`
`PFIZER EX. 1051
`
`Page 1
`
`PFIZER EX. 1051
`Page 1
`
`

`

`
`
`PFIZER EX. 1051
`
`Page 2
`
`PFIZER EX. 1051
`Page 2
`
`

`

`Journal of Molecular Biology
`
`Volume 215, Number 1
`
`Contents
`
`Communication
`
`Crystallization of the Reovirus Type 3 Dearing
`Core. Crystal Packing is Determined by the :12
`Protein
`
`K. M. Coombs, B. N. Fields and
`S. C. Harrison
`
`Articles
`
`Genetic and Molecular Analysis of Eight tRNAT"
`Amber Suppressors in Caenorhabdltls elegans
`
`K. Kondo, B. Makovec,
`R. H. Waterston and J. Hodgkin
`
`Discrimination Between Bacteriophage T3 and T7
`Promoters by the T3 and T7 RNA Polymerases
`Depends Primarily upon a Three Base-pair Region
`Located 10 to 12 Base—pairs Upstream from the
`Start Site
`
`J. F. Klement, M. B. Moorefield,
`E. Jorgensen, J. E. Brown, S. Risman
`and W. T. McAllister
`
`Identification of a Region of the Bacteriophage T3
`and T7 RNA Polymerases that Determines
`Promoter Specificity
`
`K. E. Joho, L. B. Gross,
`N. J. McGraW, C. Raskin and
`W. T. McAllister
`
`Both Genes for EF—Tu in Salmonella lypkimmium
`are Individually Dispensable for Growth
`
`D. Hughes
`
`Trantigcn is Not Bound to the Replication Origin
`of the Simian Virus 40 Late Transcription
`Complex
`
`K. G. Hadlock and L. C. Lutter
`
`DNA Replication in Escherichia coll is Initiated by
`Membrane Detachment of oriC. A Model
`
`V. Norris
`
`Sixteen Discrete RNA Components in the
`Cytoplasmic Ribosome of Euglena gracilis
`
`Fourteen Internal Transcribed Spacers in the
`Circular Ribosomal DNA of Euglena gracil’ls
`
`Higher Order Structure of Balbiani Ring
`Premessenger RNP Particles Depends on Certain
`RNase A Sensitive Sites
`
`M. N. Schnare and M. W. Gray
`
`M. N. Schnare, J. R. Cook and
`M. W. Gray
`
`T. Wurtz, A. Lonnroth and
`B. Daneholt
`
`Effect of Mutations in Domain 2 on the Structural
`
`Organization of Cocyte 5 S rRNA from Xempus
`laem's
`
`C. Brunei, P. Romby, E. Westhof,
`P. J. Romaniuk, B. Ehresmann and
`C. Ehresmann
`
`Crystallographic Analysis of Ribulose 1,5—
`Bisphosphate Carboxylase from Spinach at 24 A
`Resolution. Subunit Interactions and Active Site
`
`S. Knight, I. Andersson and
`C.-I. Branden
`
`High~resoluti0n Spot—scan Electron Microscopy of
`Microcrystals of an tat-Helical Coiled~coil Protein
`
`P. A. Bullough and P. A. Tulloch
`
`Framework Residue 71 is a Major Determinant of
`the Position and Conformation of the Second
`
`A. Tramontano, C. Chothia and
`A. M. Lesk
`
`Hypervariable Region in the VH Domains of
`Immunoglobulins
`
`21—29
`
`31—39
`
`41751
`
`53~65
`
`67—71
`
`73—83
`
`85—91
`
`937101
`
`103—111
`
`113—160
`
`161—173
`
`1757182
`
`PFIZER EX. 1051
`
`Page 3
`
`PFIZER EX. 1051
`Page 3
`
`

`

`Dynamic Monte Carlo Simulations of Globular
`Protein Folding. Model Studies of in Vivo
`Assembly of Four Helix Bundles and Four
`Member fi-Barrels
`
`Author Index
`
`A. Sikorski and J. Skolnick
`
`183—198
`
`199
`
`Cover Illustration." Bacteriophage T4, Tobacco mosaic Virus and Turnip Yellow mosaic virus, three of the
`viruses often featured in the Journal of Molecular Biology. Micrographs courtesy of Dr John Finch, MRC
`Laboratory of Molecular Biology, Cambridge.
`
`Origination by BPCC Whitefriars Ltd, Tunbridge Wells,
`and printed and bound in Great Britain by BPCC Wheatons Ltd, Exeter
`This journal is printed on acid-free paper
`
`PFIZER EX. 1051
`
`Page 4
`
`PFIZER EX. 1051
`Page 4
`
`

