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Phosphocholine binding immunoglobulin Fab McPC603
J. Mol.
Biol.
(1986) 190, 593-604
Phosphocholine
Binding Immunoglobulin
An X-ray Diffraction Yoshinori
Study at 2.7 A
Satowf-, Gerson H. Cohen, Eduardo National
Fab McPC603
A. Padlan and David R. Davies
Laboratory of Molecular Biology Institute of Arthritis, Diabetes and Digestive and Kidney Diseases National Institutes of Health, Bethesda, MD 20892, U.X.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 .& 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 hypervariable 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
Konnert, 1981). In this paper, of this investigation.
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 Kl derivative (Amzel et al., 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 (IgA,rc) protein that was previously reported (Segal et al., 1974) was an unrefined structure based on 3.1 .& diffractometer data and with only partial sequence information. This work has now been extended through‘the collection of a complete set of 2.7 d diffraction data using augmented by the oscillation photography, 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 &
2. Materials
v$O3.00/0
the results
and Methods
(a) Crystal structure analysis and preliminary rejinement at 3.1 B 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 TmCl, (American Potash & Chemical Corp.) K,Pt(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 m&r-TmCl, or 0.4 mMrespectively, for 3 to 4 weeks. An iodine K,Pt(CPU’S),, derivative was prepared by soaking crystals in 50 m;M-KI, 2.5 miv-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 d data were collected using a Picker FACS-I diffractometer (Segal et al., 1974); the TmCl, 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-
t Present address: Photon Factory, National Laboratory for High Energy Physics, Oho-machi, Tsukuba-gum, Ibaraki-ken, 305, Japan. 002%2836/86/160190-12
we report
593
0
1986 Academic
Press Inc.
(London)
Ltd.
Y. Satow eL all
594
atom refinement and phase calculation (Dickerson et al.; 1961) were then computed using local versions of the programs of Busing et aE. (1962) and Matthews (1966). Table 1 shows the refined heavy-atom para,meters. 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 the computer graphics system GRIP at the Deparbment of Computer Science, University of Pu’orthCarolina (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 8. At this point, the fit of the model to a 2F,- F, map was using examined BILDER (Diamond, 1981), as implemented on a PDP 11/70 under the RSX-11M operating system (G. H. Cohen, unpublished results). In the second stage of refinement, the protein geometry was initially less restrained and subsequently tightened, yielding a final residual of 0.24. The r.m.s. total shift from the atomic positions at the start of the second stage of refinement was 0.42 8. A sulfate ion, t’hat 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 function based on the heavy-atom phases. These regions remained poorly defined after the preliminary refinement. (b) Crystal structure analysis and re$nement at 2.7 d resolution A new set of crystals was prepared for the higher resolution, 2.7 ip, 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 1Vo-Screen Medical X-ray films (3 in a pack) using Ni-filtered CuKcc radiation from an Elliott GX-6 rotating anode X-ray generator operated at 40 kV and 40 mA. 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 oscillation about the 2 crystal axes, b and c; with an
t Abbreviations used: r.m.s., root-mean-square; F,, observed structure factor amplitude; F,, calculated structure 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; CHl, first constant domain of heavy chain; m.i.r., multiple isomorphous replacement; Ll; L2 and L3, lst, 2nd and 3rd CDR of the light chain; Hl, H2 and H3, lst, 2nd and 3rd CDR of the heavy chain.
Table B Heavy-aiom parameters Co-ordinates Heavy-atom compound
.F
Y
2
K,Pt(CNS),
0.3807
0.3936
0.5422
0.0218
TmC13 Iodine-l iodine-2 Iodine-3 Iodine-4
0.3427 0.2336 0.2409 0.2328 0.1241
0.6617 0.7083 0.6794 0.6328 0.7017
0.2523 0.5569 0.5695 0.6404 0.4838
0.0245 0.0119 0.0184 0.0125 0.0044
occupancyt
Thermal facior (AZ) 19,6 34.8 28.3 7.5 6.7 6.8
“rThe site occupancy is on an arbitrary scale in which I-he average stru&ure amplitude of the native protein is 14.3.
oscillation 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 pm 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 1 l/70 computer using the rotation program package writ,ten by G. Cornick & 11. A. Navia (unpublished results). Intensities from each pack were further processed through intra-film-pack scaling. post, refinement. scaling and averaging using programs written for these purposes specially (Y. Satow. 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 film base and emulsion, then were corrected for Lorentz and polarization factors. The measured int’ensities 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 a,E. (1979) and Rossmann et al. (1979). The total number of 147,706 intensities, including partially recorded reflections, were finally scaled and symmetry averaged. yielding 24,235 unique reflections for the resolution range of 10 f%to 2.7 d. The agreement fa,ctor:
h
j
h
where the intensity Ihj for reflection h was measured ATh times, was 0.077 or 0.054 for the structure fa,ctor amplitudes Fhj. Within a 3 A sphere, 95% of the data were retained; wit,hin 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 ,!%results. It was subjected to a number of cycles of least-squares refinement (Hendrickson & Konnert. 1981) with periodic examinat,ion and rebuilding of the model using BILDER on a VAX 111780 (R. Ladner, unpublished results). The sulfate 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 O”41. After 5 cycles of positiona, parameter refinement with an overall temperature factor; the value was reduced to 0.33. Individual temperature factors for the atoms were used in t’he succeeding cycles. The program restrains the values of
Phosphocholine
Binding
Immunoglobulin
the 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(lF,I - lF,[)’ was approximately constant over the range of the data used (80 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 AF and W-F, 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 Rudikoff et aZ. (1981) and Rudikoff & Potter (1974), with the addition of the tetrapeptide Leu-Glu-Ile-Lys, which occurs at the end of VL (Rudikoff, unpublished results). The CHl sequence given by Auffray et al. (1981), obtained by translation of the nucleotide sequence of cDNA complementary to alpha-chain mRNA from 5558 tumor cells, (sequence 60, p. 175, Kabat et al., 1983) was used. This sequence differs in 4 places from that obtained by Tucker et aZ. (1981) from BALB/c genomic DNA, while they both differ in 2 of these positions from the sequence reported by Robinson $ Appella (1980) for MOPC511, 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 1 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 l-27 28-33 34-220
Heavy chain
Kabat et al.
