JANUARY 25, 1978
`
`VOLUME 253
`
`L 8
`
`ISSN 0021·925
`JBCHA3 253(2) 319-645 (197B)
`
`THE
`ourna o
`ICQ
`10 0
`
`•
`
`•
`
`Published by The American Society of Biological Chemists, Inc.
`
`A N 0 S U S T A I N E 0 I N P A R T B Y T H E C H R I S T I A N A. H E R T E R M E M 0 R I A L f U N 0
`
`f 0 U N 0 E 0 B Y C H R I S T I A N A. H E R T E R
`
`PFIZER EX. 1083
`Page 1
`
`

`

`Vol. 253, No.2
`
`The Journal of
`Biological Chemistry
`
`January 25 , 1978
`
`Copyright IC> 1978 by the American Society of Biological Chemists, Inc., 428 East Preston St., Baltimore, Md. 21202 U.S.A.
`
`CONTENTS
`
`membranes. Evidence for structural changes occurring
`during the calcium transport cycle.
`Alexander J . Murphy
`390 Phosphodiesterase activator from rat kidney cortex.
`Gordon J . Strewler, Vincent C. Manganiello , and Mar(cid:173)
`tha Vaughan
`395 Nucleotide sequence of rainbow trout (Salmo gairdneri)
`ribosomal 5.8 S ribonucleic acid.
`Ross N . Nazar and Kenneth L . Roy
`400 Transport of a nonphosphorylated nucleoside, 5'-deoxy(cid:173)
`adenosine, by murine leukemia L1 210 cells.
`David Kessel
`404 Human erythrocyte 5'-AMP aminohydrolase. Purification
`and characterization.
`Shyun-long Yun and Clarence H . Suelter
`
`COMMUNICATIONS
`
`319
`
`Increase in hepatic tyrosine aminotransferase mRNA dur(cid:173)
`ing enzyme induction by N6,0"' -dibutyryl cyclic AMP.
`Michael J . Ernest and Philip Feigelson
`323 Use of the integrated steady state rate equation to inves(cid:173)
`tigate product inhibition of human red cell adenosine
`deaminase and its relevance to immune dysfunction .
`William R . A . Osborne , Shi-Han Chen , and C. R onald
`Scott
`326 Electrogenic behavior of synaptic vesicles from Torpedo
`californica.
`R ichardS . Carpenter and Stanley M. Parsons
`330 Specific adhesion of rat hepatocytes to JJ-galactosides
`linked to polyacrylamide gels.
`Paul H . Weigel , Eli SchmelZ, Yuan C. Lee, and Saul
`Roseman
`334 Phosphorylation of cardiac troponin by guanosine 3' :5'(cid:173)
`monophosphate-dependent protein kinase.
`Donald K. Blumenthal, James T . Stull, and Gordon N.
`Gill
`337 Purified cyclic GMP-dependent protein kinase catalyzes
`the phosphorylation of cardiac troponin inhibitory sub(cid:173)
`unit (TN-I}.
`Thomas M. Lincoln and Jackie D . Corbin
`340 Isolation and partial characterization of an endogenous
`inhibitor of ceramide glycosyltransferases from rat brain.
`Elvira Costantino-Ceccarini and Kunihiko Suzuki
`343 Sequence homology of the Ca2+-dependent regulator of
`cyclic nucleotide phosphodiesterase from rat testis with
`other Ca2+-binding proteins.
`John R . Dedman, Richard L . J ackson , William E .
`Schreiber, and Anthony R . Means
`
`347 Glycosaminoglycan sulfotransferases of the developing
`chick cornea.
`Gerald W. Hart
`354 Effect of pressure and ionic strength on the self-associa(cid:173)
`tion of apo-A-1 from the human high density lipoprotein
`complex.
`Silvestro Formisano , H . Bryan Brewer, Jr ., and James
`C. Osborne , Jr .
`359 Appendix. Evaluation of volume changes in associating
`systems by sedimentation equilibrium.
`James C. Osborne, Jr .
`361 Comparison of atypical and usual human serum cholin(cid:173)
`esterase. Purification, number of 11ctive sites, substrate
`affinity, and turnover number.
`Oksana Lockridge and Bert N. La Du
`367 Mitochondrial ATPase activities of hepatoma BW7756
`and ascites tumor cells. Influence of Mg'+ ions, free fatty
`acids, and uncouplers.
