AUGUST 1939
`
`_
`
`'
`
`__ VOLUME 86
`' NUMBER 15
`
`
`
`Proceedings
`
`OF THE
`
`Natlonal Academy
`of Sciences
`
`OF THE UNITED STATES OF AMERICA
`
`Pfizer Ex. 1016
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`$3$292
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`owE230?
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`2mmmzbz
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`
`
`3 oceedings
`-'
`-—
`_
`OF THE
`
`National Academy
`of Sciences
`
`OF THE UNITED STATES OF AMERICA
`
`
`
`Qfl‘icers
`oftite
`Academy
`
`Editorioi Board
`oftite
`Proceedings
`
`FRANK PRESS. President
`JAMES D. EBERT. Vice President
`PETER H. RAVEN. Home Secretary
`WILLIAM E. GORDON. Foreign Secretary
`ELKAN R. BLOUT. Treasurer
`
`ROBERT H. ABELEs
`GORDON A. BAYM
`RONALD BREsLow
`MICHAEL J. CHAMBERLIN
`MARY~DELL CHILTON
`
`IGOR B. DAWID. Chairman
`EDWARD E. DAvID. JR.
`RONALD L. GRAHAM
`STUART A. KORNFELD
`DANIEL E. KOSHLAND. JR.
`PHILIP W. MAJERUS
`DANIEL NATHANs
`
`HERBERT E. SCARF
`MAXINE F, SINGER
`SOLOMON H. SNYDER
`HAROLD VARMus
`THOMAS A. WALDMANN
`
`Managing Editor: FRANCEs R. ZwANZIG
`Senior Associate Editor: GARY T. COCKS
`Associate Editor: CAY BUTLER
`Associate Editor: JOHN M. MALLOY
`Associate Editor: MARILYN .l. MASON
`Associate Editor: JANET L. MORGAN
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`Assistant Managing Editor: JOANNE D'AMICO
`
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`
`
`Proceedings
`
`OF THE
`
`National Academy
`of Sciences
`
`OF THE UNITED STATES OF AMERICA
`
`August 1989
`
`Volume 86, Number 15
`
`pp. 5671—6012
`
`Table of Contents
`
`CONDENSED INFORMATION TO CONTRIBUTORS
`
`AUTHOR INDEX
`
`Physical Sciences
`
`PHYSICS
`
`Trapped individual ion at absolute zero temperature
`
`Miniature Paul Strauhel ion trap with well-defined deep potential well
`
`Nan Yu, Hans Dehmell. and Warren
`Nagourney
`
`Nan Yu. Hans Dcltmelt, and Warren
`Nagourncy
`
`Biological Sciences
`
`BlOCI-IEMISTRY
`
`One-sided polymerase chain reaction: The amplification of cDNA
`
`()samu Ohara. Robert L. Dorit, and
`Walter Gilbert
`
`The mouse homolog of the irs'rfk-FGF gene is adjacent to int-2 and is activated
`by proviral insertion in some vitally induced mammary tumors
`
`Gordon Peters, Sharon Brookcs,
`Rosalind Smith, Marysia Placzek, and
`Clive Dickson
`
`Transmembranc signaling by a chimera of the Escherichia calf aspartate
`receptor and the human insulin receptor
`
`OF 5,
`
`Gregory R. Moe, Gideon E. Bollag, and
`Daniel E. Koshland, Jr.
`
`Identification of the operator of the fax regulon from the an'ofisc
`ATCC7744
`
`iii
`
`3‘, Jerry H. Devine, Gerald S‘ Shadel. and
`strain
`”3ng 3 Thomas 0. Baldwin
`0-
`
`
`
`
`ix
`
`Str'r'l
`
`5m:
`
`Stat-'3
`
`5678
`
`5633
`
`5533
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`.Schizosacchammyces pombe and Saccirammyces cerevisine
`
` , 'fid‘enylate cyclases in yeast: A comparison of the genes from
`
`Association of charge clusters with functional domains of cellular transcription
`factors
`
`The yeast STE12 protein binds to the DNA sequence mediating pheromone
`induction
`
`Serum- and oncoprotein-mediated induction of a gene with sequence similarity
`to the gene encoding carcinoembryonic antigen
`
`Constitutive expression of c—myc oncogene confers hormone independence and
`enhanced growth-factor responsiveness on cells transformed by human
`papilloma virus type 16
`Yeast TATA-binding protein TFllD binds to TATA elements with both
`consensus and nonconsensus DNA sequences
`
`Structural characterization of a 2:1 distamycin A-dlCGCAAATTGGC) complex
`by taro-dimensional NMR
`
`Cloning of the immunological repertoire in Escherichia cot'i for generation of
`monoclonal catalytic antibodies: Construction of a heavy chain variable
`region—specific cDNA library
`
`Expression of bovine B-1,4-galactosyl1rans[eruse cDNA in COS-7 cells
`Conservation of the sequence of islet amyloid polypeptide in live mammals is
`consistent with its putative role as an islet hormone
`
`Nucleotide sequence and genomic organization of feline immunodeficiency
`virus
`
`Yuriko Yamawaki-Kataoka. Tatsuya
`Tamaoki. Hye-Ryun Choc. l'lideho
`Tanaka. and Tohru Kataoka
`
`Volker Brendel and Samuel Karlin
`
`Joseph W. Dolan. ('elia Kirkman. and
`Stanley Fields
`
`Roman Klemenz. Sylvia l-lot'l'mann. and
`Anne-Katrin Werenskiold
`
`Tim Crook. Neil Almond. Anne Murray,
`Margaret Stanley, and Lionel
`C rawlord
`Steven Hahn. Stephen Buratowski.