This work
l-27 27a-27f 28-214
1-52 53-55 56-85 86-88 89-106 107-109 110-139 140-142 143-163 164-171 1722184 185-200 201-205 206-216 217-222
Kabat
et al.
1-52 52a-52c 53-82 82a-82c 833100 lOOa-100c 101-130 133-135 137-157 162-169 171-183 185-200 202-206 208-218 220-225
Fab McPC603
595
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 preserving 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 communicati~on).
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 ~,IJJ plot for the main chain is shown in Figure 1. There are a few residues that have “forbidden” C#J,I+!J values. The quality of the map in these regions together with the low
Table 3 Summary Konnert,
of stereochemical criteria (Hendrickson & 1981; Sielecki et al., 1979; Cohen et al., 1981) Final model
Target (r
0.225 136 Interatomic 1-2 l-3 l-4
t
distances (A) 0.020 0.040 0.037
0615 0620 0.025
(A)
0.027
0920
Chiral volumes (A3) Non-bonded contacts (A) l-4 Other
0.257
0.150
0.24 0.34
O.!jO O.!jO
Planarity
Angles (deg.) a> xs (AM Xl> ‘> x4 Temperature Main chain Main chain Side chain Side chain
27.0 5.0 factors (A’) l-2 l-3 l-2 l-3
0.5 1.0 0.4 0.7
15.0 5.0 05
0.#7 0..5 0.7
The standard groups dictionary used is specified in T.able 2 of Sielecki et al. (1979). In this Table, F0 refers to the observed structure factor, F, is the calculated structure factor and AF is the quantity llF,l-lF,ll. The target 0 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 AF, was modeled by the function w = (l/d)’ with: d = 40--5OO(sin(tl)/,l-l/6). By this means, w(AF) 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. 1(1981).
Y. Satow et al.
596
xx x G 0 0
Q x x
x X
0
Q
Q
Q
x Y
:: i
Q
(II ,x
‘mo.00
m -135.00
I!fQX X -90.00
X
c) -h.
i5 IO
00 p’
u5.00
HI
Figure 1. A plot of the dihedral angles at’ each alpha-carbon 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 (1952). 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., 1977). (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., 1974). Most of these changes are to be found in the details of the loops, in particular Ll and H3. The region of Ll 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! Ll 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.
90.00
135.00
E I
atom for the refined co-ordinates.
Asterisk,
Pro; cross,
placements Three other residues have tentative due to problems in maintaining proper stereochemistry while fitting the electron density: Gln162 and Asn163 (residues 156 and 157 in the numbering used by Kabat et al., 1983) of CL and Glu202 (residue 203, Kabat et al., 1983) of CHl. 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 t,hese cases: the configuration corresponding to the stronger density was chosen for the model. The 442 residues of &PC603 Fab include 25 proline residues. Of these, five are cis-proline: residues 8 and 101 in VL, 147 in CL, and 143 and I55 in CHI. The assignment of the configuration of all five cis-proline residues was unambiguous. The structurally homologous Pro& and Pro95 of Rei (Huber & Steigemann, 1974), and Pro147 (CL) and Pro155 (CHl) of Kol (Marquart et al., 1980), also have been found in the cis conformation. Pro143 (CHl ) of McPC603 has no counterpart in previously reported antibody structures. (b) Crystal
pa&@
The McPC603 Fab crystallizes in the space group P6, (Rudikoff et al., 1972), with a = 162.53 8: c = 60.72 A. The molecules are situated in clusters
Phosphocholine Binding
Figure 2. A stereo drawing filled
circles
of the alpha-carbon show the complementarity-determining
Immunoglobulin
skeleton of McPC603. Continuous residues (Kabat et al.; 1983).
at each of the crystallographic 3-fold axes of the unit cell (B’ig. 3). Each cluster is related to neighboring clusters via the 2-fold screw situated midway between the 3-fold axes (Brigs 3 and 4). The clusters of three are maintained principally by a set
of three
Fab McPC603
lines denote
597
the heavy
chain.
