h.,. OLECVLAR BIOLOGYs · 2017. 5. 26. · This issue completes Volume 190 UNIVtRS\\1 Gr m,.,.i!N'tll

$Page 1: h.,. OLECVLAR BIOLOGYs · 2017. 5. 26. · This issue completes Volume 190 UNIVtRS\\1 Gr m,.,.i!N'tll$
This issue completes Volume 190

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OLECVLAR BIOLOGYs

Volume 190

Editors in Chief J. C. KENDREW S. BRENNER

Number 4 20 August 1986

London Orlando San Diego New York Toronto Mont real Sydney Tokyo

JMOBAK 190 (4) 519- 651 ISSN 0022-2836 PFIZER EX. 1085

Page 1

( 'el/8.·

Journal of Molecular Biology

Editors-in-Chief J . C. Kendrew . 7 All Saints Passage , Cambridge CB2 3LS, England

H. Brenner , M.R .l' . Laboratory of Molecular Biology. University Postgraduate Medical School Hills Road. Cambridge CB2 2QH . England

Gene structure } GPne modifi cation Gene expression Gene regulation

Cell deve lopment Cell fun ction

Organelle structures } Macromolecular

assemblies

{

{

Editors

S . Brenner (address above ). P. Charnbon, Laboratoire de Genetiq ue Moleculaire des Eucaryote du

CNRS. Institut de Chimie Biologique . Faculte de Medicine. II Rue Humann. 67085 Strasbourg Cedex. France .

1l1. Gottesman, Institute of Cancer Research , College of Physicians & Surgeons of Columbia Uni\•ersity . 701 \\'. I 68th Street. New York. NY 10032, U., .A.

P. von Hippel , Institute of Molecular Biology, University of Oregon, Eugene, OR 97403- 1229, U.S.A.

B. Mach . Departement de Microbiologie. C.M.ll .. 9 av . de Champel , CH -1211 Geneve 4 . Switzerland .

K . . '11at8ubara , Institute for Molecular and Cellular Biology. Osaka l ' niversity . Yamada-oka. Suita, Osaka 565. Japan .

H. E . Huxley , M.R.C. Laboratory of Molecular Biology. l ' niversitv Postgraduate Medical School. Hills Road . Cambridge CB2 2QH. England .

A . Klug . M. R.C. Laboratory of Molecular Biology , University Postgraduate Medical School. Hills Road, Cambridge CB2 2QH. England .

Molecules : Macromolecular structure { R. Hube·r . Max-Planck-Jnstitut fiir Biochemie. 8033 Martinsried bei

Miinchen . Germany.

Letter8 to the Editor:

Physical chemistry

C:eneral Preliminary X -ray data

J. C. Kendrew (address ~bove ) . G. A . Gilbert , Department of Biochemistry. University of Birmingham,

P .O. Box 363. Birmingham Bl5 2TT. England . S . Brenner (address above) . R . Huber (address above) . J. C. Kendrew (address above) .

Associate Editors

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PFIZER EX. 1085 Page 2

J. Mol . Biol. (1986) 190, 593- 604

Phosphocholine Binding Immunoglobulin Fab McPC603

An X-ray Diffraction Study at 2·7 A

Yoshinori Satowt , Gerson H. Cohen, Eduardo A. Padlan and David R. Davies

Laboratory of Molecular Biology National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases

National Institutes of Health, Bethesda, MD 20892, U.S.A.

(Received 27 December 1985)

The crystal structure of the Fab of McPC603, a phosphocholine-binding mouse myeloma protein, has been refined at 2·7 A resolution by a combination of restrained least-squares refinement and molecular modeling. The overall structure remains as previously reported, with an elbow bend angle between the variable and constant modules of 133°. Some adjustments have been made in the structure of the loops as a result of the refinement. The hypervaria.ble loops are all visible in the electron density map with the exception of three residues in the first hypervariable loop of the light chain. A sulfate ion occupies the site of binding of the phosphate moiety of phosphocholine.

1. Introduction

Direct information about the three-dimensional structure of the antibody combining site is based on the crystal structures of three Fab species: Kol, a human myeloma protein with unknown specificity (Marquart et al., 1980); New, a human myeloma protein that binds to a vitamin K1 derivative (Amzel et at., 1974; Saul et al ., 1978); and McPC603, a mouse plasmacytoma protein specific for phosphocholine (Segal et al., 1974). Although the structures of several monoclonal antibodies with known antigen binding specificities are being investigated (Mariuzza et al., 1983; Silverton et al., 1984; Colman et al. , 1981; Gibson et al. , 1985; Amit et al., 1985}, refined , high-resolution structures from them are not available.