`Phillip A. Sharp, and Leonard
`Gttarente
`
`5593
`
`5593
`
`5733
`
`5703
`
`5713
`
`Silt}
`
`Jeffrey G. Peiton and David E. Wemmer
`
`5793
`
`L. Sastry. M. Alting-Mees. W. D. Huse.
`J. M. Short. J. A. Sorgc. B. N. Hay.
`K. D. Janda. S. J. Benkovic. and
`R. A. Lerner
`
`Arni S. Masibay and Pradman K. Qasba
`Masahiro Nishi. Shu lin Chan, Shinya
`Nagamatsu. Graeme l. Bell, and
`Donald F. Steiner
`
`Randy L. Tulbott. E. Elizabeth Sparger,
`Kathleen M. Lovelace, Walter M.
`Fitch, Niels C. Pedersen, Paul A.
`Luciw, and John H. Elder
`
`5728
`
`5733
`5738
`
`5743
`
`5743
`
`5753
`
`5753
`
`5763
`
`SW35
`
`5773
`
`5778
`
`5731
`
`5735
`
`5791
`
`5795
`
`5800
`
`5305
`
`Homology of the human intestinal Na*,-’glucosc and Escherichia coii
`Naflr‘proline cotransporters
`
`Matthias A. Hedigcr, Eric Turk. and
`Ernest M. Wright
`
`A cyanobacterial mutant requiring the expression of ribulose bisphosphate
`carboxylase from a photosynthetic anae robe
`
`J. Pierce. T. J. Carlson. and J. (i. K.
`Williams
`
`Delineation of three functional domains of the transcriptional activator encoded
`by the c—myb protooncogene
`
`Hiroshi Sakura. Chit: Kanei—lsltii,
`Takahiro Nagase. Hideki Nakagoshi.
`Thomas J. Gonda. and Shunsukc Ishii
`
`Autoregulatory control of B-tubulin mRNA stability is linked to translation
`elongation
`
`David A. Gay, Sangram S. Sisodia, and
`Don W. Cleveland
`
`The iron-responsive element binding protein: A method for the affinity
`purification of a regulatory RNA—binding protein
`
`Actin polymerization induces a shape change in actin—containing vesicles
`
`Self-assembly of synthetic phytochrome holoprotein in l-‘it‘t‘O
`
`Tracey A. Rouault. Matthias W. l-lentzc.
`David J. Haile. Joe B. Harford, and
`Richard D. Klausner
`
`Jorge Daniel Cortese. Bill Schwab Ill,
`Carl Frieden, and Elliot L. Elson
`
`J. Clark Lagarias and Donna M. Lagarias
`
`Role of capsid precursor processing and myristoylation in morphogenesis and
`infectivity of human immunodeficiency virus type 1
`
`Henrich G. Gottlinger, Joseph G.
`Sodroski. and William A. Haseltine
`
`Predicting the orientation of eukaryotic membrane—spanning proteins
`
`Phorbol 12-myristate 13-acetate inhibits binding of leukotriene 84 and
`platelet-activating factor and the responses they induce in neutrophils: Site
`of action
`
`Enno Hartmann, Torn A. Rapoport, and
`Harvey F. Lodish
`
`Mttnehiro Yamazaki, Julian Gomez-
`Cambronero, Melissa Durstin.
`Thaddeus F. P. Molski, Elmer L.
`Becker, and Ramadan I. Sha‘afi
`
`Translation of glucose—regulated protein 78fimmunoglobulin heavy-chain
`.
