JANUARY 25, 1978

THE

L 8

VOLUME 253

ISSN 0021·925 JBCHA3 253(2) 319-645 (197B)

ourna o • •

10 0 ICQ

Published by The American Society of Biological Chemists, Inc.

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

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

PFIZER EX. 1083 Page 1

Vol. 253, No.2 The Journal of January 25 , 1978

Biological Chemistry Copyright IC> 1978 by the American Society of Biological Chemists, Inc., 428 East Preston St., Baltimore, Md. 21202 U.S.A.

CONTENTS COMMUNICATIONS

319 Increase in hepatic tyrosine aminotransferase mRNA dur­ing enzyme induction by N6,0"' -dibutyryl cyclic AMP.

Michael J . Ernest and Philip Feigelson

323 Use of the integrated steady state rate equation to inves­tigate 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 '­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 sub­unit (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-associa­tion 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 cholin­esterase. 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 reactiv­ity of the sulfhydryl groups of sarcoplasmic reticulum

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

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' -deoxy­adenosine, by murine leukemia L1 210 cells.

David Kessel

404 Human erythrocyte 5'-AMP aminohydrolase. Purification and characterization.

Shyun-long Yun and Clarence H . Suelter

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 Gran­kvist

437 Enzymes for RNA sequence analysis. Preparation and specificity of exoplasmodial ribonucleases I and II from Physarum polycephalum.

Daniel Pilly, Amanda Niemeyer, Maurice Schmidt, and J . Pierre Bargetzi

446 An induced aliphatic aldehyde dehydrogenase from the bioluminescent bacterium, Beneckea harveyi. Purifica­tion and properties .

Andrew L. Bognar and Edward A . Meighen

451 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

458 Intracytoplasmic membrane synthesis in synchronous cell populations of Rhodopseudomonas sphaeroides. Poly­peptide 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 Rhoda­pseudomonas 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

479 Identification of bound pyruvate essential for the activj1ty

of phosphatidylserine decarboxylase of Escherichia co '· Michel Satre and Eugene P . Kennedy

484 Independence of 1,25-dihydroxyvitamin D3-mediated c~l­cium transport from de novo RNA and protein synt~esJs.

Daniel D. Bikle , David T . Zolock , Robert L . Mornssey, and Robert H . Herman

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 he­mocyanin.

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 decar­boxylase activity by 1,1 '-[(methylethanediylidene)-dini­trilo]bis(3-aminoguanidine).

Anthony E . Pegg

5,6,7,8-Tetrahydrofolic acid. Conformation of the tetra­hydropyrazine ring.

Martin Poe and Karst Hoogsteen

Interactions of a ,-antitrypsin with trypsin and chymo­trypsin.

James W. Bloom and Margaret J . H unter

Purification and properties of a heat-stable protein inhib­itor 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

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' -triphos­phates.

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- in­fected 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. Weiss­man

621 Nucleotide sequence of t he genes for the simian virus 40 proteins VP2 and VP3.

V . Bhaskara Reddy, Ravi Dhar, and Sherman M. Weiss­man

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 . Her­man, Orlo H . Clark , and Evangeline G. Congco

641 Corticosteroid suppression of lymphocytic thyroid stimu­lator 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.

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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 se­quences 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 predomi­nant {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 com­pared. 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 ob­served 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 impre­cise 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 previ­ously 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 mea­sured 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 replace­ment, 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 de­scribed (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 deforma­tions 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 correla­tion 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 remeas­ured 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 coordi­nates 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 Lab­oratories.

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).

Len£h Filter constants

Probe (resi ues) C, C,

1 1 0.1 lQ- 4

2 2 0.1 lQ- 4

3 3 0.1 lQ- 4

• See Diamond (5) for definition of terms.

TABLE II

Conditions of real space refinement•

Zone length : 5 Margin width : 6 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: A mi n Am in/ "-mu.