The three-dimensional structure for the Fab of McPC603 (lgA,K} protein that was previously reported (Segal et al., 1974) was an unrefined structure based on 3·1 A diffractometer data and with only partial sequence information. This work has now been extended through the collection of a complete set of 2·7 A diffraction data using oscillation photography, augmented by the knowledge of the complete amino acid sequence and with the help of interactive molecular graphics procedures (Lipscomb et al., 1981; Diamond, 1981). The crystal structure has been refined using restrained least-squares procedures (Hendrickson &

t Present address: Photon Factory, National Laboratory for High Energy Physics, Oho-machi, Tsukuba-gum , Ibaraki-ken, 305, Japan.

0022- 2836/86/ 160190- 12 $03.00/0 593

Konnert, 1981 ). In this paper, we report the results of this investigation.

2. Materials and Methods

(a) Crystal structure analysis and preliminary refinement at 3·1 A resolution

Crystals were prepared from concentrated solutions of ammonium sulfate as described (Rudikoff et al., 1972). After preparation, the crystals were transferred into stabilizing solutions consisting of 50% saturated ammonium sulfate (pH 7·0) , 0·2 M-imidazole or 0·1 Msodium cacodylate. Isomorphous heavy-atom derivatives were prepared using TmC1 3 (American Potash & Chemical Corp.) K2Pt(CNS)6 (K&K Laboratories, Inc.) , and KI (Fisher Scientific Co.). The thulium and platinum derivatives were prepared by soaking crystals in solutions containing cacodylate and 30 mM-TmC1 3 or 0·4 mMK2Pt(CNS)6 , respectively, for 3 to 4 weeks. An iodine derivative was prepared by soaking crystals in 50 mM-KI, 2·5 mM-Chloramine T (Eastman Kodak Co.) for 2 weeks, after which the iodinated crystals were washed with stabilizing solution to remove excess iodine. Double and triple derivatives were prepared using these heavy-atom compounds. The native and iodinated crystals were prepared using imidazole buffer; all other derivatives were prepared in cacodylate buffer.

Most of the 3·1 A data were collected using a Picker FACS-I diffractometer (Segal et al. , 1974) ; the TmCI 3 derivative data set was collected from precession photographs. Co-ordinates for the heavy-atom sites in the thulium and platinum derivatives were obtained from difference Patterson syntheses at 4·5 A resolution (Padlan et al. , 1973). Those for the iodine derivative were obtained from a difference Fourier synthesis with phases computed using the first 2 derivatives. Alternating cycles of heavy-

© 1986 Academic Press Inc . (London) Ltd.


594 Y. Salow et a l.

atom refinement and phase calculation (Dickerson et al ., 1961) were then computed using local versions of the programs of Busing et al . (1962) and Matthews (1966). Table l shows the refined heavy-atom parameters. A " best" Fourier synthesis (Blow & Crick , 1959) was computed and a Kendrew skeletal model was fitted to the electron density using an optical comparator (Richards , 1968). The model was improved using t he computer graphics system GRIP at the Department of Computer Science, niversity of North Carolina (Tsernoglou et al .. 1977) and was then subjected to restrained least-squares refinement (Konnert, 1976; Hendrickson & Konnert, 1981 ) on the TI-ASC computer at the U.S . Naval Research Laboratory. The initial value of the conventional crystallographic R-factor was 0·41. This was reduced to 0·30 after 5 cycles with a single overall temperature factor and tight structural restraints. Further refinement using individual temperature factors for the atoms reduced the residual to 0·27. The r.m.s.t total shift from the original position was 0·76 A. At this point, the fit of the model to a 2F0 -Fc map was examined using BILDER (Diamond , 1981 ), as implemented on a PDP 11/70 under the RSX-llM operating system (G. H. Cohen, unpublished results). In the second stage of refinement , the protein geometry was initially less restrained and subsequently tightened , y ielding a final residual of0·24. The r.m.s. total shift from the atom ic positions at the start of the second stage of refinement was 0·42 A. A su lfate ion , that had been located in the hapten binding cavity (Padlan et al. , 1973; Segal et al. , 1974) , was included throughout the refinement.

The regions corresponding to residues 101 to 108 in the heavy chain and residues 31 to 35 in the light chain were not clearly defined in the original electron density fun ction based on the heavy-atom phases. These regions remained poorly defined after the preliminary refinement.