`binding protein mRNA is increased in poliovirus-infectcd cells at a time
`when cap-dependent translation of cellular mRNAs is inhibited
`
`Peter Sarnow
`
`Isolation and characterization of cDNA encoding the a subunit of Cap Zap”),
`an actin-capping protein from the 2 line of skeletal muscle
`
`Xenopus homolog of the mos protooncogene transforms mammalian fibroblasts
`and induces maturation of Xermpus ooeytes
`
`James F. Casella, Samuel J. Casella,
`Jacqueline A. Hollands, Jane E.
`Caldwell, and John A. Cooper
`Robert S. Freeman, Kathleen M.
`Pickham. John P. Kanki, Bruce A.
`Lee. Susan V. Pena, and Daniel J.
`Donoghue
`
`iv
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`Contents
`
`Low density lipoprotein receptor—related protein mediates uptake of cholesteryl
`esters derived from apoprotein E-enriched lipoproteins
`
`Robert C. Kowa]. Joachim Hera, Joseph
`L. Goldstein. Victoria Esser, and
`Michael S. Brown
`
`BIOPHYSICS
`
`Tryptophan fluorescence quenching as a monitor for the protein conformation
`changes occurring during the photoeycle of bacteriorhodopsin under
`different perturbations
`
`Enzymes work by solvation substitution rather than by desolvation
`
`Effects of lipid packing on polymorphic phase behavior and membrane
`properties
`
`Du-Jeon Jang and M. A. EI—Sayerl
`
`Arieh Warshel, Johan Aqvist. and Steve
`Creighton
`Sek-Wen Hui and Arindarn Scn
`
`BOTANY
`
`Altered regulation of ,t'l-arnylase actiVity in mutants of Arabidopsis with lesions
`in starch metabolism
`
`Timothy Caspar, Tsan-Piao Lin,
`Jonathan Monroe, Werner Bernhard.
`Steven Spilatro, Jack Preiss, and Chris
`Somerville
`
`CELL BIOLOGY
`
`Alteration in membrane protein band 3 associated with accelerated erythrocyte
`aging
`
`Anion channel blockers cause apparent inhibition of cxocytosis by reacting
`with agonist or secretory product, not with cell
`
`Purification of scatter factor, a fibroblast-derived basic protein that modulates
`epithelial interactions and movement
`
`Marguerite M. B. Kay, Naomi Flowers,
`Joseph Goodman, and Gieljan Bosman
`Jaroslav G. Vostal. Diane M. Reid.
`Charles E. Jones, and N. Raphael
`Shulman
`
`Ermanno Gherardi, Julia Gray. Michael
`Stoker, Marion Perryman. and Robert
`Furlong
`
`Cellular responses to finder-in thionin are mediated by Ca2+ influx and
`phospholipase A; activation and are inhibited by thionin tyrosine iodination
`
`Jess Evans, Yiding Wang. Kai-Ping
`Shaw, and Leo P. Vernon
`
`Identification and characterization of nuclear retinoic acid-binding activity in
`human myelohlastic leukemia HL—t‘il) cells
`
`Tumor-elicited polymorphonnclcar cells, in contrast to “normal" circulating
`polymorphonuclear cells. stimulate invasive and metastatic potentials of rat
`mammary adenocarcinoma cells
`
`Clara Nervi, Joseph F. Grippo. Michael
`I. Sherman. Margaret D. George, and
`Anton M. Jetten
`
`Danny R. Welch, Daniel J. Schissel,
`Richard P. Howrey, and Paul A. Aeed
`
`Tetruhydrohiopterin. the cofactor for aromatic amino acid Itydroxylases. is
`synthesized by and regulates proliferation of erythroid cells
`
`Keiko Tanaka, Seymour Kaufman, and
`Sheldon Milstien
`
`DEVELOPMENTAL BIOLOGY
`
`to son autorndiography and ligand-dependent tyrosine kinase activity reveal
`insulin receptors and insulin—like growth factor I receptors in prepancreatic
`chicken embryos
`
`Matias Girbau. Lluis Bassas, Jorge
`Alemany. and Flora de Pablo
`
`GENETICS
`
`Cardiac and neurological abnormalities in v-fps transgenic miCe
`
`Different a alleles of Heritage maydis are necessary for maintenance of
`filamentous growth but not for meiosis
`
`Implications of a quasispecies genome structure: Effect of frequent, naturally
`occurring amino acid substitutions on the antigenicity of foot—and—mouth
`disease virus
`
`Scanning detection of mutations in human ornithine transcarhantoylase by
`chemical mismatch cleavage
`
`Mutator phenotype of Werner syndrome is characterized by extensive deletions
`
`Siu-Pok ch, David Mock, Victor
`Maltby, Malcolm Silver, Janet
`Rossant, Alan Bernstein. and Tony
`Pawson
`
`Flora Banuett and Ira Herskowitz
`
`M. G. Mateu, M. A. Martinez, E.