`Randall L . Barbour and Samuel H . P . Chan
`377 Human blood group glycosyltransferases. I. Purification
`of N -acety lgalactosaminy ltransferase.
`Masako Nagai , Vibha Dave, Bruce E . Kaplan , and Akira
`Yoshida
`380 Human blood group glycosyltransferase. II. Purification
`of galactosyltransferase.
`Masako Nagai , Vibha Dave, Helmut Muensch, and
`Akira Yoshida
`382 Comparative studies of Hb Lepore Boston, Hb A2 , and Hb
`A.
`KazuhikoAdachi, ToshioAsakura , Frances M . Gill , and
`Elias Schwartz
`385 Effects of divalent cations and nucleotides on the reactiv(cid:173)
`ity of the sulfhydryl groups of sarcoplasmic reticulum
`
`409 Purification and properties of a third form of anthrani-
`late-5-phosphoribosylpyrophosphate
`phosphoribosyl-
`transferase from the Enterobacteriaceae.
`Michael Largen, Stanley E . Mills, Joan Rowe, and
`Charles Yanofsky
`413 On the processive mechanism of Escherichia coli DNA
`polymerase I. Quantitative assessment of processivity.
`R obert A . Bambara, Dennis Uyemura, and Theodore
`Choi
`424 Processivity of D A exonucleases.
`Kirk R . Thomas and Baldomero M . Olivera
`430 Thioredoxin from Escherichia coli. Radioimmunological
`and enzymatic determinations in wild type cells and
`mutants defective in phage T7 DNA replication.
`Arne Holmgren , Ingrid Ohlsson , and Maja-Lena Gran(cid:173)
`kvist
`437 Enzymes for RNA sequence analysis. Preparation and
`specificity of exoplasmodial ribonucleases I and II fro m
`Physarum polycephalum.
`Daniel Pilly, Amanda Niemeyer, Maurice Schmidt, and
`J . Pierre Bargetzi
`446 An induced aliphatic aldehyde dehydrogenase from the
`bioluminescent bacterium, Beneck ea harveyi. Purifica(cid:173)
`tion and properties.
`Andrew L. Bognar and Edward A . Meighen
`Intracytoplasmic membrane synthesis in synchronous cell
`populations of Rhodopseudomonas sphaeroides. Fate of
`"old" and " new" membrane.
`Donald R . Lueking, Robert T. Fraley, and Samuel
`Kaplan
`Intracytoplasmic membrane synthesis in synchronous cell
`populations of Rhodopseudomonas sphaeroides. Poly(cid:173)
`peptide insertion into growing membrane .
`Robert T . Fraley, Donald R . Lueking, and Samuel
`Kaplan
`465 Synthesis of photopigments and electron transport com·
`ponents in synchronous phototrophic cultures of Rhoda(cid:173)
`pseudomonas sphaeroides.
`Colin A. Wraight , Donald R. Lueking, Robert T . Fraley,
`and Samuel Kaplan
`472 Biosynthesis of peptidoglycan. Definition of the microen·
`vironment of undecaprenyl diphosphate-N-acetylmura·
`myl-(5-dimethylaminonaphthalene-1-sulfonyl) pentapeP"
`tide by fluorescence spectroscopy.
`William A . Weppner and Francis C. Neuhaus
`y
`Identification of bound pyruvate essential for the activj
`of phosphatidylserine decarboxylase of Escherichia co '·
`Michel Satre and Eugene P . Kennedy
`Independence of 1,25-dihydroxyvitamin D3-mediated c~l­
`cium transport from de novo RNA and protein synt~esJs.
`Daniel D. Bikle, David T . Zolock , Robert L . Mornssey,
`and Robert H . Herman
`
`1t
`
`479
`
`484
`
`451
`
`458
`
`Full Instructions to Authors will be found in THE JouRNAL, 253, 1 (1978), and reprints may be obtained on request from the editorial office.
`
`2
`
`PFIZER EX. 1083
`Page 2
`
`

`

`Reconstitution of the apoenzyme of cytochrome oxidase
`from Pseudomonas aeruginosa with heme d 1 and other
`heme groups.
`Kristina E . Hill and David C. Wharton
`A unifying mechanism for sti mulation of mammalian
`pyruvate dehydrogenase. kinase by reduced nicotinamide
`adenine dinucleotide, dihydrolipoamide, acetyl coenzyme
`A, or pyruvate.