(b) Crystal structure analysis and refinement at 2·7 A resolution

A new set of crystals was prepared for the higher resolution , 2·7 A, phase of this study. The imidazole buffer employed during crystallization was replaced by cacodylate when the crystals were transferred to stabilizing solutions. Intensity data to 2·7 A were collected by rotation photography (Arndt & Wonacott, 1977) with Kodak No-Screen Medical X-ray films (3 in a pack) using Ni-filtered CuKcx radiation from an Elliott GX-6 rotating anode X-ray generator operated at 40 kV and 40 rnA. A Franks double bent mirror system (Harrison , 1968) purchased from Brandeis University was used to focus the X-ray beam . Data were recorded on an Enraf-Nonius Arndt- Wonacott rotation camera with a nominal 87 mm crystal-to-film distance. The data consisting of 88 film packs were collected from 26 crystals by osci llation about the 2 crystal axes, b and c, with an

t Abbreviations used: r.m .s., root-mean-square; F0 ,

observed structure factor amplitude; Fe , calculated tructure factor amplitude; w, weight; CDR,

complementarity determining region (Kabat et al., 1983); VL, light chain variable domain ; VH, heavy chain variable domain ; CL, light chain constant domain; CHI, first constant domain of heavy chain; m.i.r. , multiple isomorphous replacement; Ll , L2 and L3 , l st, 2nd and 3rd CDR of the light chain ; Hl , H2 and H3, l st, 2nd and 3rd CDR of the heavy chain.

Heavy-atom compound

K2Pt(CNS) 6

TmC1 3

Iodine-I lodine-2 lodine-3 lodine-4

Table 1 Heavy-atom parameters

Co-ordinates

X y Occupancyt

0·3807 0·3936 0·5422 0·02 18 0·3427 0·6617 0·2523 0·0245 0·2336 0·7083 0·5569 0·0119 0·2409 0·6794 0·5695 0·0184 0·2328 0·6328 0·6404 0·0125 0·1241 0·7017 0·4838 0·0044

Thermal factor (A2)

19·6 34·8 28·3 7·5 6·7 6·8

t The site occupancy is on an arbitrary scale in which the average structure amplitude of the native protein is 14·3 .

osci llation range of 1·25° and an overlap of 0·25°. The time for each exposure was 14 to 21 h.

The films were scanned at 100 Jlm steps on an Optronics P-1000 film scanner. The initial film processing, which included preliminary refinement of the crystal orientations and successive evaluation of the integrated intensities , was made on a PDP 11 /70 computer using the rotation program package written by G. Cornick & M.A. Navia (unpublished results). Intensities from each pack were further processed through intra-film-pack scaling, post refinement, scaling and averaging using programs specially written for these purposes (Y. Sa tow, unpublished results). Intensities in a single pack were scaled by refining non-linear response correction factors (Matthews et al., 1972) and absorption factors for the fi lm base and emulsion, then were corrected for Lorentz and polarization factors. The measured intensities from the films were merged , reduced to a unique set and processed by a scaling and averaging program , which refines relative scale and exponential fall -off factors for individual films . This program follows closely the formalism of the established algorithms of Hamilton et al. (1965) and Rossmann et al. (1979). Post refinement of the crystal orientations, lattice constants and rocking curve parameters were done as proposed by Winkler et al. (1979) and Rossmann et al. (1979). The total num ber of 147 ,706 intensities , including partially recorded reflections, were finally scaled and symmetry averaged , yielding 24 ,235 unique reflections for the re olution range of 10 A to 2·7 A. The agreement factor:

N.,

R = L L llhj-I--;.1 /L NJh , h j h

where the intensity ]hi for reflection h was measured Nh

times , was 0·077 or 0·054 for the structure factor amplitudes Fhj · Within a 3 A sphere, 95% of the data were retained ; within the complete 2·7 A sphere, 93% were retained. The earlier diffractometer data were not included in this final set of 2·7 A film data.

The initial model for the 2·7 A work was taken from the 3·1 A results . It was subjected to a number of cycles of least -squares refinement (Hendrickson & Konnert, 1981) with periodic examination and rebuilding of the model using BILDER on a VAX 11 /780 (R. Ladner, unpublished results). The su lfate ion located in the 3·1 A analysis was not included in the early stages of leastsquares refinement. With an overall temperature factor , the initial value of the R-factor was 0·41. After 5 cycles of positional parameter refinement with an overall temperature factor , the value was reduced to 0·33. Individual temperature factors for the atoms were used in the succeeding cycles. The program restrains the values of


Phosphocholine Binding Immunoglobulin Fab McPC603 595

t he B-factors so that each is influenced by the B-factors of the atoms to which it is bound as well as the atoms 1 removed along a chain. In the final stages of least-squares refinement, particular care was taken to ensure that the stereochemistry was kept reasonable and that w(IF.I-1Fcll 2 was approximately constant over the range of the data used (8·0 A through 2·7 A).

The main-chain stereochemistry was constantly monitored by the program GEOM (G. H. Cohen, unpublished results) and points of significant departure from expected stereochemistry were examined and corrected via interactive computer graphics. In addition to a sulfate ion, 138 water molecules of variable occupancy were identified from examination of t1F and 2F0 - Fe maps and refined with the protein molecule.