The
of hydrogen-bond and van der Waals contacts between neighboring molecules. Residues 14 to 20 and 71 of VL interact with residues 1, 26, 2’7, 100, 102, 104, 105 108 and 110 of the VH Iof the neighboring molecule while residues 18, 66, 67, 69,
Figure 3. A projection of the alpha-carbon skeletons of 4 unit cells and the ab plane, illustrating that run parallel to the c axis through the crystal. The heavy chains have been drawn bolder in the 3 clustered about a crystallographic 3-fold axis of the lower left-hand cell. The 3-fold axes are indicated Z-fold screw axes are located midway between each adjacent pair of 3-fold axes; a 6, axis is located each cell.
the large channels molecules that are by the symbol A: at each corner of
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 along the long diagonal of the unit cell (see Fig. 2). It contains the two 3-fold axes and the Z-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 molecule at residues 35, 55 and 61 to 63. This pattern repeats three times around the axis. Other intermolecular contacts are found between VW of one molecule and CL and CHl of a second molecule related to the first by the 2-fold screw axis. Several additional contacts are found between VL and CHl of a neighbor via the x unit cell translation. It has been noted (Padlan et al., 1973) 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). neighboring
(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 mainchain atoms out of 456 atoms. A number of the residues of the hypervariable loops align quite well, particularly at t,he 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 (NTovotny 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 169” 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 133”, essentially unchanged from the earlier report, (Segal et al., 1974). (d) Bntramotecular contacts As noted elsewhere (reviewed by Davies & Metzger, 1983; Amzel R: Poljak, I979), VLjVH contacts involve hypervariable residues as well as framework residues. The boxed regions of Table 4 indicate hypervariable t,o hypervariable residue while other regions of the Table interactions, involve framework residues. Nearly half (46 in 105) of the interactions between VL and VH involve only hypervariable residues, with most of t,hese being located at the upper end of the interface (Fig. 2) in the vicinity of the combining site a,nd 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 AsnlOlH, and between the hydroxyl group of TyrlOOL and Glu35H. The latter interact’ion 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 loss of phosphocholine binding ability. Other interdomain, intramolecular contacts are presented in Ta’bles 5 to 7. There are no interactions
Phosphocholine Binding
E35
E i::1 Y42 Q44 P49 P50 L52 Y55 G56
Q39
R44
L45
W47
Fab McPC603
599
Table 4 between residues of VL and VH
Contacts Y33
Immunoglobulin
A50
E61
Y97
NlOl
Y103
S105 T106 W107 Y108
F109 W112 G113 All4
: : : : 4 . 1
rl
:
: 2
2 8
: F104 G105 A106
.
: 1 1 6
: 3
:
:
:
10
2
2
4
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 hypervariable/hypervariable interactions.
involving VL with CHl or VH with CL. Tables 6 and 7 show that the number of interdomain intrachain contacts, i.e. VL with CL or VH with CHl, is small.
(e) Domain structure The structures of the four domains of McPC603 by Figure 5, which shows also the location of the hydrogen bonds between the mainchain amide groups. The general tertiary structure of the domains and the distribution of the hydrogen are illustrated
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 IC chain domains of McPC603 resemble the ;1 chain domains of New, Kol and Meg, as well as the IC chains of various hu.man VL dimers (Padlan & Davies, 1975; Padlan, 1977). A significant difference from the y chain structure occurs in CHl, where there is an additional disulfide bond formed between residues 198 and 222 (residues 198 anld 225, in Kabat et aE., 1983). The Cys222 residue should therefore be regarded as forming the end of CHl
Table 5 Contacts between residues of CHl and CL Y131 1123 F124 S127 El29 Q130 s133 v139 F141 N143 L166 5168 W169 T170 S180 Ml81 S182 R217
P132
L133 i
T134
L135
4
2 5.
P136
1145 V173 5
:
F175
P176 Al78
S188 Q196
:
:
:
.
.
3
:
:
1
2 4
i 2
i 1
:
i 1 3 5
:
i 3. 14 1 2 2
i
i
:
:
: i
:
.
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
P46 A86 L89 K109 El11 1112 K113 R114
Y. Satow et al. Table 5
Table 7
Contacts between residues on VL and CL
Contacts between residues qf VJU and Cfib
Y146
El23
D171 Q172 Sl74
K175
D176
i
:
S177 Y179
2 ;
:
:
4 ; 2 1 6
:
3 5
:
:
:
s
: 1
i
j
:
The residues listed in the left column are from VL; those listed at the column heads are from CL. The numbers are as in Table 4.
Figure 5. Schematic drawings of bonds between the main-chain amide with a double line.
G9 Cl0 Lll T117 T119 s121
5124 Al25
F154 P155 G157 T158 IX11
S212 2
i ;
2
;
2
;
: 1
: 3
3 4
The residues listed in the left column are from VH; those listed at the column heads are from CHl. The numbers are aa in Table 4.
the (a) VH, (b) VL; (c) CHl residues. The circles marking
and (d) CL hypervariable
domains residues
illustrating the hydrogen in VH and VL are drawn
Phosphocholine Binding
Immunoglobulin
Fab McPC603
601
8:0
6-O
6.0
;FTFsD LVOPGGSLRCSCATS( SVSAfEAV~MCKSS OSL _LN! XNOKNFL~WYOMcP6OP~Ktt
EVKLVESG 0 IVMTOSP
10
20
30
40
SRNKGNKYTTEYS IYGA
AS”KG”Fl”6ROTSOS~LYLoMN,
~TFJESGVP ~II~T~~GSG
50
60
70
TDFTLIISS
80
(a)
8-O 7.0 6.0 5.0 4.0 3uo -
-----___
- 2.0
ESARNPTLYPLTLPPALS ADAAFVSI
120
SDPVI IGCL~HDYFPSGTM~VTWG
FPPSSE~LTSGGASVVCFLNNFYPK
130
140
KSGKOI TTVNFPPALASGG
LILNVKWKI DGS~AO
150
RVTMSN$TLPAVECP
NGVLNSW~DODSKDSTVSMSSTLTLTK~EYERH
160
170
180
190
I
EGESVKCSVOIJIS
NP VOELLI~NC
NSYTCEATN
KTSTSPIVKSFNRNEC
200
210
l,O
220
(b) Figure 6. The separation (in d) of corresponding alpha-carbon atoms of the superimposed domains of (a) VH and VL, and (b) CHl and CL. The upper sequence is that of the heavy chain and the lower sequence is of the light chain. Every 10th letter in each sequence is underlined. The numbers below the sequences refer to the residue number in th(e light chain. Where the structural alignment has matched identical residue types, the corresponding letters are shown in bold face. Caps occur when there are no corresponding residues for comparison. The horizontal broken lines indicate the r.m.s. separation.