`Rocha, D. Andrea, J. Parejo, E.
`Giralt, F. Sobrino, and E. Domingo
`
`Markus Grompe, Donna M. Muzny. and
`C. Thomas Caskey
`
`Ken-ichiro Fukuchi, George M. Martin,
`and Raymond J. Monnat, Jr.
`
`581”
`
`5515
`
`5830
`
`5834
`
`5839
`
`5844
`
`5849
`
`5854
`
`585‘)
`
`5364
`
`5868
`
`5873
`
`5878
`
`5883
`
`5888
`
`5893
`
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`
`- pit! screening of a human genomic library in yeast artificial chromosomes for
`single-copy sequences
`
`Tissue-specific genetic variation in the level of mouse alcohol dehydrogenase is
`controlled transcriptionally in kidney and posttranscriptionally tn liver
`
`Tn55upF. a 264-base-pair transposon derived from Tnj for insertion
`mutagenesis and sequencing DNAs cloned in phage A
`
`
`
`IMMUNOLOGY
`
`Developmentally restricted immunoglohulin heavy chain variable region gene
`expreSSed at high frequency in chronic lympht'icytic leukemia
`
`Decrease by chronic energy intake restriction of cellular proliferation in the
`intestinal epithelium and lymphoid organs in autoimmunity-prone mice
`
`Interleukin 7 is a T-ccll growth factor
`
`Recognition of a self major histtlcompatibility complex TL region product by
`75 T-cell receptors
`
`Haplotype-specific differences in signaling by transfected class II molecules to
`a Ly-l+ B-cell clone
`
`Structure of an antibody—antigen complex: Crystal structure of the HyHEL—ill
`Fab-lysozyme complex
`
`MEDICAL SCIENCES
`
`Antidiabetic effects of interleukin 1
`
`Fatal syndrome in mice engrafted with cells producing high levels of the
`leukemia inhibitory factor
`
`Human interleukin 6 is a direct promoter of maturation of megakaryocytes in
`virm
`
`Critical role of the 021 S55 region on chromosome 21 in the pathogenesis of
`Down syndrome
`
`Studies of the mechanisms of action of the antiretroviral agents hypericin and
`pseudohypericin
`
`Dipyridamole potentiates the inhibition by 3'-azid0-3'~deoxythymidine and
`other dideoxynucleosides of human immunodeficiency virus replication in
`monocyte—macrophages [Correction]
`
`Christopher N. Traver, Sue Klapholz.
`Richard W. Hyman. and Ronald W.
`Davis
`
`Lynda Tusscy and Michael R. Felder
`
`Suhas H. Phadnis. Henry V. Huang. and
`Douglas E. Berg
`
`Thomas J. Kipps. Eric Turnhavc. Lisa F.
`Pratt. Stuart Duffy. Pojen P. Chen.
`and Dennis A. Carson
`
`Motohiro Ogurn. I‘lirttko Ogurtt. Stistlnttt
`lkehara. My Lien Dan, and Robert A.
`Good
`
`Geoffrey D. Chuzett. Geruldo M. 13.
`Percira. Urahzmt Ict.iros. Steven
`Gillis. and Ethan M. Shcvach
`
`Marc Bonneville, Kouichi lto, Edvins (i.
`Krecko. Shigcyoshi ltohara. [)ielrrtar
`Kappes. Isao Ishidtt. Osami
`Kanagawa, Charles A. Janewuy. Jr..
`Donal B. Murphy. and SuSumu
`Tonegawa
`
`Gail Ahendroth Bishop and Jeffrey A.
`Frclingcr
`Eduardo A. Pudlan. Enid W. Silvertott.
`Steven Sheriff, Gerson H. Cohen.
`Sandra J. Smith-Gill, and David R.
`Davies
`
`Adriana del Rey and Hugo Bcsctlovsky
`
`D. Metcalf and D. P. Gearing
`
`Toshiyuki Ishihashi. Hidco Kimura.
`Tatsumi Uchida. Shigeo Kariyone.
`Paul Fricse, and Samuel A. Burstein
`
`Zohra Rahmani, Jean-Louis Blouin.
`Nicole Crew—Goldberg. Paul C.
`Watkins. Jean-Francois Maltei. Marc
`Poissonnier, Marguerite Priettr.
`Zoubida Cheltouh, Annie Nicole.
`Alain Aurias, Pierre—Marie Sinet, and
`Jean-Maurice Delabar
`
`Gad Lavie, Fred Valentine, Brandi
`Levin. Ychnda Mazur, Gloria Gallo.