`

`Journal of Molecular Biology
`
`Editor-in-Chief
`P. Wright
`Department of Molecular Biology, Research Institute of Scripps Clinic
`10666 N. Torrey Pines Road, La Jolla, CA 92037, U.S.A.
`
`Assistant Editor
`J. Karn
`MRC Laboratory of Molecular Biology
`Hills Road, Cambridge CB2 2QH, U.K.
`
`Founding Editor
`Sir John Kendrew
`
`Consulting Editor
`Sydney Brenner
`
`Editors
`P. Chambon, Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Institut de Chimie Biologique,
`Faculté de Médecine, 11 Rue Humann, 67085 Strasbourg Cedex, France.
`A. R. Fersht, University Chemical Laboratory, Cambridge University, Lensfield Road, Cambridge CB2 lEW, U.K.
`M. Gottesman, Institute of Cancer Research, College of Physicians & Surgeons of Columbia University,
`701 W. 168th Street, New York, NY 10032, U.S.A.
`P. van Hippel, Institute of Molecular Biology, University of Oregon, Eugene, OR 9740371229, U.S.A.
`R. Huber, Max-Planck-Institut fiir Biochernie, 8033 Martinsried bei Miinchen, Germany.
`A.
`[(1119, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 201,11, U.K.
`
`Associate Editors
`C. R. Cantor, Human Genome Center, Donner Laboratory, Lawrence Berkeley Laboratory, University of California,
`Berkeley, CA 94720, U.S.A.
`N.-H. Chua, The Rockefeller University, 1230 York Avenue, New York, NY 10021, U.S.A.
`F. E. Cohen, Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco,
`CA 941434l446, U.S.A.
`D. J . DeRosier, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02254, U.S.A.
`W. A. Hendricksrm, Department of Biochemistry & Molecular Biophysics, College of Physicians & Surgeons of
`Columbia University, 630 West 168th Street, New York, NY 10032, U.S.A.
`[.13. Holland, Institute de Genetique et Microbiologie, Batiment 409, Universite’ de Paris XI, 91405 Orsay Cedex 05,
`France.
`B. Ham'g, Department of Biochemistry & Molecular Biophysics, College of Physicians & Surgeons of Columbia
`University, 630 West 168th Street, New York, NY 10032, U.S.A.
`V. Luzzati, Centre de Génétique Moléculaire, Centre National de la Recherche Scientifiquc, 91 Gif—sur-Yvette. France.
`J. L. Mandel, Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, Institut de Chimie Biologique.
`Faculté de Médecine, 11 Rue Humann, 67085 Strasbourg Cedex, France.
`B. Matthews, Institute of Molecular Biology, University of Oregon, Eugene, OR 97403—1229, U.S.A.
`J. H. Miller, Department of Microbiology, University of California, 405 Hilgard Avenue, Los Angeles, CA 90024, U.S.A.
`M. F. Moody, School of Pharmacy, University of London, 29/39 Brunswick Square, London WClN IAX, U.K.
`T. Richmond. Institut fiir Molekularbiologie und Biophysik, Eidgenossische Technische Hochschule, Honggerberg,
`CH 8093 Zurich, Switzerland.
`R. Schleif, Biology Department, Johns Hopkins University, Charles & 34th Streets, Baltimore, MD 21218, U.S.A.
`N. L. Stemberg, Central Research & Development Department, E. I. du Pont Nemours & Company, Wilmington,
`DE 19898, U.S.A.
`K . R. Yamamoto, Department of Biochemistry and Biophysics, School of Medicine, University of California.
`San Francisco, CA 94143—0448, U.S.A.
`M. Yamagida, Department of Biophysics, Faculty of Science, Kyoto University, Sakyo-Ku. Kyoto 606, Japan.
`Editorial Office
`0. Harris, Journal of Molecular Biology, 10d St Edwards Passage, Cambridge CB2 3PJ, U.K.
`
`JOURNAL OF MOLECULAR BIOLOGY: ISSN 0022—2836. Volumes 211—216, 1990, published twice a month on the
`5th and 20th by Academic Press at 24—28 Oval Road, London NW1 7DX, England. Annual subscription price including
`postage: £768 U.K. and U.S.$l464 overseas. Personal subscription rate: £233 U.K. and US. $350 overseas. Subscription
`orders should be sent to Academic Press Limited, Foots Cray, Sidcup, Kent DA14 5HP, U.K. (Tel: 081—300 3322). Send
`notices of changes of address to the publisher at least 6—8 weeks in advance, including both old and new addresses.
`Second class postage rate paid at Jamaica, NY 11431, U.S.A.
`
`Air freight and mailing in the U.S.A. by Publications Expeditng 1110., 200 Meacham Avenue, Elmont, NY 11003, U.S.A.
`U.S.A. POSTMASTERS: send change of addresses to JOURNAL OF MOLECULAR BIOLOGY. c/o Publications
`Expediting, Inc., 200 Meacham Avenue, Elmont, NY 11003, U.S.A.
`Printed in U.K.
`
`PFIZER EX. 1051
`
`Page 5
`
`PFIZER EX. 1051
`Page 5
`
`