Y. Xatow et al.
602
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 MePC603 are compared in Figure 6(a) and (b). The sequence identity derived from the structural alignment 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 Asn155 (CHl) 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 Asnl60 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 An analogous poorly defined trace of moiety. density is found in the electron density map of the Fab of 5539 (Suh et aZ., 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 2 M) 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 int,eractions 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 molecule, TyrlOOL, Glu35H, another water AsnlOlH, 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
Figure 7. Part of the combining site residues of McPC603, showing the key residues that, surround the 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 Llo2 are from the light chain, the remaining residues being from the heavy chain. Potential hydrogen bonds are illustrated with thin cont,inuous 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 ver8u8 2 X lo* mol-’ in an aqueous environment without ammonium sulfate the sulfate (Rudikoff et al., 1972). Assuming eoncent’ration 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-I. This can be compared with the value of lo6 mol-l for the association constant of the sulfate ion and a sulfate binding protein quoted by Pflugrath & Quiocho (1985). Here the sulfate has a total of seven hydrogen bonds from the protein and is sufficiently different from the binding observed in McPC603 to readily account for the difference in the binding constants.
The Fabs whose structures have been elucidated all display strong lateral interactions between homologous domains in the light and heavy chains, and wesk longitudinal interactions between the variable and constant domains of the same chain. This weak longitudinal interaction results in differences between the relative dispositions of the
Phosphocholine
Binding
Immunoglobulin
variable and constant pairs in each Fab. The Fab of McPC603 and New have an elbow bend near 133”(Navia et al., 1979); the elbow bend of 5539 is 145” (Suh et aZ., unpublished results), while Kol Fab is essentially straight (Marquart et al., 1980). The bend in McPC603 results in the close approach of CHl 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, 5539, 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 CHl probably allows these Fabs to be bent in what appears to be an extreme position, as found in McPC603, 5359 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 VLjCL 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 residues in the light chain and 11 residues in the heavy chain are from hypervariable regions (Table 4). In McPC603 Fab, approximately 45% of the VL/VH atomic contacts are due to hypervariablel hypervariable interactions, while less than onethird of the contacts is contributed by framework/ framework interactions (Table 4). This large contribution of hypervariable/hypervariable 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 viable antibody structures. Alternatively, CLjCHl 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 VLjVH 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 Ll of McPC603 could not be totally ascertained, so that the
complete McPC603
structure of the is not available.
combining site of The present data
represent
the
collection
limit
of data
with
our
available equipment and we do sot plan any further refinement
of the
structure.
The
complex
with
phosphocholine has been refined independently and elsewhere (Padlan et al., will be reported unpublished results). We a,re grateful to Drs Stuart Rudikoff and Michael Potter for the gift of McPC603. We also acknowledge the
Fab McPC603
helpful advice and comments Stuart Rudikoff.
603
of Drs Elvin
Kaba,t and