As sequence data became available, the "working" sequence was updated appropriately. The VL and VH sequences are listed (sequences 12, p. 45 and 1, p . 128, respectively) by Kabat et al. (1983), quoted from R.udikoff et al. (1981) and Rudikoff & Potter (1974) , with the addition of the tetrapeptide Leu-Glu-IIe-Lys, which occurs at the end of VL (Rudikoff, unpublished results). The CHI sequence given by Auffray et al. (1981), obtained by translation of the nucleotide sequence of eDNA complementary to alpha-chain mRNA from J558 tumor cells, (sequence 60, p. 175, Kabat et al. , 1983) was used. This sequence differs in 4 places from that obtained by Tucker et al . (1981) from BALBJc genomic DNA, while they both differ in 2 of these positions from the sequence reported by Robinson & Appella (1980) for MOPC51 1, as quoted by Kabat et al. ( 1983). Examination of the final electron density map at these positions did not permit a clear distinction to be made between these sequences . The sequence of MOPC21 (sequence 23, p. 167 , Kabat et al., 1983 from Svasti & Milstein , 1972) was used for the CL domain of McPC603.

Throughout this paper, the amino acid numbering is serial, starting from number l with the 1st residue of each chain of the molecule. The correspondence between our numbering scheme and that of Kabat et al. (1983) is presented in Table 2.

As noted by Segal et al . (1974) , the molecule possesses 2 approximate local dyad symmetry axes, which relate the pair of variable domains and the pair of constant domains to each other. We examined the relationship and the

Table 2 Correspondence between the numbering scheme used

here and that of Kabat et al. (1983)

Light chain

This work

1- 27 28- 33 34- 220

Kabat et al.

1- 27 27a- 27f

28- 214

Heavy chain

This work

1-52 53-55 56-85 86-88 89-106

107- 109 110-139 140- 142 143- 163 164- 171 172- 184 185- 200 201- 205 206- 216 217- 222

Kabat et al.

1-52 52a-52c

53-82 82a-82c

83-100 100a- 100c

101- 130 133- 135 137- 157 162-169 171- 183 185-200 202-206 208-218 220-225

similarity of these pairs of domains using the program ALIGN (G. H. Cohen, unpublished results) , which iteratively rotates one set of atoms to another set to optimize their fit while preserv ing the order of the linear sequences of the 2 sets. The program uses the algorithm of Needleman & Wunsch (1970) to identify the structural homology while accounting for insertions and deletions.

Interdomain and intermolecular contacts were calculated with the aid of the program CONTAX (E. A. Padlan , unpublished results). Two atoms are defined to be in contact if their co-ordinates lie within the sum of their van der Waals radii plus 1·0 A. Intramolecular main-chain hydrogen bonds were calculated by the program EREF (M. Levitt, personal communication).

3. Results and Discussion

Table 3 shows an estimate of the quality of the stereochemical parameters for the final model. It is expressed in terms of the r.m .s. deviations of the various classes of parameters from accepted values (Sielecki et al ., 1979). The </>,1/1 plot for the main chain is shown in Figure 1. There are a few residues that have " forbidden" </>,1/1 values . The quality of the map in these regions together with the low

Table 3 Summary of stereochemical criteria (Hendrickson & Konnert , 1981; Bielecki et al., 1979; Cohen et al. ,

1981)

R = l:IJF.I-JFcll l:IF.I

Average llF

Interatomic distances (A) 1- 2 1- 3 1- 4

P lanarity (A)

Chiral volumes (A3 )

Non-bonded contacts (A) 1- 4 Other

Angles (deg.) w, Xs (Arg) x, , ... , X4

Temperature factors (A2)

Main chain 1- 2 Main chain 1- 3 Side chain 1- 2 Side chain 1- 3

Final model

0·225

136

0·020 0·040 0·037

0·027

0·257

0·24 0·34

27·0 5·0

0·5 1·0 0·4 0·7

Target u

t

0·015 0·020 0·025

0·020

0·150

0·50 0·50

15·0 5·0

0·5 0·7 0·5 0·7

The standard groups dictionary used is specified in Table 2 of Sielecki et al. (1979) . In this Table, F. refers to the observed structure factor , Fe is the calculated structure factor and !lF is the quantity IIF.I-IFcll - The target u represents the inverse square-root of the weights used for the parameters listed. The values given are the r.m.s. deviations from the respective ideal values.

t The weight chosen for the structure factor refinement, the " target u" of !lF , was modeled by the function w = (1 /d) 2 with :

d = 40-500(sin(£l)/J.-1 /6).

By this means, w(llF) was approximately constant over all of the data (w representing the weight for a given reflection). Other details of this Table are explained fully by Cohen et al. (1981).


596 Y. Satow et a!.

0 0

0 0

0

(!)

CD X 1- 80.00

(!)

X

(!)

(!)X

C!!!,

ClCj.

X (!)

(!) (!) (!) X

(!) X

(!)

(!)

(!)

(!)

(!)