`

`J. lr!ol. Biol. (1990) 215, 175-182
`
`Framework Residue 71 is a Major Determinant of the
`Position and Conformation of the Second
`Hypervariable Region in the VH Domains
`of lmmunoglobulins
`
`Anna Tramontano 1
`
`, Cyrus Chothia 2
`
`3 and Arthur M. Lesk 1 •2
`
`•
`
`1 European Molecular Biology Laboratory
`.M eyerhofstrasse 1
`Postfach 1022.09
`6900 Heidelberg, F.R.G.
`2 M RC Laboratory of 1'Jrl olecular Biology
`Carnbridge, CB2 2QH, U.K.
`3Christopher Ingold Laboratory
`University College London
`20 Gordon Street
`London WCJH OAJ, U.K.
`
`( Received 2 January 1990; accepted 18 May 1990)
`
`Analysis of the immunoglobulins of known structure reveals systematic differences in the
`position and main-chain conformation of the second hypervariable region of the VH domain
`(H2). We show that the major determinant of the position of H2 is the size of the residue at
`site 7 I, a site that is in the conserved framework of the VH domain. It is likely that for about
`two thirds of the known V.1 sequences the size of the residue at this site is also a major
`determinant of the conformation of H2. This effect can override the predisposition of the
`sequence, as in the case of the H2 loop of J539, which is an exception to the rules relating
`sequence and conformation of short hairpin loops. Understanding the relationship between
`the residue at position 71 and the position and conformation of H2 has applications to the
`prediction and engineering of antigen-binding sites of immunoglobulins.
`
`1. Introduction
`I mmunoglobulins are multi-domain proteins
`consisting of two chains, a light chain with one
`variable ( VL t) and one constant domain, and a
`heavy chain containing one variable domain ( VH)
`and three constant domains. The antigen-binding
`site is formed by six loops, three from the VL and
`three from the Vii domains. Figure l shows a simpli(cid:173)
`fied view of the antigen-binding site, indicating the
`relative positions of the loops. The variability of the
`residues in the antigen-binding site gives rise to the
`
`t Abbreviations used: V11 , variable domain of
`immunoglobulin heavy chains; VL, variable domain of
`immunoglobulin light chains; 1-12, second hypervariable
`loop of heavy drnin; r.rn.H., root-mean-square; aR, right(cid:173)
`handed a-helical conformation; IXL, left-handed a-helical
`conformation.
`
`0022-28:l(l/!l0/17017/H)8 $0:l.00/0
`
`high range of specificity achieved by antibodies (Wu
`& Kabat, 1970; Kabat et al., 1987).
`The atomic structures of several immunoglobulin
`fragments have been determined by X-ray crystal(cid:173)
`lography (Davies & Metzger, 1983; Alzari et al.,
`I 988). They show that all the domains have a very
`similar folding pattern: two {J-sheets packed face to
`face. A core of the double /J-sheet structure, called
`the framework, has a very similar conformation in
`different variable domains because of the conserva(cid:173)
`tion of internal residues and the requirements of
`internal packing. The residues that form the inter(cid:173)
`face between the VL and V.1 domains are also
`strongly conserved.
`These results have led to the view that the frame(cid:173)
`work structure plays an essentially passive role in
`the structural variation that occurs in the antigen(cid:173)
`binding site.
`175
`
`© 1!)!)0 Academic Press Limited
`
`PFIZER EX. 1051
`Page 6
`
`