`Richard L. Cate and Thomas E. Roche
`Purification and characterization of human erythrocyte
`purine nucleoside phosphorylase and its subunits.
`Vassilis Zannis , Deborah Doyle , and David W. Martin ,
`Jr .
`Interaction between DNA and Escherichia coli Protein w.
`Formation of a complex between single-stranded DNA
`and w protein.
`Richard E . Depew , Leroy F . Liu, and James C. Wang
`Preparation and properties of the major copper-binding
`component in human fetal liver. Its identification as
`metallothionein.
`Lars Ryden and Harold F . Deutsch
`Molecular weights of aggregation states of Busycon he(cid:173)
`mocyanin.
`Sharon Quitter, Laurel A. Watts, Carol Crosby, and
`Robert R oxby
`Radioimmunoassay and ·characterization of enkephalins
`in rat tissues.
`Richard J . Miller, Kwen-Jen Chang, Barret Cooper, and
`Pedro Cuatrecasas
`Inhibition of mammalian S-adenosylmethionine decar(cid:173)
`boxylase activity by 1,1 '-[(methylethanediylidene)-dini(cid:173)
`trilo]bis(3-aminoguanidine).
`Anthony E . Pegg
`5,6,7,8-Tetrahydrofolic acid. Conformation of the tetra(cid:173)
`hydropyrazine ring.
`Martin Poe and Karst Hoogsteen
`Interactions of a ,-antitrypsin with trypsin and chymo(cid:173)
`trypsin.
`James W. Bloom and Margaret J . H unter
`
`Purification and properties of a heat-stable protein inhib(cid:173)
`itor of phosphoprotein phosphatase from rabbit liver.
`Ramji L . Khandelwal and Soni M. Z inman
`Gene 4 protein of bacteriophage T7. Purification, physical
`properties, and stimulation of T7 DNA polymerase during
`
`3
`
`the elongation of polynucleotide chains.
`Richard Kolodner, Yukito Masamune , J . Eugene Le(cid:173)
`Clerc , and Charles C. Richardson
`574 Gene 4 protein of bacteriophage T7. Characterization of
`the product synthesized by the T7 DNA polymerase and
`gene 4 protein in t he absence of ribonucleoside 5' -triphos(cid:173)
`phates.
`Richard Kolodner and Charles C. Richardson
`585 Preliminary refinement and structural analysis of the
`Fab fragment from human immunoglobulin New at 2.0 A
`resolution.
`Frederick A . Saul, L . Mario Amzel, and Roberto J .
`Poljak
`598 Purification of interferon from mouse Ehrlich ascites
`tumor cells.
`Masao Kawakita , Bartolome Cabrer, Hideharu Taira,
`Moacyr Rebello, Elizabeth Slattery, Hansjorg Weideli , and
`Peter Lengyel
`603 Cellular RNA sy nthesis in normal and mengovirus-in(cid:173)
`fected L-929 cells.
`James W. Apriletti and Edward E . Penhoet
`612 Nucleotide sequence of t he DNA encoding t he 5'-terminal
`sequences of simian virus 40 late mRNA.
`Ravi Dhar, V . Bhaskara Reddy, and Sherman M . Weiss(cid:173)
`man
`621 Nucleotide sequence of t he genes for the simian virus 40
`proteins VP2 and VP3.
`V . Bhaskara Reddy, Ravi Dhar, and Sherman M . Weiss(cid:173)
`man
`631 Production of a non-immunoglobulin t hyroid stimulator
`by human lymphocytes during mixed culture with human
`thyroid cells.
`Basil Rapoport, Rao J . Pillarisetty , Elizabeth A . Her(cid:173)
`man, Orlo H . Clark , and Evangeline G. Congco
`641 Corticosteroid suppression of lymphocytic thyroid stimu(cid:173)
`lator production.
`Elizabeth A . Herman , Rao J . Pillarisetty , and Basil
`Rapoport
`
`ADDITIONS AND CORRECTIONS
`
`645 DNA polymerase III holoenzyme of Escherichia coli.
`Purification and resolution into subunits. Vol. 252 (1977)
`6478-6484.