`David Lavie. David Weiner. and
`Daniel Meruclo
`
`Janos Szehcni, Sharon M. Wahl. Mikulas
`Popovic. Larry M. Wahl, Suzanne
`Gartner, Robert L. Fine, Uros
`Skuleric, Robert M. Friedman. and
`John N. Weinstcin
`
`MICROBIOLOGY
`
`Megabase-sized linear DNA in the bacterium Bone-fin bmgdmfen‘, the Lyme
`disease agent
`
`Mehdi S. Ferdows and Alan G. Barbour
`
`5898
`
`5903
`
`5908
`
`5913
`
`5918
`
`S923
`
`5928
`
`5933
`
`5938
`
`59143
`
`5948
`
`5953
`
`5958
`
`5963
`
`5963
`
`5969
`
`S974
`
`Tumor necrosis factor a activates human immunodeficiency virus type 1
`through induction of nuclear factor binding to the NF-xB sites in the long
`terminal repeat
`
`Elia .l. Duh, Wendy J. Maury, Thomas
`M. Folks, Anthony S. Fauci, and
`Arnold B. Rabson
`
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`

`Contents
`
`Characterization of a RNA virus from the parasite Leishmania
`
`Giovanni Widmer, Anne Marie Comeau,
`Deirdre B. Furlong, Dyann F. Wirth,
`and Jean L. Patterson
`
`NEUROBIOLOGY
`
`Neuromuseular recovery using calcium protease inhibition after median nerve
`repair in primates
`
`Dynamic properties of the Ca3+{calmodulin-dependent protein kinase in
`Dmsopln'la: Identification of a synapsin l-Iiko protein
`Vasoactive intestinal polypeptide stimulates cell proliferation and adenylate
`Cyclasc activity of cultured human keratinocytes
`
`Primary structure of four allatostatins: Neuropeptide inhibitors of juvenile
`hormone synthesis
`
`Mario A. Badalamente, Lawrence C.
`Hurst. and Alfred Stracher
`H. Mitnchulat
`
`A. Haegcrstrand, B. Jonzon. C.-J.
`Dalsgaard, and J. Nilsson
`A. P. Woodhead, B. Stay, S. L. Scidel,
`M. A. Khan, and S. S. Tobe
`
`PHYSIOLOGICAL SCIENCES
`
`()smurcgulatory changes in myo—inositol transport by renal cells
`
`Renal epithelial cyst formation and enlargement in virro: Dependence on cAMP
`
`Takeshi Nakanishi, R. James Turner.
`and Maurie: B. Burg
`
`Roberto Mangoo-Karim, Marie Uchic.
`Claude txchenc, and .laretl J.
`Grantham
`
`5979
`
`5983
`
`5988
`
`5993
`
`5997
`
`6002
`
`600?
`
`vii
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`

`

`Proc. Natl. Acud. Sci. USA
`Vol. 36. pp. 5938—5942. August 1939
`Immunology
`
`Structure of an antibody—antigen complex: Crystal structure of the
`HyHEL-IO Fab—lysozyme complex
`tit-ray crystallography/comptementarity/discontinuous epitom)
`
`EDUARDO A. PADLAN*. ENID W. SILvearon". STEVEN SHERIFF“, GERSON H. COHEN3,
`SANDRA J. SMITH-Gm}, AND DAvtD R. DAVIES“
`
`"Laboratory of Molecular Biology. National Institute of Diabetes. Digestive and Kidney Diseases. and iLaboratory of Genetics. Nat ionsl Cancer Institute.
`National Institutes of Health. Bethesda. MD 30892
`
`Contributed by David R. Diaries. April 24. I 989
`
`The crystal structure of the complex of the
`ABSTRACT
`anti-lysozyme HyHEL-IO Fab and hen egg white lysozyme has
`been determined to a nominal resolution of3.0 A. The antigenic
`determinant {epitope} on the lysozyme is discontinuous, con-
`sisting of residues from four different regions of the linear
`sequence. it consists of the exposed residues of an tit-helix
`together with surrounding amino acids. The epitope crosses the
`active-site cleft and includes a tryptophan located within this
`cleft. The combining site of the antibody is mostly flat with a
`protuberance made up of two tyrosines that penetrate the cleft.
`Alt six complemenlarity-determining regions of the Fab con-
`tribute at least one residue to the binding: one residue from the
`framework is also in contact with the lysozyme. The contacting
`residues on the antibody contain a disproportionate number of
`aromatic side chains. The antibody-antigen contact mainly
`involves hydrogen bonds and van der Waals interactions; there
`is one ion-pair interaction but it is weak.
`
`The interaction of antibodies with protein antigens has been
`the subject of several recent crystallographic investigations.