Scale factor and background 0.0001 0.01 Translational refinement 0.001 0.01 Rotational refinement• 0.001 0.001

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 refine­ment. 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 coordi­nates for all atoms of Fab New after refinement, given in the "Appendix," are filed with the Protein Data Bank at Brook­haven 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 coordi­nates in "Appendix" and an overall temperature factor' (B =

I8.0), is R = 0.46. This value is reasonable given the refine­ment 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 refine­ment 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).

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 super­imposed on the corresponding electron density of the multiple isomorphous replacement 2 A resolution map.

VL 360 . • . \ . .... I • . ";·,f·· • I •

~· coo I . ... I

300 . •"' ... • I .. .. • I •

0 .. -I / 1 •

• . / / / I• I : I 240 • I I I I I I '1 I I I ' \ •

'------ ~~· ._I . • • "'

0 180

/- --.-, I ~ oo - /

/ ., -- -- . I

120 .:• ----- -- - - -- - -

60

0 ----- - ...... -. - -,

0 60 120 180 240 300 360

"'

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 poly­peptide chain are clearly seen.

CL 360 .. .

·' . . . . . '"'-,· . <f'•;¥-":• • o l

• • I •. I 300 .· : • I •. . . • I , .. / /l

/

I I " I

I 1 I 240 I I I I I I I

' I . I I I _______ I ,_1

"' 180

r -----/ I . . / •• I

I I 120

f----------1

. . 60

. --------- ' . ' 0

0 60 120 180 240 300 360

"' 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 homol­ogy 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 poly­peptide 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 equiva­lent 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 super­impose 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 proce­dure 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)

Number of Average Number of Average c(l pairs minimum Ca pairs minimum

Subunits equiva- base change equiva- base change lenced with per codon for lenced with per codon for d c.-cAS 1.5 dca-caAs 1.5 dc. -cAS 3.0 dca-caAs 3.0

VwVL 56 0.98 81 0.97 CHI-CL 60 0.71 82 0.80 cL.vL 40 1.03 66 1.23

CL-VH 29 1.04 59 1.28 cHJ.vL 27 1.04 58 1.24 CHI-VH 25 1.29 49 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 duplica­tion mechanism which gave rise to the different homology regions of immunoglobulins.

The Cas of the homologous sequences, -Phe-Gly-Gly-Gly­(99 to 102) in VL and -Trp-Gly-Gln-Gly- (107 to 110) in VH, can be closely superimposed as can Ca atoms immediately preced­ing 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 se­quences 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(I06 to 108) corresponds to the COOH terminus of VL and the residues -Gln-Pro-Lys- (110 to 112) constitute the NH2 termi­nus 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 be properly considered as the COOH terminus of VL.

In agreement with the Gm(4-) serological specificity oflgG New (17), a lysine residue is placed at position 2I4 in ~ 1. This residue, corresponding to the Gm(17) allotype provides a better fit with the Fourier map than an arginine residue (which correlates with Gm(4)) at that position. This interpre­tation is reinforced by the Gm(l +) serological specificity of IgG New (I7) which has been verified by amino acid sequence analysis (12).

Quaternary Structure

Contacts between Homology Subunits- The closer contacts between the homology subunits of Fab New are diagrammat­ically represented in Fig. 9 by lines joining Ca atoms sepa­rated by a distance of 8 A or less. This figure provides a description of regions of VH, VL, ~ 1, and ~ in which there are higher density of contacts. Inspection of Figs. 8 and 9 indicates that the interactions between VH and VL and be­tween Ct1 I and ~ are more extensive than those between VH and ~I and those between VL and~ · The fact that the VH and ~ 1 subunits (whose major axes make an angle smaller than 90°) interact more extensively than VL and ~ (whose major axes makes an angle larger than 90°) is also reflected in Figs. 8 and 9.