~ X

(!)(!)

(!)

X

(!) X

X

X

X'-X X(!)

X

X

X

00

Figure 1.· A plot of the dihedral angles at each alpha-carbon atom for the refined co-ordinates. Asterisk , Pro; cross, Gly ; circle, any other residue.

resolution thwarted the assignment of more satisfactory geometry. The estimate of error in this model is 0·3 A as determined by the method of Luzzati (I952). The final crystallographic R-factor is 0·225 for 23,737 reflections in the range of 8·0 A to 2·7 A. The diffraction data, co-ordinate data, temperature factors and solvent occupancies for the Fab of McPC603 have been deposited in the Protein Data Bank at Brookhaven National Laboratory (Bernstein et al., I977).

(a) General structure of the Fab

Several changes were observed in the molecule as a consequence of this refinement , although the overall structure remained similar to that described earlier (Segal et al., I974) . Most of these changes are to be found in the details of the loops, in particular LI and H3. The region of LI is poorly defined in the electron density maps, even with the higherresolution data. We have now rebuilt the neighboring H3, whose density has become less ambiguous and, concurrently, have altered the conformation of Ll. In Figure 2, LI may be noted to protrude from the general surface of the molecule. The direction of this protrusion is reasonably correct but the electron density is too weak to define the positions of the three outermost amino acid residues.

Three other residues have tentative placements due to problems in maintaining proper stereochemistry while fitting the electron density : Glni62 and Asni63 (residues 156 and I57 in the numbering used by Kabat et al. , I983) of CL and Glu202 (residue 203, Kabat et al., I983) of CHI. In these cases, we permitted the stereochemical constraints to dictate the final placement. Also, several disordered side-chains were observed within the entire molecule. In all these cases, the configuration corresponding to the stronger density was chosen for the model.

The 442 residues of McPC603 Fab include 25 proline residues. Of these, five are cis-proline: residues 8 and lOI in VL, I47 in CL, and I43 and I55 in CHI. The assignment of the configuration of all five cis-proline residues was unambiguous. The structurally homologous Pro8 and Pro95 of Rei (Huber & Steigemann, I974) , and Proi47 (CL) and Proi55 (CHI) of Kol (Marquart et al. , I980) , also have been found in the cis conformation. Prol43 (CHI) of McPC603 has no counterpart in previously reported antibody structures.

(b) Crystal packing

The McPC603 Fab crystallizes in the space group P63 (Rudikoff et al ., 1972), with a= I62·53 A, c = 60·72 A. The molecules are situated in clusters


Phosphocholine Binding I mmunoglobulin Fab McPC603 597

Figure 2. A st ereo drawing of t he a lpha-carbon skeleton of McPC603. Continuous lines denote the heavy chain . The filled circles show t he complementari ty-determining residues (K abat et al. , 1983).

of three at each of t he crysta llographic 3-fold axes of the uni t cell (Fig. 3). Each cluster is related t o neighboring clusters via the 2-fold screw situated midway between t he 3-fold axes (Figs 3 and 4) . The clusters of three are maintained principally by a set

of hydrogen-bond and van der Waals contacts between neighboring molecules. Residues 14 to 20 and 71 of VL interact with residues 1, 26, 27 , 100, 102, 104, 105 108 and 110 of t he VH of the neighboring molecule while residues 18, 66, 67 , 69,

Figure 3. A p rojection of the a lpha-carbon skeletons of 4 unit cells and t he ab plane, illustrating t he large cha1mels t hat ru n parallel to t he c ax is t hrough t he crystal. T he heavy chains have been drawn bolder in t he 3 molecules that are clustered about a crystallographic 3-fold axis of t he lower left-hand cell . The 3-fold axes are indicated by the symbol .A. : 2-fold screw axes are located midway between each adj acent pair of 3-fold axes; a 63 axis is located at each corner of each cell.


598 Y. Satow et al.

Figure 4. The projected structure viewed perpendicular to the ac plane, illustrating the effect of the dyad screw axis. The plane of projection is a long the long diagonal of the unit cell (see Fig. 2) . It contains the two 3-fold axes and the 2-fold screw midway between them. For clarity, only the 4 molecules closest to the plane have been drawn . The heavy line corresponds to the heavy chain of the molecule.

73 and 82 of the same VL meet the VL of the neighboring molecule at residues 35, 55 and 6I to 63 . This pattern repeats three times around the axis. Other intermolecular contacts are found between VH of one molecule and CL and CHI of a second molecule related to the first by the 2-fold screw axis . Several additional contacts are found between VL and CHI of a neighbor via the z unit cell translation. It has been noted (Padlan et al., I973) that roughly 70% of the cell volume is occupied by solvent, thus permitting the diffusion of hapten to the molecule in the crystal. The long axis of the molecule makes an angle of approximately 50° with the xy plane of the unit cell (Fig. 4).