`Charles McHenry and Arthur Kornberg
`
`NOTE: EFFECTIVE J ANUARY 1, 1978, THE MANUSCRIPT HANDLING CHARG E IS 0 LO GER REQUIRED.
`
`THE JOURNAL OF BIOLOGICAL CHEMISTRY
`
`GENERAL INFORMATION
`
`MANUSCRIPT SUBMISSION AND PAGE CHARGES
`
`Submit manuscripts of full papers in duplicate and Communi(cid:173)
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`
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`on matters of general editoria l policy, requests for re(cid:173)
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`tion fulfillment, and orders for back copies to: THE JouRNAL OF
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`
`PFIZER EX. 1083
`Page 3
`
`

`

`THB JOURN AL OF BIOLOGICAL CHEMISTRY
`Vol. 253, No. 2, Issue of Janua ry 25, pp. 585-597, 1978
`Printed in U.S.A.
`
`Preliminary Refinement and Structural Analysis of the Fab
`Fragment from Human Immunoglobulin New at 2.0 A
`Resolution*
`
`(Received for publication, July 7, 1977)
`
`FREDERICK A. SAuL, L. MARIO AMZEL, AND RoBERTO J. PowAK:J:
`From the Department of Biophysics, The Johns Hopkins University School of Medicine , Baltimore,
`Maryland 21205
`
`The three-dimensional structure of t he Fab fragment
`from human myeloma lgG New has been refined using
`"model building" and " real space" procedures. By these
`techniques, the correlation between the amino acid se(cid:173)
`quences and the 2.0 A resolution multiple isomorphous
`replacement Fourier map has been optimized. The average
`shift of all atoms during real space refinement was 0.62 A. A
`list of the refined atomic coordinates for the 440 amino acid
`r-esidues in the structure is given. Ramachandran plots
`prepared using the refined coordinates show a distribution
`of c/> , 1/J angular values which corresponds to the predomi(cid:173)
`nant {l-pleated sheet conformation present in the structure.
`The structures of the homology subunits VH, VL, CHI, and
`CL were superimposed by pairs and quantitatively com(cid:173)
`pared. The closest similarities were observed between VH
`and V L and between CHI and CL. Amino acid sequence
`alignments obtained from this structural superposition are
`given. The closest sequence homology in Fab New is ob(cid:173)
`served between CHI ('Y heavy chain) and CL (A. light chain).
`In addition, there is considerable homology between the
`variable and constant regions.
`The distances of close contacts between the homology
`subunits of Fab New have been determined. The closer
`contacts, those between atoms at a distance :5 1.2 times their
`van der Waals radii , are analyzed in relation to the constant,
`variable, and hypervariable nature of the immunoglobulin
`sequence positions at which they occur. Most of the residues
`which determine the closer contacts between V Hand VL and
`between CHI and CLare structurally homologous and highly
`conserved or conservatively replaced in immunoglobulin
`sequences.
`The relation between idiotypic determinants, antigen
`combining site and hypervariable regions, is discussed in
`terms of the refined model.
`
`In this paper we present the results of a preliminary
`crystallographic refinement and a list of atomic coordinates of
`
`• This work was supported by Research Grant AI 08202 from the
`National Institutes of Health and by Research Grants NP-141B and
`IM-105C from the American Cancer Society. The costs of publication
`of this article were defrayed in part by the payment of page charges.
`This article must therefore be hereby marked "advertisement" in
`accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
`t Recipient of United States Public Health Service Research
`Career Development Award AI-70091.
`
`Fab New.' Some features of the refined structure are discussed
`in relation to the genetic control and physiological function of
`immunoglobulins.
`It is generally accepted (see review in Ref. 1) that electron
`density maps calculated by multiple isomorphous replacement
`techniques contain significant errors which may lead to impre(cid:173)
`cise determination of structural details such as the location of
`amino acid side chain atoms, bond angles, cp and t/J values, cis
`or trans character of proline residues, etc. This refinement
`project was undertaken with the aim of obtaining more
`accurate coordinates which can be applied to structural studies
`of other immunoglobulins and Fab ·hapten complexes (2). The
`starting atomic coordinates were those of the structure previ(cid:173)
`ously described (3, 4) obtained using multiple isomorphous
`heavy atom replacements. Since the complete refinement of
`the structure of Fab New is a complex undertaking, we
`present here initial results obtained after application of two
`consecutive refinement techniques. In the first step we have
`applied a "model building" procedure (5) in which the mea(cid:173)
`sured atomic coordinates were adjusted to impose standard
`bond lengths and bond angles. In a second step a "real space"
`procedure (6, 7) was used to optimize the correlation between
`the Fab New model and the multiple isomorphous replace(cid:173)
`ment, electron density Fourier map.