`These include complexes of hen egg white lysozymc with the
`Fab fragments olthe monoclonal anti-lysozymes D1.3 l1) and
`HyHEL-S (2] and a Fab complex with influenza neuramini-
`close (3). From these data a common pattern ofinteraction is
`emerging [4) in which there is a high degree of complemen—
`tarity between the interacting surfaces of the antibody and
`antigen; the cpitopc is made up of several small. discrete
`segments of the polypeptide chain: and relatively small
`conformational changes occur in the antigen as a result of
`binding. Here we report the x-ray analysis of HyHEL-Il]
`Fab—lysozyme, in which the antigenic site differs from the
`two previous examples. The results complement the previous
`studies but differ from them in several ways.
`HyHEL-Ifl is an IgGltxJ antibody specific for hen egg
`white lysozyme. The affinity of HyHEL-Iu for hen egg white
`lysozyme. as estimated by PEG immunoprecipitation. is 1.5
`x 109 M‘1 {M. E. Benton and H. A. Scheraga. personal
`communication). slightly lower than that of HyI-IEL-S, thus
`making Hyi-lEL-lfl intermediate in affinity between HyHEL-
`5 and D13.k
`
`HyHEL-lO expresses a member of the VHJtS-oti variable
`gene segment family. the DQ52 diversity gene segment, and
`the JHJ joining gene segment in the heavy (H) chain and a
`14,23 gene and J..2 in the light it.) chain (9). Thus. HyHEL-lo
`15 structurally distinct
`from HyHEL-S {which expresses
`55%?)in I44} and D1.3 (which expresses VHQ52 and
`__________________________
`_
`_
`part b
`a e h'
`The publication costs ofthis article were defrayed in '
`payment. This article must therefore be hereby marked "atl’l'srtfisefngiff
`In accordance with 18 U.S.C. §1734 solely to indicate this fact.
`
`MATERIALS AND METHODS
`
`Crystals ofthe complex of HyI—lEL-lo Fab with hen egg white
`lysozyme, grown as described (11), exhibit the symmetry of
`s ace group P212121 with n = 57.4“}. b = 118.13. c =131.68
`{and one Fab-lysozyme complex per asymmetric unit.
`Intensity data were collected with the Mark II multiwire
`detector system at the University of California. San Diego
`{12}. The R factor relating the intensities ofsymmctry-relaled
`reflections [121 was 0.066. The data set used in the structure
`analysis had 12.501 reflections beyond 10.0-A spacings withF
`; 3olF]. These constitute about 78% of the theoretically
`observable reflections between 10.0- and 3.1-.51 spacin s: an
`additional 5% of the reflections between 3.1 and 3.0
`are
`present in this data set.
`The structure was determined by molecular replacement
`{13) using a predecessor of the program package MERLDT
`[14). Rotation and translation searches were performed in-
`dependently (15) forthe lysozyme. Fv (module containinglr'H
`and VL. the variable domains of the H and L chains}, and
`CLICHl [constant domain ofL chain/first constant domain of
`H chain) portions of the structure.
`In the search for the
`orientation of the lysozyme and the Fv. the highest peaks in
`the rotation function turned out to be the correct peaks. The
`correct peak in the rotation search for the CLKCHl was onllr
`the seventh highest. The translation search gave unambigU-
`ous results in all
`three cases. Details of the molecular
`replacement analysis will be published elsewhere (3-5--
`E.A.P.. G.H.C.. and D.R.D.). The molecular probes that
`proved useful in the analysis were hen egg white llISC'Zl’mE
`from the refinement analysis of Diamond {16) [Protein Data
`Bank (PDBl File 6LYZ], the Fv of McPC603 or) (PDB File
`1MCP], and the CLICHl of I-IyI-IEL-S t2) (PDB File ZHFLli
`The orientations and positions of the various parts oilht
`complex Were refined with CORELS {18} allowing the V1,. Vii.
`CL and CH1 domains and Iysozymc to move independently.
`The structure was then subjected to restrained least-55M“?s
`________—__________._..—
`
`Abbreviations: H, heavy; L. light: VI. and VH- “ariab'e gmmflf
`L and H chains; CL and CH1. constant domain of L chain and III‘SI.
`constant domain of H chain: Fv. module containing Vt and
`"‘
`CDR. complementarity-determining region; CDRN'L or CDRHIH'
`luh CDR of L or H chain.
`Present address: Squibb Institute for Medical Research. P-O‘ BM
`4000. Princeton, NJ 08543-4000.
`§Using l’EG immunoprecipitation at pH 7.2. Denton and Scherasg
`dclfifmtned association constants of 1.5 X llil9 Mr] and 2'5 x 15}
`M for HyHEL-It} and HyHEL-S. respectively. Lavote H ”L [ID
`delfirmlned association constants of =4 X 10" ”1‘1 and 31'” ' n
`M- at pH 3.2 by the method or Friguet ct at. (6). The “Squaw:
`constant for 01.3 Fab. also determined by the method of much!