`

`176
`
`A . T ramontano et a l.
`
`Fig ure 2 sho ws t he gc11cral sLru ct u l'a l co n Lex t o f'
`H 2 within t he V11 d o m a in .
`
`2. Co-ordinates and Calculations
`
`Protei11 st ru ct ures w;cd in thi s wo l'k a l'e listed in
`T ab le I. Th e ato mi e eo -ordin atcs o f" t hese st ru ct u res
`by
`th e
`l'l'0 Lei11 D ata B a11k
`are distributed
`( Bern st ei11 et al. , I 977 ), ex ce pL fo r th e l'c fi ned co (cid:173)
`ordin at es o f" .J!i:39 w hi ch arc a pl'i v a.t.e c:o mmuni <'n (cid:173)
`Lion f"rom Drs K A. Pad Ian an d I). H,. Da v ies. Th e
`SLl'u ct ures were di splayed usin g In sig ht ( D ay rin ge r
`et al. , 1986) on a11 E va,ns & SuLh<• f'l a 11d
`l'S:rno .
`Program s writlc n by A.M. L . (Lcs k , lfl8fi) wNe uscct
`fo l' ana lys is o f"
`th e strn <:L 111·<·s a11d
`d atabase
`sear ching.
`Thro ug hout t he pa pc: 1' res idu e numbers l'efer Lo
`t he heavy -<;hain 11umhe1"i11g sc· henw o f" l(ahaL et rrl .
`( I 987) . f n V11 dom a i11 s, t he c:on ser vcd //-sheet. f"ranw(cid:173)
`wo rk eonsisU, of" residues :3 Lo 12, 17 Lo 2!i , :l:3 Lo !i:Z,
`5(i to UO , li8 Lo 82 , 88 Lo !l!i a nd I 02 Lo 11 2 (Cho t hi a
`in Lil<'
`I !l87). Th ese n :sid,ws w<· r·< · used
`& Lesk ,
`superposi t ions o f" V1.1 d o m a in s.
`
`3. The Conformations of H2 Loops
`Tn V11 sequ cnc:cs t,lw s<'l'otl<I hy pel' Val'i a bl e region
`rcs id11 ('s 50
`eo nsist s o f" a
`/J-h a il'pin , compri sing
`through fi!i (Wu & K a ba t , 1!)70; I<ahaL d n.l. , I !)87).
`In t he know11 V11 sLrucL11n!s Llw m a.i11 -c:ha in c:onfol'(cid:173)
`m aLion s o f resid ll <!s !iO Lo fi2 and 5(i Lo Wi al'l' Lhl'
`sam e: fol' hig h -rnso l11 Lion we ll -re fin ed stru ct ures Lhe
`hack bone ato m s o f rcsid 11es 50 t.o !i2 a nd !iG to (i4 fit
`with a root -mean -sq ua re (1'.m .s. ) d ev iatio n bnLwcen
`0·4 and ()-7 A. Thi s region is illt1 stra.Lcd in Fi g 111"e :l:
`
`Figure 1. Outli11 e structure of t he ant igen-bi11di11g sit e.
`ThP site is forrn ed by fi loops of poly pept ido (/IN) li11lrnd
`to strand s i 11 /J-sheets ( 0 ).
`
`T n th e i rn mu nog lobul in s of kn ow n stru etu re t he
`co nform atio ns of the second hy per variahl e region in
`vl·I (H2) differ. Th e posi t ion or th e H2 with res pect,
`t o t he co nse r ved fra m ework is also vari abl e. For
`in t he V1.1 dom ain s of imrnunog lob ulin s
`exampl e,
`.)5:39 and H y .H EL-5 , th e .F-12 regions have th e sam e
`nu rn ber of residu es . ff t he fram ew ork strn etu res a re
`supel'irnposed , th e (f ato m s in l'esidu e 5:3, at Lh e Lip
`fr~ A
`of H 2, al'e found to differ
`in posi t ion by
`(I A=O· I nm). H ere, w e show that, th e va ri ations in
`two stl'u t:t ural featu l'es o f H2, i ts posi t ion and its
`co nform ation , a.re co upl ed , a nd th at t hey d epend in
`la rge pa r t on th e nature of th e a min o acid residue
`t hat occupi es posi t ion 71 m
`th e heavy- diain
`fram ew ork.
`
`Hl
`
`H2
`
`Hl
`
`H2
`
`Figure 2. Th e stru eturnl <.:ontext of H2 within th e 1111 domai n of l<'ab ,1 5:Jtl. H 2 is shaded rnlativoly darkl y, HI i~
`shaded relatively light ly . Th e t hi ek broken eirele indi cates t he guanidinium gmup of" i\rg:n J.
`
`PFIZER EX. 1051
`Page 7
`
`

`

`Position and Conformation of the fl2 Loop
`
`177
`
`Table 1
`I mmnnoglo{Jl(Z,:n heavy chain 1,aria/Jle domains of known atornic structure