`The coordinates obtained by these proc~ures have provided
`an improved model which has been used to compare the
`tertiary structure of the homology subunits, to calculate
`interatomic contacts that define the quaternary structure of
`Fab, and to re-examine the conformation of the combining
`site.
`
`METHODS
`Measurement of Model Coordinates -Atomic coordinates were
`measured on the 2 A (nominal) resolution model previously de(cid:173)
`scribed (4). A two-pointer device was used for this purpose: a
`horiwntal pointer (50 inches long) was brought to touch atom
`centers by displacing the device on the base of the model, adjusting
`the height of the pointer along a graduated scale (z coordinate)
`while a second, parallel, fixed pointer of equal length gave the x, y
`coordinates on a grid at the base of the model. The use of a level and
`leveling screws at the base of the two-pointer device was essential
`for obtaining reproducible coordinates. While these measurements
`were made, the image of the model and the atom centers were
`
`' The abbreviations used for immunoglobulins, their polypeptide
`chains, and fragments are as recommended in (1964) Bull. W H 0
`30, 447.
`
`585
`
`PFIZER EX. 1083
`Page 4
`
`

`

`586
`
`Structural Refinement of Fab New
`
`projected on the corresponding sections of the Fourier map using an
`optical comparator (8) to verify that their location and the coordinate
`values corresponded with the Fourier map.
`Model Building Procedure-The set of measured coordinates for
`the 3185 non-hydrogen atoms in the structure provided the starting
`point for this procedure. In general , these coordinates are subject to
`errors due to measurement uncertainty and to mechanical deforma(cid:173)
`tions of the skeletal brass model. Consequently, the mathematical
`model building procedure of Diamond (5) programmed for a digital
`computer was used in order to impose standard bond lengths and
`bond angles in the model. The measured coordinates were used to
`provide a guide point for each (non-hydrogen) atom in the structure.
`Some conditions used in this part of the refinement process are given
`in Table I. All the varied angles are dihedral; the T0 (TN-{;<>-C) angle
`was allowed to vary since this condition gave a much closer correla(cid:173)
`tion with input coordinates without introducing large distortions in
`the idealized, model-built geometry of the molecule. Residues for
`which the model-built coordinates differed considerably from the
`input coordinates or which had an abnormal T0 value were remeas(cid:173)
`ured and checked for correspondence with the Fourier map. These
`discrepancies could always be traced to errors in the measurement
`of coordinates or to distortions of the brass model. When necessary
`the coordinates were measured again after rebuilding distorted
`regions and the remeasured coordinates were submitted to the model
`building procedure as guide points. The final average value of
`t:rr.(t:rr. = IT. - 109.3°1) for the 440 residues in the model-built
`structure was 5.43°. Pro 151 in (;,;1 was built and refined in the cis
`conformation. The root mean square shift from the initial coordi(cid:173)
`nates for all non-hydrogen atoms in the structure was 0.2 A.
`Real Space Refinement- The model-built coordinates were used
`as a starting set for real space refinement (6). The 2.0 A electron
`density map used for the automatic fitting of the atomic coordinates
`was calculated using multiple isomorphous replacement phases as
`described before (3, 4). The electron density function was calculated
`at intervals of 1/160 along x, (a = 111.43 Al, 1/80 along y (b = 56.68
`Al, and 1/130 along z (c = 90.30 Al in sections of constant y . A
`computer program incorporating a fast-Fourier transform algorithm
`was used for this calculation. Five cycles of real space refinement
`were carried out using the conditions defined in Table II. Values of
`atomic radii for carbon, nitrogen, oxygen, and sulfur that gave
`fastest convergence in trials using a small part of the structure were
`adopted and kept constant during refinement. Progress in the
`refinement process was followed by inspection of the root mean
`square shifts in coordinates, shifts in the coordinates of individual
`atoms, and adjustments of amino acid scale factors. Unusually large
`shifts in coordinates were checked using the optical comparator and
`the Fourier map. All refinement calculations were carried out using
`computer programs implemented at the Brookhaven National Lab(cid:173)
`oratories.