(c) Intramolecular pseudosymmetry

Analysis of the approximate 2-fold axes relating pairs of domains yielded the following results: for the VLJVH relationship, we find a 173° rotation with a r.m.s. deviation of 1·3 A (3·5 A for poorest agreement) between 385 matched pairs of main chain atoms out of 456 atoms. A number of the residues of the hypervariable loops align quite well , particularly at the beginning and end of each loop. The departure from a more exact 2-fold symmetry is probably due to the fact that the interface residues can be described as fitting a cylinder containing four strands from the light chain and five strands from the heavy chain (Novotny et al. , 1983). This asymmetry of the VHJVL interaction distinguishes it from most of the VLJVL interactions observed in Bence- Jones dimers which, in many cases, display exact 2-fold symmetry. The dyad symmetry of the CLJCHl pair is not as close. The best alignment for the constant modules uses

358 of the possible 400 pairs of main-chain atoms to achieve a r .m.s. deviation of 2·0 A (5·1 A for poorest agreement) between the pairs of matched atoms. The best fit between CL and CHI involves a rotation of I69° together with a translation of 2·6 A along the rotation axis. A comparison of these two axes of rotation yields an elbow bend of I33°, essentially unchanged from the earlier report (Segal et al. , I974) .

(d) Intramolecular contacts

As noted elsewhere (reviewed by Davies & Metzger, 1983; Amzel & Poljak, 1979), VLJVH contacts involve hypervariable residues as well as framework residues. The boxed regions of Table 4 indicate hypervariable to hypervariable residue interactions, while other regions of the Table involve framework residues. Nearly half (46 in 105) of the interactions between VL and VH involve only hypervariable residues, with most of these being located at the upper end of the interface (Fig. 2) in the vicinity of the combining site and with the framework interactions at the other end. A number of good hydrogen-bonded contacts occur, notably two between the highly conserved residues Gln44L and Gln39H, between Gln39H and Tyr93L, between the CDR residues Asp97L and AsnlOJH , and between the hydroxyl group of TyrlOOL and Glu35H. The latter interaction appears to be important in maintaining the integrity of the phosphocholine binding pocket (Rudikoff et al., 1981) as it has been observed (Rudikoff et al. , 1982) that a mutation of Glu35H to Ala results in a Joss of phosphocholine binding ability.

Other interdomain , intramolecular contacts are presented in Tables 5 to 7. There are no interactions



Table 4 Contacts between residues of V L and V H

Y33 E35 Q39 R44 IA5 W47 A50 E6l Y97 NlOl Yl03 Sl05 Tl06 Wl07 Yl08 Fl09 Wll2 Gll3 All4

K36 F38 Y42 Q44 4 P49 P50 L52 Y55 G56 Y93

Q95 0 ~~:X, 6,·

PlOI LI02 Fl04 Gl05 AJ06

l l 6

2

3

5 3

l 9

5 2

2 3

Residues listed in the left column are from VL; the residues listed above the columns are from VH. The numbers in the Table correspond to the number of pairs of atoms from 2 residues that are within potential van der Waals

contact distance, as calculated by the program CONTAX (see Materials and Methods, section (b)). A dot indicates no contacts. The boxed regions delineate possible hypervariablefhypervariable interactions.

involving VL with CHI or VH with CL. Tables 6 and 7 show that the number of interdomain intrachain contacts, i.e. VL with CL or VH with CHI, is small.

(e) Domain structure

The structures of the four domains of McPC603 are illustrated by Figure 5, which shows also the location of the hydrogen bonds between the mainchain amide groups. The general terti!;try structure of the domains and the distribution of the hydrogen

bonds are very like those of other antibody classes and subgroups. Thus, the a. heavy-chain domains of McPC603 Fab resemble those of the y chain domains of the human proteins New and Kol, and the K chain domains of McPC603 resemble the A. chain domains of New, Kol and Meg, as well as the K chains of various human VL dimers (Padlan & Davies, I975; Padlan, I977). A significant difference from the y chain structure occurs in CHI, where there is an additional disulfide bond formed between residues I98 and 222 (residues 198 and 225, in Kabat et al., I983). The Cys222 residue should therefore be regarded as forming the end of CH I

Table 5 Contacts between residues of C H 1 and C L

Yl3 1 Pl32 Ll33 Tl34 Ll35 Pl36 1145 Vl73 Fl75 Pl76 Al78 Sl88 Ql90

1123 2 F l 24 4 5 5 Sl27 3 El29 3 Ql30 14 Sl33 l Vl39 2 Fl41 2 3 2 Nl43 4 Ll66 Sl68 l Wl69 2 Tl70 SISO I Ml8l 3 Sl82 5 R217 3

Residues listed in the left columns are from CHI; the residues listed above the columns are from CL.