`
`l\loll'<'llil'
`
`:'\EW:\I
`llyll EL-10
`
`llyll EL-ii
`KOL
`.Jii:rn
`
`:\lei'( '(iO:I
`•l-·1-20
`
`II 2 Sl'(jlll'IH'P
`
`HPsid11P 71
`
`Referpnce
`
`y
`y
`
`II D
`s
`(l
`
`I' u
`I)
`I)
`I'
`I)
`
`s
`(l
`s
`(l
`s u
`
`'."i
`N
`
`K
`K
`
`() N
`y
`I'
`
`K
`N
`
`y
`y
`
`V
`I{
`
`A
`H
`H
`
`R
`R
`
`S,utl et al. ( 1!178)
`Pad Ian et al. ( I !l8!l)
`
`Sheriff el al. (l!J87)
`l\larquart d al. ( l!lSO)
`Suh el al. (l!JS(i)
`
`Satow et al. ( l!l8li)
`lfrrron fl al. ( l!lS!J)
`
`Th<' 112 n•sid11ps an• (hosp hP!.WPPn positions ii:! and ii(i (seP text).
`
`the main-chain atoms of' residuPs f>{i to (j() form
`hyd mgcn bonds to thos(' of residues ..J.8 to 52 to form
`a /J-hairpin. Scq11c11ce variations in these residues
`have little or no effect 011 the main-chain conforma(cid:173)
`tion, because the side-chains are 011 the s11rface. The
`turn
`that
`links
`these
`two strnnds, comprising
`rcsid ucs f>2a to f>f> or f>:~ to f>f>, we re for t.o as Uw H 2
`region. 111 the known strnct11res it differs in length
`and co11fonnatio11.
`Hairpin strudures have been classified according
`to t.hcir length and (:011forrnatio11 (Vcnkat.achalam,
`1 !Hi8; Efirnov, 1 !l8(i; Sihanda &. Thornton, 1!)8f>;
`Silianda el al., 1!)8!)). Partic11lar co11formatio11s are
`usually associat.<:d with characteristic sequence
`patterns. The posit.ions of Cly, Asn, Asp and Prn
`r·Psidues arc important. because these residues allow
`rnai11-chai11 conformations that. in other residues
`ca11sc st.rain.
`
`(a) 'l'hre1'-rr!sirl111'. f/2 reuion8
`
`In NJ•:w;v1 and Hyll EL-10, the 112 loop is a
`t.!11·<:<:-rnsidue hairpin, n:sidues f>:3 to f>5. The NEVITM
`1-12 loop is shown as <"onforrnation I in Figure :t The
`usual sequerwe rcquircnwnt for this conformation is
`a C:ly (or Asn or Asp) at. Uw t.hird position (residue
`f>f>), which can take up a + + conformation (that is,
`
`4>>0, v1>0) (Sibandaetal., Hl8!l). Both NEWM and
`HyHEL-10 have a glycine at this position:
`
`NEWl\l
`Hyl-rnL-10
`
`ii:l
`
`Tyr
`Tyr
`
`[i.j.
`
`His
`Ser
`
`55
`
`Gly
`Clly
`
`71
`
`Val
`Arg
`
`and in both cases the Gly is in a + + conformation.
`
`(b) Four-re8id·ue II2 reuions
`loop of HyHEL-5 is a four-resi(~ue
`The H2
`hairpin, residues 52a to f>5. This is shown as confor(cid:173)
`mation 2 in Figmc :t The conformation is close to
`the one most commonlv observed in four-residue
`turns, in which the first" three residues arc in an cxR
`conformation and the fourth in an CXL conformation.
`These tmns normally require Gly in the fourth
`position (Efimov, I!)8(i; Sibanda & Thornton, 1!)85;
`Sibanda et al., H)89), as observed in HyHEL-5.
`The H2 regions in KOL and J53H form four(cid:173)
`residue turns with a conformation different from
`TiyHEL-f>. They both have the third residue (?4) in
`the cxL conformation and the first, second and fourth
`in the cxR conformation. This is shown as conforma(cid:173)
`tion :~ in Figure:~.
`
`I
`D1·3
`NEWM
`HyHEL-10
`Figure 3. Tlw main-chain <·onformations of the 2nd hyp<:rvarinhlP region in 1'11 domains in the immunoglobulins of
`known structure. The <·onfonnations are numlH'r<'d I to 4. The i11111111noglob11li11s in which these conformations are found
`an• li;;trnl undPr Pach nun1h!'r.
`
`2
`HyHEL-5
`NC41
`
`3
`KOL
`J539
`NQIO
`
`4
`McPC603
`4-4-20
`
`PFIZER EX. 1051
`Page 8
`
`