Intersubunit contacts between side chain and main chain atom~ situated at a distance not larger than 1.2 times their van der Waals radii are given in Table IV. This table lists contacting residues and the number of close contacts that atoms from a given residue make with atoms of other residues. Evidently , amino acids with larger side chains have a poten­tial to make more contacts with other amino acids, thus for example, VH Trp 107 makes 29 intersubunit contacts, Trp 47

PFIZER EX. 1083 Page 7

Structural Refinement of Fab New 589

10 20 27obc 30 CO 50 ----SSVLTQPPSVSGAP-GQRVTISCTGSSSMJGAG.BVKWYQQLPGTAPK-LLIPBW.AR*

10 20 30 co 50 60 ----ZVQLZQSGPGLVRP-SQTLSLTCTVSGSTPSMD-YYTWVRQPPGRGLBWIGYVPYBGTIDTD

110 120 130 leo 150 CL QP~AAPSVTLPPPSSBBLQAN~ATLVCLISDPYPGAV-TVAW~--Aoss-----------------

120 130 uo 150 160 ASTKGPSVPPLAPSSJtSTSGGTAALGCLVI.DYPPIPV-TVSWif---SG------------------

61 70 80 90 100 109 ---PSVSJtSG----------SSATLAITGLQABDBADYYCQSYDRSLR**-VPGGGTJtLTVLR

70 80 90 100 110 117 TPLRSRVTIILVRT-S-------JtWQPSLRLSSVTAAOTAVYYCARNLIAG-CIDVWGQGSLVTVSI

160 170 180 190 no -PVXA--GVBTTTPSXQS •• XYAASSYLSLTPBQWXSBXSYSCQVTB--BGST-VBXT-VAPTBCS

170 180 190 200 no 220 CH1 -ALTS--GVBTPPAVLQSSGLYSLSSVVTVPSSSLGT-QTYic•v•ax•s•Tx-vox~-v••xsc

FIG. 4. Amino acid sequences of the VL , c._ , VH, and C, 1 homology regions ofFab New aligned by comparison of their three-dimensional Btructures. - - - indicate gaps introduced to maximize alignment of the three-dimensional structures. * indicate deletions in the VL sequence. See Ref. 11 for the VL and Cc sequences, Ref. 12 for VH, and Ref. 13 for C., 1. Abbreviations for amino acids are as given in Ref. 14.

makes 28 contacts and Arg 43 makes 24 contacts. . The contacts between VH and VL are of particular interest hl view of the fact that different H and L immunoglobulin chains can form structurally viable pairs. Three types of Vw

FIG. 5. Diagram of hydrogen bond­ing (broken lines) between main chain atoms for the VL, Cc, VH , and C,1 homology regions of Fab New. The hydrogen-bonded clusters correspond to the two ,8-sheet structures of each subunit. Cysteine residues that partic­ipate in intrachain and interchain di­sulfide bonds are underlined.

VL contacts will be considered in this discussion: first, the contacts which are at the core of the contacting region, made by residues which are invariant or semi-invariant in VH and VL sequences; second, the contacts made by invariant or semi-

PFIZER EX. 1083 Page 8

590 Structural Refinement of Fab New

FIG. 6. Stereo pair drawings of the a carbon backbones of the VL (top) and VH (bottom ) subunits. The subunits are viewed here in similar orientations.

FIG. 7. Stereo pair drawing of the a carbon backbones of the C,. ( top ) and C,l (bottom ) subunits viewed in simi­lar orientations.

PFIZER EX. 1083 Page 9

Structural Refinement of Fab New 591

FIG. 8. Stereo pair drawing of the a backbone of Fab New.

ln1~ariar1t residues with hypervariable residues; and finally , made between hypervariable residues.

The core of the VwVL contacting region can be described as ~ten111ii1ed by residues Val 37, Gin 39, Leu 45, Tyr 94, and

107 in VH and by residues Tyr 35, Gin 37, Ala 42, Pro 43, and Phe 99 in VL . These residues are structurally

~)JJlt3Io1gmis with the exception that VL Ala 42 has no clear r:corresJ)(md.ence in VH due to a structural "insertion" (see Fig.