References Amit, A. G., Mariuzza, R. A., Phillips, S. E. V. & Poljak, R. J. (1985). Nature (London), 313, 156-158. Amzel, L. M. & Poljak, R. J. (1979). Annu. Rev. B&hem. 48, 961-997. Amzel, L. M., Poljak, R. J., Saul, F., Varga, J. M. & Richards, F. F. (1974). Proc. Nat. Acad. Sci., U.S.A. 71; 1427-1430. Arndt, U. W. & Wonacott, A. J. (1977). The Rotation in Method Crystallography, North-Holland, Amsterdam. Auffray, C., Nageotte, R., Sikarov, J.-L., Heidmann, 0. & Rougeon, F. (1981). Gene, 13, 365-374. Bernstein, F. C., Koetzle, T. F., Williams, G. (J. B., Meyer, E. F., Brice, M. D., Rogers, J. R., Kennard, 0.; Shimanouchi, T. & Tasumi, M. (1977). J. Mol. Biol. 112, 535-542. Blow, D. M. & Crick, F. H. C. (1959). Acta Crystallogr. 12, 794-802. Busing, W. R., Martin, K. 0. & Levy, H. A. (1962). ORFLS, a Fortran Crystallographic Least-Squares Program. Report ORNL-TM-305, Oak Ridge National Laboratory, Oak Ridge, TN. Cohen, G. H., Silverton, E. W. & Davies, D. R. (1981). J. Mol. Biol. 148, 449-479. Colman, P. M., Gough, K. H., Lilley, G. G., Blagrove, R. J., Webster, R. G. & Laver, W. G. (1981). jr. Mol. Biol. 152, 609-614. Davies, D. R. & Metzger, H. (1983). Annu. Rev. Immunol. 1, 87-117. Diamond, R. (1981). In Biomolecular Structure, Function, Conformation and Evolution (Srinivasan, R. : ed. ) , vol. 1, pp. 567-590, Pergamon Press, Oxford. Dickerson, R. E., Kendrew, J. C. & Strandberg, B. E. (1961). Acta Crystallogr. 14, 1188-1195. Gibson, A. L., Herron, J. N., Ballard, D. W., Voss, E. W. Jr, He, X. M., Patrick, V. A. & Edmundson, A. B. (1985). Mol. Immunol. 22, 499-502. Hamilton, W. C., Rollet, J. S. & Sparks, R. A. (1965). Acta Crystallogr. 18, 129-130. Harrison, S. C. (1968). J. AppZ. Crystallogr. 1, 84-90. Hendrickson, W. A. & Konnert, J. A. (19811). In Biomolecular Structure, Function, Conformation and Evolution (Srinivasan, R., ed.), vol. 1, pp. 43-47, Pergamon Press, Oxford. Hill, T. L. (1985). Co-operatiwity Theory in Biochemistry, Springer-Verlag, New York, Berlin, Heidelberg and Tokyo. Huber, R. & Steigemann, W. (1974). FEBS Lettecrs, 48, 235-237. Kabat, E. A., Wu, T. T., Bilofsky, H., Reid-Miller, M. & Perry, H. (1983). Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, Public Health Service, Nabtional Institutes of Health, Washington. Kohnert, J. H. (1976). Acta Crystallogr. sect. A, 32, 614617. Luzzati, V. (1952). Acta Crystallogr. 5, 802-810. Mariuzza, R. A., Jankovic, D. L., Boulot, G., Amit, A. G.: Saludjian, P., LeGuern, A., Mazie, J. C. & Poljak, R. J. (1983). J. Mol. Biol. 170, 1055-1058. Marquart, M., Deisenhofer, J., Huber, R. & Palm, W. (1980). J. Mol. Biol. 141, 369-391. Matthews, B. W. (1966). Acta CrystaZZogr. 20, 82-86.
604
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Matthews, B. W., Kopfenstein, C. E. $ Colman, P. ;\I. (1972). ./. Phys. E. Sci. Instr. 5, 353-359. Navia, M. A., Segal, D. M., Padlan, E. A., Davies, D. R., Rao, D. S., Rudikoff, S. & Potter, M. (1979). Proc. Nat. Acad. Xci., U.S.A. 76, 4071-4074. Needleman, S. B. & Wunsch, C. D. (1970). J. MoZ. Biol. 48, 443-453. n’ovotny, J. & Haber, E. (1985). Proc. Nat. Acad. Sci., U.S.A. 82, 4592-4596. Piovotny, J., Bruccoleri, R., h’ewell, J., Murphey, D.: Haber, E. & Karplus, M. (1983). J. Biol. Chem. 258, 14433-14437. Padlan, E. A. (1977). Quart. Rev. Biophys. 10, 35-65. Padlan, E. A. & Davies, D. R. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 819-823. Padlan, E. A., Segal, D. M., Spande, T. F., Davies, D. R., Rudikoff, S. & Potter, M. (1973). Nature New BioE. 245, 165-167. Pflugrath, J. W. & Quiocho, F. A. (1985). Nature {London), 314, 257-260. Richards, F. M. (1968). J. Mol. BioZ. 37, 225-230. Robinson, E. A. & Appella, E. (1979). J. BioZ. Chem. 254, 11418-11430. Robinson, E. $. & Appella, E. (1980). Proc. Nat. Acad. Sci., U.S.A. 77, 4909-4913. Rossmann, M. G., Leslie, A. G. W., Abdel-Meguid, S. & Tsukihara, T. (1979). J. AppZ. Crystallogr. 12, 570581. Edited
Rudikoff, S. & Potter, M. (1974). Biochemistry, 13, %t334038. Rudikoff, S., Potter, M., Segal, D. M., Padlan, E, A. & Davies, D. R. (1972). Proc. Xa.t. Acad. Sci., U.S.A. 66. 3689-3692. Rudikoff, S., Satow, Y., Padlan, E. A., Davies, D. R,. c?r Potter, M. (1981). Mol. Immunol. 18! 705-711, Rudikoff, S., Giusti, A. M., Cook, W. D. & Scharff. M. I). [1982). Proc. Nat. Acad. Sci., U.S.A. 79, 1979-1983. Saul: F., Amzel, L. M. & Poljak, R. J. (1978). J. Riol. Chem. 253, 585-597. Segai, D. M., Padlan, E. A., Cohen, G. H.. Rudikoff: S.. Potter, M. & Davies, D. R.. (1974). Proc. *Vat. Acad. Sci., U.S.A. 71, 4298-4302. Sielecki, A. R..; Hendrickson, W. 9., Broughton, C. G.. Delbaere, L. T. J.: Brayer, 6;. D. & James, M. X. G. (1979). J. Mol. Biol. 134; 781-804. Silverton, E. W.; Padlan, E. A., Davies, D. R., SmitbGill, S. & Potter, M. (1984). J. Mol. BioZ. 180, 761765. Svasti, J. & M&stein, C. (1972). Biochem. J. 128. 427-444. Tsernoglou, D.. Petsko, 6. A.; McQueen, J. E. & Hermans, J. (1977). Science, 197, 1378-1381. Tucker, P. W., Siightom, J. R. & Blattner, F. R. (1981). hoc. Nat. Acad. Sci., U.S.A. 78, 7684-7688. Winkler, F. K., Schutt, C. E. & Harrison, S. 6. (1979). Acta Crystallogr. sect. A, 35, 901-911.
by R. Huber
Biol.
