�iLion
`of the th ree regions gil't's th<• following alignment:
`
`�E\\'�I
`KOL
`RHE
`
`91
`92
`93
`Tvr Asp Arg
`T;.p Asn St'r
`Trp Asn ..\'I'
`
`93a
`
`93b 9-1 95 96
`
`. .\�n $er Trr
`Leu A'l?
`Ser
`Ser Asp
`Ser Leu ..\>p
`C:lu Pio
`
`32
`
`J539
`
`MCPC 603
`REI
`Figure S. The C'Onforma111111 of the LI regions of \', ;\l('P<'603. \'• REI and v. J539. Residues 26 to 29 and 32 hav�:
`:<ame c·onformation i n the 3 strudurt>s. Tht' sidf'-C'hain o f residue :!() is buried within the framework structure,
`,,. .. 1·tion -L
`
`9 of 18
`
`BI Exhibit 1062
`
`

`

`The SfrucfurP of llyprn·ari((b/e Region.,
`
`909
`
`52
`
`KOL L2
`Figure 6. The C'Onformation of thP L2 region of \'�
`KOL. This region packs against framework residues Ile47
`and Glyti4.
`
`REI L3
`Figure 7. Tht: conformation of t.he L3 region of \'.
`REI. The conformation is stabili2ed by the hyclrogPn
`bonds made ll\' the framework residuP Gln90 and b\' the
`ri.� conformati�n of the peptide of Pro95.
`·
`
`In a.II three \'i structures. residues 91 to !12 and 95
`to 96 form an extension of the P-sheet framework
`with main-chain hydrogen bonds between residues
`92 and 95:
`
`the sequence and conformation of two-residue t nrn,.;
`(Sibanda & Thornton. 198!); F.fimov, 1986) art>
`given in Table 2. Similarly, in L3 regions wit.h eight
`residues 1rt- would <'Xpe1·t 9 1 to fl:! and !I;) to 96 to
`continue the P-sheet framework and 93. !I:�''· 93b
`and 94 to form a four-residue turn.
`
`(b) J'K d1J//lflilt8
`The L3 regions in RF.I. �l('J>( '@:� and .J539 art:
`the same size:
`
`91
`
`Tyr
`A�p
`
`Trp
`
`!I:!
`
`<:In
`HiR
`Thr
`
`93
`:"'\er·
`St-r
`T.n
`
`!I+
`Leu
`Tyr
`Pro
`
`9fi
`
`Pro
`Pro
`Leu
`
`!lti
`.. r,·r
`l.�tt
`II<'
`
`REI
`)!<'PC 'fill:l
`.)!):)9
`
`In R.EI and :\l<'P('fiO:l. tlw L3 regions han· the
`same conformation: the r.m.s. differente in the
`positions of the main-chain 11t111ns of residues 91 to
`96 is O·.J.:J A. L3
`in J5a9 ha;; a e;onforma.tion
`different from that in REI and :\!( 'P< 'lill:l.
`Normally, for six-residue looµs, w1· might <·xp('<·t
`tht- main-chain a.toms of rt:sidul"s !l2 and !)!') to form
`hydrogen bonds. and residues 93 and 94 to form a
`turn (><t-t" t.he di,.;<·ussion of L3 in tht> \"i c·hains,
`;;('('(ion 6{a). above). This conformation is prevented
`in the two \'� ,.;t rnd.11 f(',.; R.ET and :\H 'I'( '()(I:� Ii.\' a
`thest•
`Pro residue at position 95. In
`two \'.­
`:-;tnwt.ur<:'s. rt-�idue 92 has an 1XL c:onformation and
`Pro95 has a ris peptide. This puts residues 93 to 96
`in an extended c·onformation (Fig. 7). Important
`determina.nt.s of this particular L3 con format.ion are
`the hydrogen lionds formed to its main-cha.in a.toms
`by the side-chain of framework residue 90. Though
`the side-chains at po:-;it ion 90 are not identical (Rl<:J
`
`93A.-- 93h
`I
`I
`93 __ 94
`93
`94
`I
`I
`I
`I
`92= = =95
`92= = = 95
`I
`I
`I
`I
`91
`96
`96
`91
`I
`I
`I
`I
`90= = =97
`90= = =97
`Residues 93 and 94 in NR\\':11 form a t11·0-n:.,<idut"
`type II' turn (see Table :?). Residues 93. 93a, 93b
`and 94 in RHE and KOL form a four-residue turn