These homologous VH and VL residues make numerous eontacts with each other (about 50% of those listed in Table IV) or with other , nonhypervariable residues. The rings ofTrp 107 (VH) and Pro 43 (VL), at the center of the VL-VH contacting

'l'egi.on, are nearly parallel and stacked on each other. The contact residues listed above are invariant or are replaced by homologous residues in VL (K and A.) and VH sequences from different animal species. For example, Tyr 35, Gin 37, Pro 43, and Phe 99 appear constant in human L chains (K or>..), and Gin 39, Tyr 94 (replaced by Phe in a very few cases), and Trp 107 (replaced by Phe or Tyr in a very few cases) appear nearly constant in human H chains. Ala 42, Tyr 86 in VL and Val37 in VH are more frequently replaced by homologous residues: Ser 42, Phe 86, and Ile 37. The invariant or nearly invariant nature of these residues of the main VwVL contacting area provides a structural basis (together with interactions be­tween 4t1 and 4,, see below) for the property of different H and L chains to recombine into new immunoglobulin mole­cules (see References 18, 19, and in particular 20, for a recent review and experimental data on this topic).

A second type of contact listed in Table IV is made between constant or nonhypervariable residues and hypervariable res­idues. For example, the side chain atoms of VH Trp 47 , a constant residue in human, mouse, guinea pig, and in most rabbit immunoglobulin sequences, make close contacts with Ser 93, Leu 94, and Arg 95 in the third hypervariable region of VL. However, a large number of these contacts involve the peptide chain atoms of the VL residues. Replacements in the VL side chains will not necessarily alter the nature of these contacts. Similar contacts appear to be made by VL Leu 45 (invariant or semi-invariant in human L chains) with the peptide chain at VH hypervariable position 104. Contacts of this type could also be made from VL Tyr 35 to the peptide chain atoms of the fourth hypervariable region ofVH in chains of different length than VH New.

The third type of contact to be discussed here is that made between hypervariable residues, such as those made between VH Asn 98 and VL Arg 95. These contacts are more difficult to evaluate in general terms (a) because the location of some of the residues involved might be changed by further refinement to a larger extent than those of most other residues in the Bequence, and (b) because it is possible that in other immu­noglobulins, replacements by different amino acid side chains

at these positions could be accommodated by small displace­ments of the hypervariable peptide loops. These "idiotypic" interactions are consequently more difficult to assess. How­ever, they could perhaps explain the preferred reassociation observed between complementary H and L chains derived from a single immunoglobulin molecule (20). Most of the contacts discussed above consist of van der Waals interactions between hydrophobic side chains. However, a few hydrogen bonds can be indicated: VH Gln 39 to VL Gin 37, and VH Asn 98 to VL Tyr 90 and/or VL Arg 95. Also, an ion pair is formed between VH Arg 43 and VL Asp 84.

In the Fab New model the contacts between VH and VL are very close (Table IV), giving rise to a compact dimer. No haptens or even solvent molecules can be accommodated between VH and VL beyond the combining site, a situation which is different from that described for an L-chain dimer (21) .

As shown in Table IV the interactions between 4t 1 and 4, are extensive. The core of the contact area between 4t 1 and 4, is defined by 4t1 residues Leu 128, Ala 129, Gly 143, and Leu 145 and the structurally homologous 4, residues Phe 120, Pro 121 , Val 135, and Leu 137. These residues appear to be invariant or nearly invariant in the H and L sequences from different animal species. Most of the other contact residues such as ~1: Phe 126, Pro 127, Thr 139, Lys 147, Phe 170, Pro 171 , Val173 , Gin 175, Ser 181, Val185, Lys 218, and CL: Thr 118, Ser 123, Glu 125, Glu 126, Lys 131 , Thr 133, Thr 164, Ser 177, Tyr 179, Lys 206, are also invariant or replaced by homologous residues in the immunoglobulin chains from dif­ferent animal species. In the contact area the central location of~ 1 Leu 128 and 4, Phe 120 is reflected in the large number of contacts (20 contacts) they make with each other and with many other residues (see Table IV). As pointed out by Novotny and Franek (22) the amino acid sequence of the four-stranded {3-pleated sheet is more conserved than the rest of the CA. regions in different animal species, leading to a dendrogram (or genealogic tree) of distorted evolutionary distances. This observation can be analyzed in terms of the structural model presented here as follows . The four-stranded {3 sheets of ~ 1 and 4, contain side chains which make intrasubunit contacts and in particular, they contain all or nearly all of the contact residues between Ct11 and 4, (discussed above). Evidently, mutational events leading to amino acid replacements at these positions would have to occur in a complementary pattern in both ~ 1 and 4, in order to preserve tertiary and quaternary immunoglobulin structure , and consequently they would be expected to occur at a slower rate than mutations in other regions of 4t 1 and 4, .