(1986) 190, 593-604
Phosphocholine
Binding Immunoglobulin
An X-ray Diffraction Yoshinori
Study at 2.7 A
Satowf-, Gerson H. Cohen, Eduardo National
Fab McPC603
A. Padlan and David R. Davies
Laboratory of Molecular Biology Institute of Arthritis, Diabetes and Digestive and Kidney Diseases National Institutes of Health, Bethesda, MD 20892, U.X.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 .& 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 hypervariable 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
Konnert, 1981). In this paper, of this investigation.
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 Kl derivative (Amzel et al., 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 (IgA,rc) protein that was previously reported (Segal et al., 1974) was an unrefined structure based on 3.1 .& diffractometer data and with only partial sequence information. This work has now been extended through‘the collection of a complete set of 2.7 d diffraction data using augmented by the oscillation photography, 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 &
2. Materials
v$O3.00/0
the results
and Methods
(a) Crystal structure analysis and preliminary rejinement at 3.1 B 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 TmCl, (American Potash & Chemical Corp.) K,Pt(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 m&r-TmCl, or 0.4 mMrespectively, for 3 to 4 weeks. An iodine K,Pt(CPU’S),, derivative was prepared by soaking crystals in 50 m;M-KI, 2.5 miv-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 d data were collected using a Picker FACS-I diffractometer (Segal et al., 1974); the TmCl, 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-
t Present address: Photon Factory, National Laboratory for High Energy Physics, Oho-machi, Tsukuba-gum, Ibaraki-ken, 305, Japan. 002%2836/86/160190-12
we report
593
0
1986 Academic
Press Inc.
(London)
Ltd.
Y. Satow eL all
594
atom refinement and phase calculation (Dickerson et al.; 1961) were then computed using local versions of the programs of Busing et aE. (1962) and Matthews (1966). Table 1 shows the refined heavy-atom para,meters. 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 the computer graphics system GRIP at the Deparbment of Computer Science, University of Pu’orthCarolina (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 8. At this point, the fit of the model to a 2F,- F, map was using examined BILDER (Diamond, 1981), as implemented on a PDP 11/70 under the RSX-11M operating system (G. H. Cohen, unpublished results). In the second stage of refinement, the protein geometry was initially less restrained and subsequently tightened, yielding a final residual of 0.24. The r.m.s. total shift from the atomic positions at the start of the second stage of refinement was 0.42 8. A sulfate ion, t’hat 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 function based on the heavy-atom phases. These regions remained poorly defined after the preliminary refinement. (b) Crystal structure analysis and re$nement at 2.7 d resolution A new set of crystals was prepared for the higher resolution, 2.7 ip, 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 1Vo-Screen Medical X-ray films (3 in a pack) using Ni-filtered CuKcc radiation from an Elliott GX-6 rotating anode X-ray generator operated at 40 kV and 40 mA. 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 oscillation about the 2 crystal axes, b and c; with an
t Abbreviations used: r.m.s., root-mean-square; F,, observed structure factor amplitude; F,, calculated structure 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; CHl, first constant domain of heavy chain; m.i.r., multiple isomorphous replacement; Ll; L2 and L3, lst, 2nd and 3rd CDR of the light chain; Hl, H2 and H3, lst, 2nd and 3rd CDR of the heavy chain.
Table B Heavy-aiom parameters Co-ordinates Heavy-atom compound
.F
Y
2
K,Pt(CNS),
0.3807
0.3936
0.5422
0.0218
TmC13 Iodine-l iodine-2 Iodine-3 Iodine-4
0.3427 0.2336 0.2409 0.2328 0.1241
0.6617 0.7083 0.6794 0.6328 0.7017
0.2523 0.5569 0.5695 0.6404 0.4838
0.0245 0.0119 0.0184 0.0125 0.0044
occupancyt
Thermal facior (AZ) 19,6 34.8 28.3 7.5 6.7 6.8
“rThe site occupancy is on an arbitrary scale in which I-he average stru&ure amplitude of the native protein is 14.3.
oscillation 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 pm 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 1 l/70 computer using the rotation program package writ,ten by G. Cornick & 11. A. Navia (unpublished results). Intensities from each pack were further processed through intra-film-pack scaling. post, refinement. scaling and averaging using programs written for these purposes specially (Y. Satow. 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 film base and emulsion, then were corrected for Lorentz and polarization factors. The measured int’ensities 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 a,E. (1979) and Rossmann et al. (1979). The total number of 147,706 intensities, including partially recorded reflections, were finally scaled and symmetry averaged. yielding 24,235 unique reflections for the resolution range of 10 f%to 2.7 d. The agreement fa,ctor:
h
j
h
where the intensity Ihj for reflection h was measured ATh times, was 0.077 or 0.054 for the structure fa,ctor amplitudes Fhj. Within a 3 A sphere, 95% of the data were retained; wit,hin 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 ,!%results. It was subjected to a number of cycles of least-squares refinement (Hendrickson & Konnert. 1981) with periodic examinat,ion and rebuilding of the model using BILDER on a VAX 111780 (R. Ladner, unpublished results). The sulfate 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 O”41. After 5 cycles of positiona, parameter refinement with an overall temperature factor; the value was reduced to 0.33. Individual temperature factors for the atoms were used in t’he succeeding cycles. The program restrains the values of
Phosphocholine
Binding
Immunoglobulin
the 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(lF,I - lF,[)’ was approximately constant over the range of the data used (80 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 AF and W-F, 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 Rudikoff et aZ. (1981) and Rudikoff & Potter (1974), with the addition of the tetrapeptide Leu-Glu-Ile-Lys, which occurs at the end of VL (Rudikoff, unpublished results). The CHl sequence given by Auffray et al. (1981), obtained by translation of the nucleotide sequence of cDNA complementary to alpha-chain mRNA from 5558 tumor cells, (sequence 60, p. 175, Kabat et al., 1983) was used. This sequence differs in 4 places from that obtained by Tucker et aZ. (1981) from BALB/c genomic DNA, while they both differ in 2 of these positions from the sequence reported by Robinson $ Appella (1980) for MOPC511, 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 1 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 l-27 28-33 34-220
Heavy chain
Kabat et al.