`

`178
`
`A. 1'ramontano et al.
`
`Table 2
`Res·ults of a database search for main-chain conformations the same as that of the JJ2
`loop of I( 0 L
`
`I',.
`(A)
`
`0·18
`0· I!)
`0·22
`0·22
`o-->->
`0·2:l
`0·24
`0·28
`0·2!)
`0·2!)
`
`Molecule (Protein Data Bank code)
`
`Rhizopuspepsin (3APR)
`Nubtilisin Carlsberg (2NEC)
`Ribonuclease A (7RNA)
`Pepsinogen ( I PSG)
`4:14 reprcssor protein ( 1 IW!l)
`Calmodulin (3CLN)
`Calmodulin (:!CLN)
`Adenylate kirnise (:JADK)
`Fab ,Jr,:J!l
`Cytochrome ciiiil (4iilC)
`
`Ntarting
`residue
`
`1-1;";
`JO
`32
`142
`:ir,
`m
`21
`l(j(j
`:ir,:3
`!)
`
`Nequ!·nce
`
`s
`L
`Q G
`I
`L
`K A
`N R N
`L
`I) Q
`L
`(l
`E N
`K
`G
`A
`(l N
`I)
`K
`1) G
`l)
`K
`I
`0
`R
`I' D N G
`N K
`C
`G
`
`/!,., root-mean-square deviation of N, C", C and O atoms of residues ii:! to ii(i of the V11 domain of KOL
`and well-fitting regions from other known structures.
`
`type of turn has not been described
`This
`previously, but we find that it occurs fairly often in
`proteins. vVe searched the database of solved struc(cid:173)
`tures for regions similar in main-chain conformation
`to the H2 loop of KOL. Table 2 lists the closest
`matches: ten loops, including ,J5:39 H2, for which
`the r.m.s. difference in position of main-chain atoms
`is less than 0·3 A. There arc (i I such loops with
`r.m.s. deviation less than 0·5 A. For KOL and ,J5:3n
`H2 and the nine best-titting non-homologous loops,
`the average values of the conformational angles and
`their standard deviations are:
`
`(see Fig. 5(b)). The r.m.s. deviation of all N, c•, C
`and O atoms is (H)(j A. The McPCGO:l Il2 loop is
`shown as conformation 4 in Figure :1. The sequences
`in these regions arc:
`
`f>2a
`
`r,21,
`
`ii2c
`
`r,:1
`
`[i.1
`
`55
`
`71
`
`.McPC!iO:l
`4-4-20
`
`Asn
`Asn
`
`Lys Tyr Arg
`Lys Gly Asn
`Pro Tyr Asn Tyr Arg
`Lys
`
`In both structures residue 54 is in Uw cxL confor(cid:173)
`mation. I II the other V11 sequences with six-residue
`
`Angle
`Mean (deg.)
`Standard deviation (deg.)
`
`<p 1
`-(ii
`12
`
`t/11
`-:ir,
`8
`
`,/J2
`-!Jii
`12
`
`V'2
`77
`1-1
`
`<p 3
`(if,
`11
`
`22
`11
`
`Of the nine loops in Table 2, excluding ,J5:m, seven
`have a Gly in the third position, like KOL, one has
`Asn and one has Lys. Of all the loops with r.m.s.
`deviation Jess than 0·5 A, none is like J5:39 in having
`Gly at only the fourth position.
`These results show that H2 in ,J5:39 is an excep(cid:173)
`tion to the rules relating sequence and structure in
`short hairpins. Both HyHEL-5 and J539 have Gly
`in the fourth position of the loop:
`
`KOL
`,J5:l!l
`HyHEL-ii
`
`52a
`
`Asp
`Pro
`Pro
`
`53
`
`Asp
`Asp
`Gly
`
`54
`
`Gly
`Ser
`Ser
`
`55
`
`Ser
`Gly
`Gly
`
`The position of Gly in J539 should imply a confor(cid:173)
`mation of H2 similar to that of HyHEL-5. Instead
`the conformation observed in ,J5:39 is the same as in
`KOL (see Fig. 4(b) and Fig. 5(a)). The r.m.s. devia(cid:173)
`tion in the position of the H2 main-chain atoms in
`,T5,39 and HyHEL-5 isl·!} A; for J539 and KOL it is
`0·:3 A. The residues of H2 in ,J5:39 make no non(cid:173)
`bonded contacts to residues other than those in H 1
`and Arg71 and Asn73 (see Fig. 2).
`
`( c) Six-residue 112 regions
`
`In McPCHo:3 and 4-4-20, the H2 loops are six(cid:173)
`residue hairpins. Their conformations arc similar
`
`H2 loops, the residues found at this position are
`Gly, Asn or Asp (Kabat et al., 1!)87). It is interesting
`to note in this context that the Lys at position 54 in
`McPC{j{)3 is the result of a somatic mutation from a
`germ-line gene that contains a Gly.
`
`The Interactions of H2 with the Framework
`l(xamination of the interactions of the H2 loops
`with the rest of the V. 1 domain shows that the
`determinants of the conformations of four-residue
`I-12 loops arc not entirely within the sequence of the
`loop itself, but involve the packing of the loop
`against the rest of the structure.
`In Figures 4 and 5 we show, for pairs of anti(cid:173)
`bodies of known structure, the relative positions of
`the HI and H2 loops and the contacts made by
`certain side-chains. The relative positions of these
`loops in these Figures are those induced by the
`superposition of the framework structures. The
`Figures show that the J-11
`loops occupy rather
`similar positions with respect to the framework in
`all the known structures. But the positions of the
`H2 loops are in some cases very different. These
`differences are related to the siw of the residue at
`position 71.
`KOL and ,J53!) have four-residue F-i2 loops in very
`similar positions and conformations (Fig. 4(b)). The
`residue at position 71 is Arg in both structures. The
`
`PFIZER EX. 1051
`Page 9
`
`

`

`Position nnd Conformation of the !12 Loop
`
`179
`
`H2 ,--------------
`
`.~
`··-1 •,,
`
`~ Phe 29/!le 29
`
`Arg 71/ Val 71
`
`(a)
`
`HI
`
`Arg 71
`
`( b)
`
`/
`
`/
`
`. ~igure 4. The relative positions of the H 1 and II2 hypervariablc regions and of framewori, residue 71, in different pairs
`of 1mrnunoglobulim,. The HI and H2 regions are represented by their C" atoms. The positions shown here are those found
`after the superposition of the V11 framework residues (see text). (;t) NKWM (continuous lines) and HyHEL-10 (broken
`lines). (b) KOL (continuous lines) and J53!) (broken lines).
`
`side-chains of these argmme residues are buried.
`They form hydrogen bonds to main-chain atoms of
`residues in the HJ and H2 loops and pack against
`the Phe at position 29.
`The superposition of J53B and Hyl-IEL-5 shown
`in Figure 5(a) illustrates the case of two immuno(cid:173)
`glohulin structures with H2 loops of the same length
`but different conformation and position. In J539, in
`which residue 71 is an Arg, residue Pro52a in the H2
`loop is on the smfaee. In HyHEL-5, in which
`residue 71 is an Ala, Pro52a is buried, filling the
`cavity that would be created by the absence of a
`long side-chain at position 71. The manner in which
`these H2 loops pack against the r·cst of the Vi,
`domain explains why the H2 region of J539 does not
`have the conformation that we would expect from
`Gly at position 56. If it did have the expected
`conformation, like that in HyHEL-5, the Pro52a
`side-chain would occupy the same space as the side(cid:173)
`chain of Arg7l (Fig. 5(a)). The set of torsion angles
`
`that move the side-chain of Pro52a away from
`Arg71 require an H2 conformation different from
`that in HyHEL-5.
`In both McPC603 and 4-4-20, H2 is a six-residue
`turn, and residue 71 is an Arg. In McPC603 Arg7l
`has its side-chain buried, and is hydrogen bonded to
`the main-chain of Hl and H2, as in KOL and J539
`(Fig. 5(b)). The Tyr at the sixth position (55) packs
`against Arg7l.
`
`5. The Role of Residue 71
`These observations can be summarized as follows.
`(l) Position 71 contains a small or medium-sized
`residue. For three and four-residue I-12 loops the
`residue at position 53/52a packs against residues at
`positions 71 and 29. This brings the HI and H2
`loops close together and puts four-residue H2 loops
`in conformation 2 (Fig. 3).
`(2) Residue 71 is an arginine. The side-chain of
`
`PFIZER EX. 1051
`Page 10
`
`