As can be seen in Table IV the region immediately preced­ing the interchain disulfide bond does not provide close con­tacts between 4t 1 and 4, . In addition, the two strands of

PFIZER EX. 1083 Page 10

592 S tructural R efinement of Fab New

VH 9

10

11

112

113

114

VL VH

110

CH1

7· 120 ·==r 151

152

153

205

206

CL CHl

127

116

117

118

119

120

121

122

123

124

125 140

126 141

142

143

162

163

164 170

165 171

166 172

167 173

174

175

175 176

176

177

212 218

213 219

214 220

CL VL 38

38

40

167

168 79

169 80

171 82

172

FIG. 9. Intersubunit a carbon contacts at distances of 8 A or less. Contacts are indicated by lines joining the corresponding amino acid residue numbers . Numbers on the lines indicate the contact distance (in Angstroms). Note the extensive VL -V" and Cc -C,1 1 interactions.

polypeptide chain that come together at the interchain disul­fide bond do not closely interact with the rest of C..l or <::1_. This region can be described as having a loose conformation, with a lower electron density in the Fourier map. These structural features are in agreement with the notion of seg-

mental flexibility residing around this part of the immuno­globulin structure and in the immediately adjacent hinge region of the H chain.

Hyperuariable R egions , Idiotypes, and Combining S ite­The results of several experimental approaches (see Chapter II

PFIZER EX. 1083 Page 11

Structural Refinement of Fab New 593

in Ref. 23 for a comprehensive review) strongly suggested that bypervariable residues in the amino acid sequences of H and L chains, idiotypic determinants, and combining site residues of an antibody molecule partially overlap. X-ray crystallo­graphic analyses (2-4, 21 , 24, 25) provided an unequivocal confirmation of these conclusions and three-dimensional models of different immunoglobulin molecules in which the structural bases of these operational concepts could be further defined.

The hypervariable regions of the Hand L chains are located on exposed bends of the polypeptide chains, in regions which can be folded without a defmed secondary structure (such as a regular f:l-bend) and which consequently appear capable of accommodating sequence variations without major structural constraints. From analyses of the structural bases of the lnv allotypic antigenic determinants of human K chains (26) and of the Oz, Kern isotypic antigenic determinants of human A. chains (3, 27, 28) it is known that a variation in a single exposed immunoglobulin amino acid side chain is sufficient to generate a specifically recognizable antigenic determinant. There are regions of immunoglobulin molecules such as those around residues 85 to 90 in human H chains (29) or around residues 1 to 3 in rabbit L chains (30) where such specific unique antigenic determinants (idiotypes) could arise. How­ever, since most of the sequence variation occurs at an exposed end, where VH and VL hypervariable regions join in determining the conformation of the combining site, most of the idiotypic determinants would be expected to occur at this site or immediately adjacent to it (see Fig. 10). Two questions arise: (a) are all antigen contact (complementarity-determin-

SER 26

ing) residues idiotypic antigenic determinants?; (b) do all idiotypic determinants contribute directly to antigen binding? These questions have been explored before in some ligand­antibody systems (see review in Ref. 23). For example , idi­otypic determinants of anti-phosphorylcholine antibodies from inbred mice can be divided into site-associated determinants and other idiotypic determinants which are not site-associated (31 , 32). In order to provide answers to these questions on the basis of the three-dimensional model of an immunoglobulin presented in this paper, it is necessary to make some assump­tions which will be briefly reviewed in the following para­graph.