This work
l-27 27a-27f 28-214
1-52 53-55 56-85 86-88 89-106 107-109 110-139 140-142 143-163 164-171 1722184 185-200 201-205 206-216 217-222
Kabat
et al.
1-52 52a-52c 53-82 82a-82c 833100 lOOa-100c 101-130 133-135 137-157 162-169 171-183 185-200 202-206 208-218 220-225
Fab McPC603
595
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 preserving 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 communicati~on).
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 ~,IJJ plot for the main chain is shown in Figure 1. There are a few residues that have “forbidden” C#J,I+!J values. The quality of the map in these regions together with the low
Table 3 Summary Konnert,
of stereochemical criteria (Hendrickson & 1981; Sielecki et al., 1979; Cohen et al., 1981) Final model
Target (r
0.225 136 Interatomic 1-2 l-3 l-4
t
distances (A) 0.020 0.040 0.037
0615 0620 0.025
(A)
0.027
0920
Chiral volumes (A3) Non-bonded contacts (A) l-4 Other
0.257
0.150
0.24 0.34
O.!jO O.!jO
Planarity
Angles (deg.) a> xs (AM Xl> ‘> x4 Temperature Main chain Main chain Side chain Side chain
27.0 5.0 factors (A’) l-2 l-3 l-2 l-3
0.5 1.0 0.4 0.7
15.0 5.0 05
0.#7 0..5 0.7
The standard groups dictionary used is specified in T.able 2 of Sielecki et al. (1979). In this Table, F0 refers to the observed structure factor, F, is the calculated structure factor and AF is the quantity llF,l-lF,ll. The target 0 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 AF, was modeled by the function w = (l/d)’ with: d = 40--5OO(sin(tl)/,l-l/6). By this means, w(AF) 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. 1(1981).
Y. Satow et al.
596
xx x G 0 0
Q x x
x X
0
Q
Q
Q
x Y
:: i
Q
(II ,x
‘mo.00
m -135.00
I!fQX X -90.00
X
c) -h.
i5 IO
00 p’
u5.00
HI
Figure 1. A plot of the dihedral angles at’ each alpha-carbon 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 (1952). 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., 1977). (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., 1974). Most of these changes are to be found in the details of the loops, in particular Ll and H3. The region of Ll 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! Ll 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.
90.00
135.00
E I
atom for the refined co-ordinates.
Asterisk,
Pro; cross,
placements Three other residues have tentative due to problems in maintaining proper stereochemistry while fitting the electron density: Gln162 and Asn163 (residues 156 and 157 in the numbering used by Kabat et al., 1983) of CL and Glu202 (residue 203, Kabat et al., 1983) of CHl. 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 t,hese cases: the configuration corresponding to the stronger density was chosen for the model. The 442 residues of &PC603 Fab include 25 proline residues. Of these, five are cis-proline: residues 8 and 101 in VL, 147 in CL, and 143 and I55 in CHI. The assignment of the configuration of all five cis-proline residues was unambiguous. The structurally homologous Pro& and Pro95 of Rei (Huber & Steigemann, 1974), and Pro147 (CL) and Pro155 (CHl) of Kol (Marquart et al., 1980), also have been found in the cis conformation. Pro143 (CHl ) of McPC603 has no counterpart in previously reported antibody structures. (b) Crystal
pa&@
The McPC603 Fab crystallizes in the space group P6, (Rudikoff et al., 1972), with a = 162.53 8: c = 60.72 A. The molecules are situated in clusters
Phosphocholine Binding
Figure 2. A stereo drawing filled
circles
of the alpha-carbon show the complementarity-determining
Immunoglobulin
skeleton of McPC603. Continuous residues (Kabat et al.; 1983).
at each of the crystallographic 3-fold axes of the unit cell (B’ig. 3). Each cluster is related to neighboring clusters via the 2-fold screw situated midway between the 3-fold axes (Brigs 3 and 4). The clusters of three are maintained principally by a set
of three
Fab McPC603
lines denote
597
the heavy
chain.