`

`180
`
`A. 'l'mmontano ct al.
`
`Pro 52a
`
`.........
`
`'···.... Arg 71
`
`A Ala 71
`
`(a)
`
`HI
`
`}t
`
`·--~
`
`c.
`
`Phe 29
`
`Arg 71
`
`( b)
`
`Figure 5. The relative prn,itiorn; of the HI and 112 hypervariahle n,gions and of f"rnrnework nesiduc 71, in pain, of
`known immunoglolrnlin stmctures. The HJ and H2 regions arn rnJ>rnscnted hy their C" atoms. Tlw positions shown hen•
`are thrn,e found after the superposition of the VH framework rcsidtws (sl'e text). (a) IJyIIEL-ii (t,ontinuous Jines) and
`,Jr;:rn (brokfm Jines); (b) 1\foPCuO:~ (continuous line,;) and 4-4-20 (brolwn lines).
`
`the argmmc is buried between H l and H2, and
`forms hydrogen bonds with the main-chain in both
`loops. The H2 loop is displaced from HI with
`residue 52a on the surface. Four-residue H2 loops
`have conformation :J (Fig. 3).
`In Fab NC41 residue 71 is a Leu, intermediate in
`In
`size.
`the
`structme of
`the Fab NC41-
`(Colman et al.,
`neuraminidase complex
`]!)87;
`Chothia et al., 198H), H2 has the HyHEL-5 confor(cid:173)
`mation. Residue 52a in NC41 is a Thr, smaller than
`the Pro at the corresponding position in HyHEL-5;
`as a result the shift in H2 produced by the Leu is
`reduced.
`For six-residue H2 loops we have information for
`McPC60:J and 4-4-20, in which residue 71 is Arg. All
`known Vi, sequences that contain six-residue H2
`loops have Arg at position 71 (sec below).
`
`6. Applications to Structure Prediction. The H2
`Regions in Immunoglobulins of
`Unknown Structure
`
`To see if the results reported here arc useful for
`predicting the strnctmcs of antigen-binding sites of
`immunoglobulins we must find out whether the
`determinants of the known conformations arc
`commonly present in sequences of Vii domains of
`( l !)87) have
`unknown strncturc. Kabat el al.
`collected the known immunoglobulin sequences. \Ve
`found in this collection :w2 V11 sequences for which
`all, or almost all, residues in the region 50 to 75 arc
`known. Of these sequences, 54- are from humans and
`248 are from mice.
`There arc 47 sequences with three-residue H2
`regions. All these have Uly or Asp at position 55.
`
`PFIZER EX. 1051
`Page 11
`
`