`
`RESULTS AND DISCUSSION
`
`The root mean square shifts of atomic coordinates after five
`cycles of real space refmement converged to an average value
`of0.09 A. The values after each cycle were: 0.46 A, 0.20 A, O.I3
`A, 0.10 A, and 0.09 A. The average shift of all atoms during
`real space refinement was 0.62 A. In most parts of the
`molecule, the coordinates after refinement are in very good
`agreement with the features of the electron density map (Fig.
`
`TABLE I
`Conditions of model building refinement"
`Parameters varied were: cf> , .p , x, T (N Ca C). Flexible proline
`residues were used . In addition, T(Ca C{3 Cy) was allowed to vary in
`Cys, His, Phe, Trp, and Tyr residues. A list of the sources of the
`amino acid groups used in model building is given in Table I of
`Diamond (7).
`
`TABLE II
`Conditions of real space refinement •
`
`5
`Zone length:
`6
`Margin width :
`Fixed atomic radii : 1.4 A
`Relative atomic weights; C:6, 0 :7, N:8, S:16
`Relative softness of angular parameters that were allowed to vary:
`</>, .p: 4.0
`x: 3.2
`Filter levels:
`
`Am in/ "-mu.
`A mi n
`0.01
`0.0001
`Scale factor and background
`0.01
`0.001
`Translational refinement
`0.001
`0.001
`Rotational refinement•
`Electron density map grid; 111.43/160, 56.68/80, 90.30/130 along cell
`edges
`" See Diamond (6, 7) for definition of terms.
`• Nonlinear constraints were used to preserve chain continuity.
`
`I). Many carbonyl groups of the main polypeptide chain can
`be reliably positioned (Fig. 2) . In general, coordinates of atoms
`in regions of strong, well defined electron density converged
`rapidly in the first cycle of refinement and moved very little
`in subsequent cycles. Atoms in regions of lower density
`converged more slowly. A few residues in poorly defined
`regions of low density showed little convergence, although
`their total shift from the starting coordinates was small. The
`movement of main chain atoms tended to be smaller than
`those of side chains, presumably due to their better defmed
`electron density and to greater constraints on their positions.
`The progress of refinement was checked after each cycle by
`inspecting the fit of atoms whose shifts were substantially
`greater than the average. Some side chain groups which had
`been shifted by the Diamond model building procedure were
`moved back to their original positions by real space refine(cid:173)
`ment. ln the fifth cycle of refinement, an average shift greater
`than I A occurred for three consecutive amino acid residues
`(Gly I66, Val I67, and His I68) in the CHI region. Inspection
`of the position of these atoms showed that they had moved to
`a conformation that appears to be in better agreement with
`the electron density map than the original model. The coordi(cid:173)
`nates for all atoms of Fab New after refinement, given in the
`"Appendix," are filed with the Protein Data Bank at Brook(cid:173)
`haven National Laboratory. No major features of the map
`remain unexplained, although a number of possible solvent
`molecules are found on the surface of the molecule . The
`conventional R factor! based on Fe obtained with the coordi(cid:173)
`nates in "Appendix" and an overall temperature factor' (B =
`I8.0), is R = 0.46. This value is reasonable given the refine(cid:173)
`ment approximations outlined in Table 11 and the fact that no
`solvent atoms were included in the model. Further refinement
`using observed structure factors and calculated phases is
`currently underway.
`The S-S distances in the five Fab disulfide bonds were
`allowed to vary without constraints. At the end of the refine(cid:173)
`ment these distances were found to be: V ,., 2.00 A; V L• 2.46 A;
`CHI, 2.30 A; CL, 2.30 A; CHI-CL, 2.43 A.
`
`Ramachandran Plots
`
`The Ramachandran plots of the V L and CL homology sub-
`
`2 R = i I F. - Fe I / iF., where F. is observed structure amplitude
`and Fe is calculated structure amplitude.
`3 The isotropic temperature factor (B ) used in the expression exp
`(- B sin2 6/"A 2).
`
`Probe
`
`Len£h
`(resi ues)
`
`Filter constants
`C,
`C,
`0.1
`1
`1
`0.1
`2
`2
`0.1
`3
`3
`• See Diamond (5) for definition of terms.