The first assumption is, necessarily, that the conformation of an Fab fragment in the crystalline state is the same as in the parent immunoglobulin in solution. Also, it is assumed that the combining site is limited to the crevice or cavity delineated by the VH and VL hypervariable regions as previ­ously described (3) . In addition, it will be assumed that antigen binding will not induce major conformational changes at the combining site although by anaiogy with enzyme-ligand systems (see for example Ref. 33) smaller movements of amino acid side chains and polypeptide backbone chains can be expected and have to be allowed for. Since the definition of idiotypic determinants relies on serological procedures, the mode of interaction between the recognized immunoglobulin and the recognizing anti-idiotypic antibody is important. Al­though no suitable structural model is available to describe them they can be expected to be complex. A minimal assump­tion that can be made is that all amino acid side chains which are accessible to solvent are potential antigenic determinants

FIG. 10. View of some of the amino acid residues at the combining site of IgG New.

PFIZER EX. 1083 Page 12

594 Structural Refinement of Fab New

that can be recognized by an anti-idiotypic antibody. Within the assumptions outlined above we conclude that

most (not necessarily all) complementarity-determining resi­dues are idiotypic determinants. The set of possible idiotypic determinants (exposed amino acid side chains) is larger than the set of complementarity-determining residues. The number of hypervariable region residues is larger than the number of idiotypic determinants since some of the hypervariable resi­dues are not exposed to solvent. In lgG New, on the basis of their location at the combining site, the following amino acid side chains can be indicated as most likely complementarity­determining: in VL, Ala 28, Asn 30, Tyr 90, Ser 93 , Arg 95; in VH, Trp 47, Tyr 50, Phe 52, lle 100, Ala 101. Other side chains which could also make contacts include VH Thr 56 and Asp 98. Atoms along the main peptide chain of the residues listed above and of several other residues will also add to contacts with antigens or haptens. VL Tyr 90 and VH Trp 47 are fairly conserved residues in VL and VH sequences, however, they line up the bottom of the combining site and should be expected to contribute to antigen contacts. In other immuno­globulin molecules having different lengths of polypeptide chains at the hypervariable regions, the complementarity­determining residues will not be expected to occur at the same positions as those listed above for lgG New. All the residues listed above, except VH Trp 47 and VL Tyr 90, are possible idiotypic determinants.

It should be emphasized here that the list of complementar­ity-determining residues discussed above is based on the characterization of the combining site as a cavity or pocket between VH and VL hypervariable regions. This characteriza­tion has been made on the basis of its conformation described as a "shallow groove" for IgG New (3) and on the basis of the binding of ligands that can be considered as haptens (or cross­reacting haptens) with defined stoichiometry and association constant (2, 24). Thus, there is no doubt that a central region of the combining site has been identified and characterized. However, no suitable structural models have yet been ob­tained to exclude the possibility that the combining site extends to regions around this central cavity. For example, there is a "side" cavity or pocket in IgG New, surrounded by VL residues 2, 3, 25, 27, 27a, 91, 92, 93 , 94, and 98, and VH residues 57 through 61. It could be assumed that this is also part of the combining site or a compartment of it. In support of this view (a) hypervariable region residues modulate the conformation of this region to make it unique for every immunoglobulin molecule, (b) affinity labeling experiments with anti-2,4-dinitrophenyl and anti-arsonate guinea pig an­tibodies (34) have tagged VH residues Tyr 60 and Lys 59, respectively, which occur in this side site. A symmetrical side site is bound by residues of the second hypervariable region of VL and by hypervariable regions around positions 30 and 95 in VH. As in the symmetrical side site described above, constant or less variable residues of VH and VL also contribute to the conformation of this subsite. If this view of an extended combining site is correct, it will have implications such as (a) more amino acid side chains in the hypervariable regions contribute to the definition of antigen binding specificity; thus, sequence hypervariability would have a bigger influence in the physiological process of antigen recognition and bind­ing; (b) antibodies are highly reactive proteins utilizing a great contact surface for antigen recognition; (c) because of the sequence diversity and the varied conformational compo­nents of such an extended site it appears inescapable that a given antibody molecule should be able to react with different

TABLE IV

lntersubunit contacts

The number of interatomic distances not larger than 1. 2 times the van der Waals radii (C-C s 4.32 A; 0-0 s 3.65 A; N-N s 3. 72 A­C-D s 3.98 A; and C-N s 4.02 Al are listed. '