The numbers are as in Table 4.


600 Y. Satow et al.

Table 6 Contacts between residues on VL and CL

Yl 46 D l 71 Q172 8 174 Kl75 Dl76 8177 Yl79

P46 2 A86 7 L89 4 Kl09 E ll! 3 111 2 2 5 K113 I Rll4 6 6 5

The residues listed in the left column are from VL; those listed at t he column heads are from CL.


Table 7 Contacts between residues of V H and C H 1

El23 8 124 Al25 Fl54 PI 55 Gl 57 Tl58 D211 8212

09 2 010 L11 2 2 : I Tl17 3 Tll9 3

121 4

The residues listed in the left column are from VH ; those listed at the column heads are from CH I.


(c)

(d)

Figure 5. Schematic drawings of the (a) VH, (b) VL, (c) CH l and (d) CL domains illustrating the hydrogen bonds between the main-chain amide residues . The circles marking hypervariable residues in VH and VL are drawn with a double line.


Phosphocholine Binding I mmunoglobulin Fab M cPC603 601

8·0 8:0

7·0 7·0

6·0 6·0

5·0 5:0

4·0 4·0

3·0 3·0

2·0 2·0

1·0 1·0

D I VMTOIPS~LSYSAIERV!MitKSS OSL ~NSGNO KNFL~WYOGK~COP~Klll YGA ST_f!ESGVP DRFT~SGSG TDFT L! I SSVOAEDL~VYYCONDHS !PLTF GAGTK~E I KR

10 20 30 40 50 60 70 80 90 100 110

(a)

8·0 8·0

7·0 7·0

6·0 6·0

5·0 5·0

3·0 3·0

- --- - ---------- - ---- --- -- -

2·0 2·0

IIIII II lui IIIII I 111111 II ldl 1·0 1-0

ESARNPT .!._ YPI. TLPPAL~ SDPV I IGCL.!._HDYFPSGTM!!VTWG KSGKD .!._ TTVNFPPAL~SG& RYTMSNQL TLPAVECP ~GESVKCSVQ!!DS NP VOELDYNC ADAA'!VS I FPPSSEQLTIGGASVV~FLNNFYPK 0 .!._NVKWKI DGS~RO NGVLNSW!DODSKDSTY~MSSTL TL TKQE YERH NSYT~EATH KTSTS~ I VKSFNRNEf.

120 130 140 150 160 170 180 190 200 210 220 (b)

Figure 6. The separation (in A) of corresponding alpha-carbon atoms of the superimposed domains of (a) VH and VL, and (b) CH I and CL. The upper sequence is t hat of the heavy chain and the lower sequence is of the light chain . Every lOth letter in each sequence is underlined. The numbers below the sequences refer to t he residue number in the light chain. Where t he structural alignment has matched identical res idue types, the corresponding letters are shown in bold face . Gaps occur when there are no corresponding residues for comparison. The horizontal broken lines indicate the r.m .s. separation.


602 Y. Satow et al.

rather than the beginning of the hinge, consistent with the gene structure observed by Tucker et al. (1981).

The structures of the light and heavy chain domains of McPC603 are compared in Figure 6(a) and (b). The sequence identity derived from the structural a lignment is 25% for the variable pair and 22% for the constant pair, excluding gaps. In the variable domains, the framework residues superimpose rather well, except for the region from residues 60 to 74. Large differences are observed in the CDRs, but these are frequently the result of differences in the lengths of the hypervariable loops. In the constant domains, the differences between the light and heavy chains are comparable to those found between the variable domains.

(f) Bound carbohydrate

Robinson & Appella (1979) noted the presence of carbohydrate attached to Asnl55 (CHI) during their sequence analysis of the heavy chain of MOPC 47A. In electron density maps of McPC603 there is suggestive density in the vicinity of the homologous Asn l60 that indicates the possibility of carbohydrate here as well. This density is, however , insufficiently defined to permit fitting of any carbohydrate chain. It appears, therefore , that carbohydrate does not occupy a fixed position on the surface of the molecule . The lack of any nearby contacts from neighboring domains could also contribute to the delocalization of the carbohydrate moiety. An analogous poorly defined trace of density is found in the electron density map of the Fab of J539 (Suh et al. , unpublished results).