`
`lQ- 4
`lQ- 4
`lQ- 4
`
`PFIZER EX. 1083
`Page 5
`
`

`

`Structural Refinement of Fab New
`
`587
`
`FIG. 1 (upper). Stereo view of some main chain and side chain
`groups of Fab New after five cycles of real space refinement super(cid:173)
`imposed on the corresponding electron density of the multiple
`isomorphous replacement 2 A resolution map.
`
`FIG. 2 (lower). Stereo view of some amino acid residues of Fab
`New superimposed on the corresponding density of the multiple
`isomorphous replacement map. Carbonyl groups of the main poly(cid:173)
`peptide chain are clearly seen.
`
`•
`coo
`
`\
`I
`• I •
`I
`
`•
`
`/
`
`• I
`I •
`-I
`
`I
`
`• I
`
`•
`
`. •
`.
`. ....
`. ";·,f··
`. ... I
`. •"' ...
`..
`..
`~·
`0 ..
`. /
`I
`.
`I
`I
`'
`'----- - ~~·
`/- --.-,
`-- -- .
`.,
`-
`.:•
`----- -- - - -- - -
`
`0
`
`I
`
`/
`
`/
`
`~
`
`I
`
`----- - ...... -. - -,
`
`0
`
`60
`
`120
`
`360
`
`300
`
`240
`
`180
`
`"'
`
`120
`
`60
`
`0
`
`VL
`
`/ 1 •
`/
`I•
`:
`I
`I
`I
`'1
`I
`I
`I
`\
`._I
`
`•
`
`•
`•
`
`oo
`
`360
`
`300
`
`240
`
`180
`
`"'
`
`120
`
`60
`
`.. .
`. . . . . '"'-,· .
`·'
`<f'•;¥-":• • o l
`• •
`I •.
`I
`I , ..
`.· : •
`I
`•. . . •
`I .
`
`I
`I
`I
`I
`'
`I _______ I
`
`/
`
`r -----
`I
`••
`I
`I
`I
`f----------1
`
`/
`
`/
`
`. .
`
`180
`
`240
`
`300
`
`360
`
`0
`
`0
`
`.
`
`-- ---- -- - '
`
`.
`
`'
`
`60
`
`120
`
`CL
`
`/ /l
`I "
`I
`I
`1
`I
`I
`I
`I
`I
`I
`,_1
`I
`
`. .
`
`180
`
`240
`
`300
`
`360
`
`"'
`"'
`F.IG. 3. Ramachandran plots of the VL and c,_ homology subunits ofFab New. The distribution ofc/>, 1/J angles indicates the predominant
`antlparallel /3-pleated sheet structure of the subunits. Glycine residues are indicated by 0 .
`
`Units (see Fig. 3) show a distribution of cp,
`tjJ angles which
`COrresponds with the predominant anti parallel {3-pleated sheet
`structure present in V H• V L• CHl, and CL. As observed in
`several other protem structures, the cp, tjJ angles for glycine
`
`residues are scattered in the plot, frequently appearing in
`nonallowed regions for L-amino acids. Several other residues
`which occur outside areas of allowed conformation are found
`in bends of the polypeptide chains; it is expected that further
`
`PFIZER EX. 1083
`Page 6
`
`

`

`588
`
`Structural Refinement of Fab New
`
`refinement will improve the angular values observed for these
`residues.
`
`Structural Comparison of Homology Subunits
`The structural homology of V H> V L• CHI, and CL has been
`described before (3, 4). A quantitative analysis of this homol(cid:173)
`ogy using the method of Rao and Rossmann (9) is presented
`here. A similar analysis has been made by Richardson et al.
`(10) comparing the structures of superoxide dismutase and
`the murine Fab McPC 603 fragment .
`Initial matrices relating theCa coordinates of the homology
`subunits were obtained from a small number of structurally
`equivalent amino acids. The number of equivalences was
`then extended by an automatic search for stretches of poly(cid:173)
`peptide chain for which the distances between putatively
`equivalent Cas was smaller than 3.8 A. Based on the extended
`equivalences new matrices were calculated and the process
`was iterated until no changes i~ equivalences were observed.
`A summary of the results is presented in Table lll which lists
`the number of Cas occurring at distances of less than 1.5 A
`and less than 3.0 A, for all the six possible pairings of
`subunits which were superimposed and compared by this
`process. The average value of the minimum base change
`necessary to exchange the codons of the structurally equiva(cid:173)
`lent amino acids is also given in Table lll.