`

`Position and Cm~formation of the I/2 Loop
`
`181
`
`This implies that they have conformations similar
`to that of 112 in NEWM and Hylll•:L-10: an impli(cid:173)
`cation supported hy the prediction of the conforma(cid:173)
`tion of the H2 region in Dl .:~ (Chothia et al., 1 !)8(1).
`At position 71, Arg or Lys occurs in 43 sequences
`and Val or Leu in four.
`There are 1 !}4 sequences with four-residue H2
`regions. Of these, :Hi seqmmees have Arg or Lys at
`position 71 and Gly, Asn Of' Asp at position fi4. For
`these we have the clear expectation that the H2
`regions have conformation :~ of Figme :3 and a
`position close to that found in KOL and ,Hi:lH.
`Another !)!) sequences have J>rn at position 52a; Gly,
`or in a few cases Asn or Asp, at position fiii; and Val,
`Leu or Ala at 71. Again we have the clear expec(cid:173)
`tation these domains have 112 loops in conformation
`2 of Figure
`:~ and
`in a position
`like that of
`Hy JI I•:L-ii.
`l\lost, of the 41 other four-residue I--12 regions do
`not have a Cly, Asn or Asp at either position 54 or
`Gf>. The expectation that these have conformations
`like that in KOL/.J5:rn or HyH EL-fi, depending 011
`the residue at position 71, is more tentative. The
`structure of Fah NQ IO has recently hcen deter(cid:173)
`mined (Spinelli d al., unpublished results). Tn N(~lO,
`the sequence of H2 is S-G-S-S, with Arg in position
`71 (Berck d al., l!l8ii). The occu1Tencc ofGly at the
`second position of a four-residue hairpin is very
`unusual; it docs not occur in any of the loops
`surveyed by Chothia & Lesk ( 1 !)87) and Sihanda et
`al. (l!l8!l). (KOL has Gly at the third position of the
`loop; and HyH EL-f> has Gly at tlw fourth position
`of the loop). The conform at.ion and position of H2 in
`NQIO are t,11(' :,;ame as in KOL: The r.m.s. deviation
`of all N, C\ C and O atoms of I--12 is 0·:3!) A; the
`r.m.:,;. deviation of all N, (?, C and O atoms of T-11
`and 112 together is 0·4:1 A (Chothia et al., Hl8!l). This
`then confirms the importance of the residue at
`position 71 in determining the conformation of the
`loop in these ca:-;()S.
`Tlwrc arc
`(i 1 sequences with six-residue T-12
`regions. All have Tyr at position iiii, Arg at 71 and
`all hut two have Gly, Asn 01· Asp at ri4. The conser(cid:173)
`vation at the:,;() sit\)s suggests thc:,;e II~ regions have
`<·onf'onnations close to that in i\lcPC(i0:1 and 4-4-20.
`
`7. Applications to Antibody Engineering
`The ability to trnnsplant hypervariahl<~ regions of
`non-human origin
`to human frameworks
`is of
`medical importance (g.cichnrnnn d al., 1 !)88). For
`the binding :-;itc of' the synthetic product to he the
`same a:,; that in the original antibody, the frame(cid:173)
`works should have the same residues at those sites
`important. for the positions and conformations of
`the hypervariahlc regions. However, binding sites
`do have a limiU)d intrinsic flexibility. The main(cid:173)
`chain portions of close-paclrnd segll!ents of proteins
`can move relative to each other by I to ~ A, with
`little expenditun\ of e1wrgy (Chothia el al., 1!)8:~),
`and the apices of loops may wPII he able to lllove hy
`larger amounts. Thus the effect on antigen binding
`of changing t.lw conf'ornrntion, or Uw position and
`
`orientation, of a hyp_erv1~ri.able region will depend
`upon whether the region 1s mvolved in bindinrr ·tnd
`if it is, on ho,~ much energy is requirnd f;r' the~
`strnctur'.11 read~ustments necessary to form
`the
`correct mtemct1011s. The most serious effects will
`occur when the framework contains a large residue,
`rather than a small one, as compression energies arc
`large.
`. ,Jo_nes et al . . ( 1 H86)
`transplanted the antige11-
`h111d111g loops from the heavy chain of a mouse
`antibody on to the framework of a human one. They
`observed that the synthetic product, when bound to
`~he original mouse light chain, had the same affinity
`for the. hap~en as the original mouse antibody.
`Tnspect1on of the two sequences used by ,Jones et al.
`( l!l8(i) shows that both the mouse aml' lrnman an ti(cid:173)
`bodies have Val at position 71, and therefore we
`should expect the f'onr-residne H2 loop from the
`mouse antibody Lo retain its conformation and posi(cid:173)
`tion on transfer to the human framework.
`Verhocycn et al. ( l!l88) transferred the hyper(cid:173)
`variable regions of the heavy chain of the 1;10use
`anti-lysor,yme antibody Dl .:~ to the framework of
`the human antibody NEWM. An affinity for lyso(cid:173)
`r,yme was retained, although
`reduced apprnxi(cid:173)
`mately tenfold. Both Dl .:i and NE\VM contain a
`Gly at position 55 of the heavy chain; at position 71
`Dl .:i contains Lys and NI~\Vl\l contains Val. This
`would suggest that in the synthetic antibody I-12
`has the correct conformation but is displaced from
`the position in Dl.3. Tn the Dl .3~1ysor,yme complex,
`the contacts made by H2 (residues 5:l to 5,5) to the
`antigen involve residues Glyfi:J and Aspii4 (Amit et
`al., 1!)8H). We cannot determine to what extent the
`slight loss of affinity by the synthetic antibody is
`associated with
`the molecular·
`readjustments
`required to retain these contacts.
`Reichmann et al. ( IH88) rnshaped an antibody by
`transplanting all six hypervariable regions from 11
`rat antibody on Lo a human framework for both V1,
`and J/i1 domains. In this case H2 had six residues, as
`does l\foPCGO:t The parent mt antibody has Arg at
`position 71, but the human framework has Val.
`There is no known I' H sequence with the com bi na(cid:173)
`tion a six-residue l-12 and Val at position 71 (see
`above). The synthetic antibody has an afTinity close
`to that of the mt original. vVhethcr this is because
`the cavity created by the smaller residue docs not
`significantly affect the conformation of the six(cid:173)
`residue T-12, or because this 1-12 makes only a
`marginn,I contribution to affinity, is unclear.
`
`8. Conclusion
`Previously we reported that framework residues
`are an important determinant of the conformation
`of first hypervariable region of V1, (Lesk & Chothia,
`1!)82; Chothia & Lcsk, Hl87). Tn that case the natme
`of the frnmew01·k residues is related directly to the
`class of the light chain: K or ).. The analysis
`presented here demonstrates that a f