Val 37 Gin 39 Arg 43 Arg 43 Arg 43 Leu 45 Leu 45 Glu 46 Trp 47 Trp 47 Trp 47 Asp 58 Asp 60 Thr 61 Tyr 94 Asn 98 Leu 99 Ala 101 Ala 101 Ala 101 Gly 102 lie 104 lie 104 lie 104 Trp 107 Trp 107 Trp 107 Trp 107

Ala 118 Ser 119 Thr 120 Phe 150 Phe 150 Pro 151 Pro 151 Glu 152 Pro 153

Gin 110 Lys 168 Asn 172

Phe 99 Gin 37 Asp 84 Tyr 86 Gin 37 Tyr 86 Phe 99 Phe 99 Arg 95 Leu 94 Ser 93 Ser 93 Leu 94 Leu 94 Ala 42 Arg 95 Arg 95 Tyr 90 His 31 Lys 33 Lys 33 Tyr 35 Gin 88 Leu 45 Pro 43 Ala 42 Phe 99 Tyr 35

Leu 11 Leu 11 Leu 11 Leu 11 Thr 114 Leu 11 Thr 114 Leu 112 Leu 112

Glu 82 Pro 39 Glu 82

No . of contacts

1 5

14 7 3 4

2 3

19 8 1 2 3 1

6 8 3 6 7 2 6 3 3 2

21 2 4

2

2 2 3 3

2 2 4 7

5 7

Phe 126 Phe 126 Phe 126 Leu 128 Leu 128 Leu 128 Ala 129 Ala 129 Lys 133 Thr 139 Thr 139 Ala 141 Leu 142 Gly 143 Leu 145 Leu 145 Lys 147 Lys 147 Lys 147 Phe 170 Phe 170 Phe 170 Pro 171 Pro 171 Val 173 Gin 175 Ser 176 Leu 182 Ser 183 Ser 183 Ser 183 Val 185 Val 185 Lys 218 Ser 219 Ser 219 Cys 220

Glu 126 Glu 125 Ser 123 Phe 120 Val 135 Pro 121 Phe 120 Pro 121 Glu 212 Thr 118 Lys 206 Phe 120 Phe 120 Phe 120 Tyr 179 Val 135 Glu 126 Lys 131 Thr 133 Leu 137 lie 138 Ser 177 Ser 167 Ala 175 Tyr 179 Glu 162 Glu 162 Tyr 179 Tyr 179 Val 135 Leu 137 Leu 137 Phe 120 Cys 213 Glu 212 Cys 213 Cys 213

No. of contacts

14

3 20 2 1 8 2 1

3 2 6 4

5

1 1 3 2

10 4 4 2 1 6 7 8 2 6

1 3 3 2 2 6

ligands; substances such as 2,4-dinitrophenyl which are highly reactive toward proteins would be bound by many different antibody molecules, thus an anti-2,4-dinitrophenyl response would have the potential to be extremely heterogeneous.

It should also be mentioned here that if the antigen combin­ing site is extended as described above, amino acid variations in the BALB/c murine A chain system (35) will contribute to antigen binding, at variance with our previous analysis (36). Positions 25, 91, and 94 (numbered following the VL sequence given in Fig. 4) in which variations have been detected in the murine A chain sequences contribute to the conformation of the side cavity or pocket described above. Irrespective of this putative antigen binding role, these chain sequence van~­tions, which have been interpreted as products of somatlc mutation mechanism (35), are likely to give rise to new

PFIZER EX. 1083 Page 13

Structural Refinement of Fab New 595

idiotypic specificities and to an altered regulatory idiotype network (37).

In order to further map and characterize the combining site it might be necessary to investigate chemical interactions in other hapten-antibody or antigen-antibody systems. At pres­ent this appears to be a task of considerable magnitude.

Ackrwwledgments- We thank Dr. H. Bernstein and the staff of the CRYSNET project for their assistance in imple­menting programs at Brookhaven National Laboratories.

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