`As can be seen in Table Ill, there is an even closer structural
`homology between VH, VL, CHI, and CL in Fab New than that
`observed for McPC 603 Fab (IO), probably reflecting the
`higher resolution of the Fab New model. Presumably the Ca
`distances given in Table UI could become smaller with further
`crystallographic refinement. The number of Cas which super(cid:173)
`impose with distances shorter than 1.5 A and 3.0 A is larger
`when comparing V H to V L and CHI to CL. Also, there is good
`(inverse) correlation between the number of Cas that are
`structurally equivalent and the average minimum base
`change per codon. Furthermore, when a restrictive condition
`for structural equivalence is imposed ( dca-cu ~ 1. 5 AJ the
`average base change per codon becomes smaller, reflecting a
`higher degree of conservation of amino acid sequences.
`It should be emphasized here that amino acid sequence
`information is not used in the quantitative three-dimensional
`alignment procedure described above. However, this proce(cid:173)
`dure leads to amino acid sequence alignments that clearly
`reflect the well established homologies between the VH and
`VL , and between the ~ and Ct. regions of immunoglobulins
`(see Figs. 4 and 5). The closest sequence similarity in Fab
`New occurs between ~l(y) and ~(A), although, as shown in
`Table Ill, the structural similarity between VH and VL is close
`to that between ~I and ~ (see Figs. 6, 7, and 8). In addition,
`
`TABLE III
`Alignment of a·carbon coordinates of four homology subunirs of Fab
`(New) using method of Rao and Rossmann (9)
`Average
`Average
`Number of
`Number of
`c(l pairs
`minimum
`Ca pairs
`minimum
`equiva-
`base change
`base change
`equiva-
`lenced with per codon for
`lenced with per codon for
`dca-caAs 3.0
`dca-caAs 1.5
`dc. -cAS 3.0
`d c.-cAS 1.5
`
`Subunits
`
`VwVL
`CHI-CL
`cL.vL
`CL-VH
`cHJ.vL
`CHI-VH
`
`56
`60
`40
`29
`27
`25
`
`0.98
`0.71
`1.03
`1.04
`1.04
`1.29
`
`81
`82
`66
`59
`58
`49
`
`0.97
`0.80
`1.23
`1.28
`1.24
`1.40
`
`Table III shows that there is considerable homology between
`the V and C regions. These results can be interpreted to
`indicate that all the homology regions contain a basic core of
`amino acid residues with highly preserved three-dimensional
`structure. The chemical nature of these residues is also
`preserved as indicated by the correspondingly lower values of
`the average base change per codon. As stated before (3) these
`findings strongly support the postulate (15) of a gene duplica(cid:173)
`tion mechanism which gave rise to the different homology
`regions of immunoglobulins.
`The Cas of the homologous sequences, -Phe-Gly-Gly-Gly(cid:173)
`(99 to 102) in VL and -Trp-Gly-Gln-Gly- (107 to 110) in VH, can
`be closely superimposed as can Ca atoms immediately preced(cid:173)
`ing and following those residues. This conserved conformation
`gives no evidence supporting the postulate (16) that the
`glycine residues could serve as a pivot to allow for optimal
`contacts between an antibody and its ligands. An alternative
`explanation for these constant, homologous VH and VL se(cid:173)
`quences has been proposed (4) in terms ofintersubunit (VH to
`VL, see below) and intrasubunit contacts.
`The limits between the V and C homology regions can be
`defined from the model. The sequence -Val-Ser-Ser- (115 to
`117, Fig. 4) which is shared byy and p.. human H chains marks
`the COOH terminus ofVH, and following a sharp bend in the
`polypeptide chain the sequence -Ala-Ser-Thr (118 to I20)
`marks the NH2 terminus of~l. The sequence -Thr-Val-Leu(cid:173)
`(I06 to 108) corresponds to the COOH terminus of VL and the
`residues -Gln-Pro-Lys- (110 to 112) constitute the NH2 termi(cid:173)
`nus of ~· Thus, in the three-dimensional model Arg I09
`(usually assigned to VL) could be considered either as the end
`of VL or as the beginning residue of~. By the structural
`alignment described here however, Arg 109 can