`at. has been reported in as 4.5 x 10T M-1 at P“ 7‘4; "We ”a" l'
`an association constant of 1.3 x 10" M" was deterrninfld by “um
`cence quenching t8).
`
`5938
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`

`Immunologw Pad'an H 6““-
`
`Proc. Natl. Acad. Sci. USA 86 (i989)
`
`5939
`
`refinement using the program PROLSO (19.20) and model
`rebuilding 011 the 133515 0‘ 0“” maps (21) ”5mg “16 graphlcs
`
`program FRODO (22). The final R value was 0.24 with devi»
`ations from ideality of0.011 A for bond lengths and of 0.034
`
`
`
`tul
`FIG. 1. Stereo diagrams.
`a-Carbon trace of the Hyl—lEL-IO
`Fab—lysozyme
`complex. Lyso-
`zytne is shown in white. VL in
`yellow. VH in light blue. CL in red.
`and CH1 in dark blue. {h} Same as
`a and showing the interacting sur-
`faces:
`the surface covering the
`epitope in green and the surface
`covering the contacting residues
`from the Fab in magenta. At left.
`the complex is as it is in the crystal
`structure:
`in the middle and at
`right. the lysozyme has been sep—
`arated from the Fab by 7 A and by
`14 A. respectively. tt‘) Backbone
`of HyHEL-lt] Fv and lysozyme
`with the contacting side chains
`from HyHEL-lfl shown in red and
`those from the lysozyme shown in
`yellow. The rest of the helical re-
`gion [lysozyme residues 88—99}
`and VL are shown in light blue.
`and VH is shown in dark blue. tdl
`HyHEL-lll Fv showing the CDRs
`in yellow and the contacting resi-
`dues in red. V1, is on the left (light
`blue) and V” is on the right {dark
`blue). {6) The HyHEL-lfl epitope
`on lysozyme showing the contact-
`ing residues in red. The helical
`region 88-99 is shown in yellow
`and the rest of the lysozyme in
`. h bl
`.
`"g l
`”'3
`Pfizer EX. 1016
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`

`5940
`
`Immunology: Padlan et at.
`
`A for angle distances and with a deviation from planarity of
`0.004 it. The refined coordinates have been deposited in the
`Protein Data Bank (23) (File 3HFM). The error in atomic
`positions was estimated (24) to be 0.4 A.
`_
`Molecular surface representations were computed With the
`program MS (25) using a probe radius of 1.5 A and standard van
`der Waals radii (26). Atomic contacts were defined according
`to the criteria of Sheriff e! at. (2?). The various dornalns of
`HyHEL~10 Fab were compared with the following immuno-
`globulin structures: McPC603 and J53? (28) (PDB File IVFBJ}.
`HyHEL—fi and D1.3 (courtesy of R. Poljak, Pasteur Institute}.
`KOL {29] (PDB File 1FB4). NEW (30} (PDB File 3FA_B_}. and
`RE] {31] (PDB File IREIJ. Least-squares superposition of
`structures was accomplished with the program ALIGN (wntten
`by G.H.C.); only a carbons were used in the superpositions.
`ALIGN reports the individual deviations and the rms deviation
`between structurally equivalent pairs of atoms. The number-
`ing scheme used here for the HyHEL-ltl residues follows the
`convention of Kabat at at. (32).
`
`RESULTS
`
`Overall Structure. Fig. It: shows the a—carbon trace of the
`HyHEL-lt} Fab—lysozyme complex. The contact between
`lysozyme and HyHEL—IU involves the complementarity—
`determining regions [CDRsl of the antibody with the exterior
`ofthe lysozyrne helix (residues 88—99} and some surrounding
`amino acid residues. The two interacting surfaces (Fig. 1b} are
`strikingly complementary so that solvent is completely ex»
`cluded from the interface. The helix in the epitope is oriented
`diagonally across the combining site so that its N terminus
`interacts with the second CDR of the L chain (CDRZ-Ll
`whereas its C terminus and the segment beyond it interact
`mainly with CDRl-H and CDRZ-H (Fig. 1 c and 0'; Table 1).
`The Epitope. The lysozyme epitope for HyHEL-lO is quite
`discontinuous. consisting of residues coming from distant
`parts of the linear sequence but made contiguous by the
`folding ofthe protein. The area of lysozyme that is in contact
`with the antibody is 7T4 A3.