(g) The sulfate ion in the combining site

Figure 7 illustrates the combining site of McPC603 as seen in these crystals. In addition to several water molecules , the m.i.r. density map contains a large peak that has been assigned to a sulfate ion (Padlan et al. , 1973). This occupies the same location as the phosphate group of phosphocholine when the latter binds to the site, and its presence is presumably due to the high concentration of ammonium sulfate (about 2M} in the crystal. This sulfate ion is in contact with a number of atoms that interact with it through salt bridges and hydrogen bonds. These include the interactions proposed for the binding of the phosphate of phosphocholine; namely, Tyr33 and Arg52 of the heavy chain (Segal et al., 1974) . In addition , there is a water molecule within hydrogen-bonding distance of another of the sulfate oxygen atoms. This water is part of a network of hydrogen bonds involving another water molecule, TyrlOOL, Glu35H, AsnlOIH, Asp97L and Trpl07H as illustrated in Figure 7. There are, therefore, a total of four hydrogen bonds to three of the sulfate oxygen atoms which, combined with the charge attraction to Arg52, could provide sufficient attractive force to keep the sulfate in this position. The binding

F38

~

Figure 7. Part of the combining site residues of McPC603, showing the key residues that surround t he phosphocholine binding site. The sulfate ion that occupies the place of the phosphate group of phosphocholine is indicated by thick lines, and the 2 larger isolated circles are water molecules that partly fill the choline binding site . Residues F38 and D97 to Ll02 are from the light chain , the remaining residues being from the heavy chain . Potential hydrogen bonds are illustrated with t hin continuous lines.

constant of the sulfate cannot be very large, because it is displaced in the crystal by phosphocholine, which itself does not bind very strongly. The association constant for phosphocholine in the crystal is 4 x 104 mol - 1 versus 2 x 105 mol - 1 in an aqueous environment without ammonium sulfate (Rudikoff et al. , 1972). Assuming the sulfate concentration in the crystal to be 2 M, and that the difference in the binding constants for phosphocholine is due only to the presence of the sulfate, these numbers lead to a value of the sulfate binding constant (Hill , 1985) of only 20 mol- 1

. This can be compared with the value of 106 mol - 1 for the association constant of the sulfate ion and a sulfate binding protein quoted by Pflugrath & Quiocho (1985). H ere the sulfate has a total of seven hydrogen bonds from the protein and is sufficiently different from the binding observed in McP C603 to readily account for the difference in the binding constants.

4. Conclusion

The Fabs whose structures have been elucidated all display strong lateral interactions between homologous domains in the light and heavy chains, and weak longitudjnal interactions between the variable and constant domains of the same chain. This weak longitudinal interaction results in differences between the relative dispositions of the



variable and constant pairs in each Fab. The Fab of McPC603 and New have an elbow bend near I33°(Navia et al. , 1979) ; the elbow bend of J539 is 145° (Suh et al., unpublished results), while Kol Fab is essentially straight (Marquart et al ., 1980). The bend in McPC603 results in the close approach of CHI to VH (Fig. 2). The contact between these two domains is not extensive (Table 7) and cannot be very strong. A comparison of the homologous residues in this contact in McPC603, J539, Kol and New reveals no major structural dissimilarities between these amino acids that could account for the very different elbow bend in Kol. The weak interaction between VH and CHI probably allows these Fabs to be bent in what appears to be an extreme position , as found in McPC603, J359 and New, or to be nearly straight as in Kol. The bent configuration buries the glycine residue at position 10 in VH; this configuration may not be feasible for some mouse heavy chains (belonging mainly to subgroups 2 and 3; Kabat et al ., 1983), which have Glu in this position. An analysis of the VLfCL contact (Table 6 and Fig. 2) suggests also that the interaction probably imposes little or no restriction on the extent of bending of the Fab.

The VL/VH contact (Fig. 2 and Table 4) is extensive, involving 18 residues from the light chain and 19 from the heavy chain . Of these, eight re idues in the light chain and 11 residues in the heavy chain are from hypervariable regions (Table 4). In McPC603 Fab, approximately 45% of the VLfVH atomic contacts are due to hypervariable/ hypervariable interactions, while less than onethird of the contacts is contributed by framework/ framework interactions (Table 4). This large contribution of hypervariablefhypervariable associations in the known Fab structures (Novotny & Haber, 1985) suggest that particular light and heavy chain pairs may be preferred in the assembly of an antibody molecule. It is likely that only those chains whose hypervariable region interactions do not disrupt the framework interactions can produce viab le antibody structures. Alternatively, CLfCHl contacts, which are also strong (Fig. 2 and Table 5) , might serve to overcome unfavorable hypervariable region interactions so that the resulting pair could still conform to the canonical VLfVH structure.

The present structural refinement of McPC603 Fab has led to a more satisfactory structure for the H3 loop and its interaction with the light chain. Because of disorder, the structure of L1 ofMcPC603 could not be totally ascertained, so that the complete structure of the combining site of McPC603 is not available. The present data represent the limit of data collection with our available equipment and we do not plan any further refinement of the structure. The complex with phosphocholine has been refined independently and will be reported elsewhere (Padlan et al., unpublished results).

We are grateful to Drs Stuart Rudikoff and Michael Potter for the gift of McPC603. We also acknowledge the

helpful advice and comments of Drs Elvin Kabat and Stuart Rudikoff.

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Edited by R . Huber


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