`The lysozyme residues that contact the antibody are His-
`15. Elly-16. Tyr-EO. and Arg-El. which are on one side ofthe
`helix: Thr-89. Assn—93. Lys-96. Lys—Q'l. and lie-98. which
`
`Table 1. HyH EL-10 residues in contact with lysozyme
`HyHEL-ll} residue“
`Lysozyme residuelsl
`
`Vt.
`Sly-30
`Asn-31 (h)
`Asa-32 (hl
`Tyr-SO
`Gin-53 (h)
`Ser-91 (rn)
`Asa-92 lm.hl
`Tyr~96 (hi
`VH
`Tbs-30"
`Set-31 {h}
`Asp-32 ls)
`
`Tyr—33 (h)
`Tyr-SO (h)
`Star-52 {h}
`Tyr-53 {h}
`Set-54
`Ber-56
`Tyr—SB (h)
`Tip-95
`
`Gly-lb
`His-15. Sly-16. Lys-96
`Gly~16. Tyr-Zt)
`Asa-93. Lys-96
`Tilt-89. Asa-93
`Tyr-Ell
`Tyr—EU. Are-21
`Arg-El
`
`Arg-TS
`Arg-TJ. Len-75
`Lys-Sl?
`
`Trp-63. Lys-W. lie-98. Set-100. Asp—101
`Arg—El. Star-100
`Asp-101
`Tip-63. Len-75. Asp-101
`Asp—101
`Asp-101. (Ely-102
`Arg-Zl. Ber-100. (Ely-102
`Arg-Zl. Lys-97, Ber-100
`
`*Nature of interaction l5 indicated in parentheses: tn. main-chain
`atoms only; h. hydrogen bonding: 5. salt bridge.
`iFramework residue.
`
`Proc. Natl. At‘ad. Sci. USA 36 U989)
`
`constitute the external surface ofthe helix; Ser—100. Asp-101.
`and Gly-102. which extend beyond the helix; Trp-63. Which
`is in the active-site cleft: and Arg-73 and Lou-75. which are
`on the other side of the cleft (Fig. la). in addition. Asn-19,
`Asn-lOJ. and Ala-107 are partly buried by the interaction with
`the antibody. although not in actual contact by the criteria we
`have used. Four of these residues participate in the contact
`with the antibody only through their main-chain atoms (His-
`15. Sly-16.
`lie-98. and Gly-ltlll. Most of the contacting
`residues are polar and five of them are charged.
`Structure of the Combining Site. The surface of HyHEL~1|J
`that
`interacts with lysozyme is unusual
`in that
`it
`is not
`noticeably concave and contains no pronounced grooves or
`cavities. On the contrary. the surface has a large protrusion.
`which fits into the active-site cleft of lysozyme. This pron-u.
`sion is formed by the side chains of Tyr-33 from CDR1~H and
`Tyr—S3 from CDRE-H (Fig. 1b). The interacting surface oflhe
`antibody contains a disproportionate number ofaromatic side
`chains that point outward and that interact with the antigen
`(Fig. 10: Table 1}. Large numbers of aromatic residues have
`also been observed in the combining sites of McPC603 (17]
`and D1.3 t1] and in the presumed binding site of the human
`class I major histocompatibility antigen A3 (33).
`All six CDRs participate in the interaction with the lyso-
`zyme. The CDRs of the L chain contribute 8 residues to the
`contract and those of the H chain contribute 10. One addi»
`tional residue from the H chain. Thrill). comes from the
`framework. CDRZ-H has the largest number of contacting
`residues with 6. while CDR3-H has only 1 [Table 1}. For 3 of
`the residues {Sly-30. Set-91. and Asa-92. all from the L
`chain). only their main-chain atoms are involved in the
`contact. Seven of the contacting residues have aromatic side
`chains: Tyr—SO and ~96 from the L chain; Tyr-33. —50. -53 and
`-58 and Trp-QS from the H chain. Only one side chain. that of
`Asp-32 of the H chain.
`is charged.
`In addition to the 19
`contacting residues. Ser-93 and Trp—94 of the L chain are
`partly buried by the interaction with the antigen. The surface
`area on the antibody that is buried by the interaction withthe
`lysozyrne is 1'20 A3.
`Conformational Changes in the Antigen. No major confor-
`mational changes occur in the structure ofthe lysozyme when
`it binds to HyHEL-IO. Comparison of the complexed lym-
`Zyme with the uncomplexed structure (coordinates of tetrag-
`003' IYSOZYMe courtesy ol'D. C. Phillips) gives a rms deviation
`of 0.47 A for corresponding at carbons. with significant differ-
`ences occurring at positions 47. 101. and 102 having deviations
`of1.44. 1.80. and 2.13 A. respectively. Larger differences are
`found for the side chains.