The
of hydrogen-bond and van der Waals contacts between neighboring molecules. Residues 14 to 20 and 71 of VL interact with residues 1, 26, 2’7, 100, 102, 104, 105 108 and 110 of the VH Iof the neighboring molecule while residues 18, 66, 67, 69,
Figure 3. A projection of the alpha-carbon skeletons of 4 unit cells and the ab plane, illustrating that run parallel to the c axis through the crystal. The heavy chains have been drawn bolder in the 3 clustered about a crystallographic 3-fold axis of the lower left-hand cell. The 3-fold axes are indicated Z-fold screw axes are located midway between each adjacent pair of 3-fold axes; a 6, axis is located each cell.
the large channels molecules that are by the symbol A: at each corner of
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 along the long diagonal of the unit cell (see Fig. 2). It contains the two 3-fold axes and the Z-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 molecule at residues 35, 55 and 61 to 63. This pattern repeats three times around the axis. Other intermolecular contacts are found between VW of one molecule and CL and CHl of a second molecule related to the first by the 2-fold screw axis. Several additional contacts are found between VL and CHl of a neighbor via the x unit cell translation. It has been noted (Padlan et al., 1973) 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). neighboring
(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 mainchain atoms out of 456 atoms. A number of the residues of the hypervariable loops align quite well, particularly at t,he 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 (NTovotny 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 169” 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 133”, essentially unchanged from the earlier report, (Segal et al., 1974). (d) Bntramotecular contacts As noted elsewhere (reviewed by Davies & Metzger, 1983; Amzel R: Poljak, I979), VLjVH contacts involve hypervariable residues as well as framework residues. The boxed regions of Table 4 indicate hypervariable t,o hypervariable residue while other regions of the Table interactions, involve framework residues. Nearly half (46 in 105) of the interactions between VL and VH involve only hypervariable residues, with most of t,hese being located at the upper end of the interface (Fig. 2) in the vicinity of the combining site a,nd 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 AsnlOlH, and between the hydroxyl group of TyrlOOL and Glu35H. The latter interact’ion 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 loss of phosphocholine binding ability. Other interdomain, intramolecular contacts are presented in Ta’bles 5 to 7. There are no interactions
Phosphocholine Binding
E35
E i::1 Y42 Q44 P49 P50 L52 Y55 G56
Q39
R44
L45
W47
Fab McPC603
599
Table 4 between residues of VL and VH
Contacts Y33
Immunoglobulin
A50
E61
Y97
NlOl
Y103
S105 T106 W107 Y108
F109 W112 G113 All4
: : : : 4 . 1
rl
:
: 2
2 8
: F104 G105 A106
.
: 1 1 6
: 3
:
:
:
10
2
2
4
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 hypervariable/hypervariable interactions.
involving VL with CHl or VH with CL. Tables 6 and 7 show that the number of interdomain intrachain contacts, i.e. VL with CL or VH with CHl, is small.
(e) Domain structure The structures of the four domains of McPC603 by Figure 5, which shows also the location of the hydrogen bonds between the mainchain amide groups. The general tertiary structure of the domains and the distribution of the hydrogen are illustrated
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 IC chain domains of McPC603 resemble the ;1 chain domains of New, Kol and Meg, as well as the IC chains of various hu.man VL dimers (Padlan & Davies, 1975; Padlan, 1977). A significant difference from the y chain structure occurs in CHl, where there is an additional disulfide bond formed between residues 198 and 222 (residues 198 anld 225, in Kabat et aE., 1983). The Cys222 residue should therefore be regarded as forming the end of CHl
Table 5 Contacts between residues of CHl and CL Y131 1123 F124 S127 El29 Q130 s133 v139 F141 N143 L166 5168 W169 T170 S180 Ml81 S182 R217
P132
L133 i
T134
L135
4
2 5.
P136
1145 V173 5
:
F175
P176 Al78
S188 Q196
:
:
:
.
.
3
:
:
1
2 4
i 2
i 1
:
i 1 3 5
:
i 3. 14 1 2 2
i
i
:
:
: i
:
.
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
P46 A86 L89 K109 El11 1112 K113 R114
Y. Satow et al. Table 5
Table 7
Contacts between residues on VL and CL
Contacts between residues qf VJU and Cfib
Y146
El23
D171 Q172 Sl74
K175
D176
i
:
S177 Y179
2 ;
:
:
4 ; 2 1 6
:
3 5
:
:
:
s
: 1
i
j
:
The residues listed in the left column are from VL; those listed at the column heads are from CL. The numbers are as in Table 4.
Figure 5. Schematic drawings of bonds between the main-chain amide with a double line.
G9 Cl0 Lll T117 T119 s121
5124 Al25
F154 P155 G157 T158 IX11
S212 2
i ;
2
;
2
;
: 1
: 3
3 4
The residues listed in the left column are from VH; those listed at the column heads are from CHl. The numbers are aa in Table 4.
the (a) VH, (b) VL; (c) CHl residues. The circles marking
and (d) CL hypervariable
domains residues
illustrating the hydrogen in VH and VL are drawn
Phosphocholine Binding
Immunoglobulin
Fab McPC603
601
8:0
6-O
6.0
;FTFsD LVOPGGSLRCSCATS( SVSAfEAV~MCKSS OSL _LN! XNOKNFL~WYOMcP6OP~Ktt
EVKLVESG 0 IVMTOSP
10
20
30
40
SRNKGNKYTTEYS IYGA
AS”KG”Fl”6ROTSOS~LYLoMN,
~TFJESGVP ~II~T~~GSG
50
60
70
TDFTLIISS
80
(a)
8-O 7.0 6.0 5.0 4.0 3uo -
-----___
- 2.0
ESARNPTLYPLTLPPALS ADAAFVSI
120
SDPVI IGCL~HDYFPSGTM~VTWG
FPPSSE~LTSGGASVVCFLNNFYPK
130
140
KSGKOI TTVNFPPALASGG
LILNVKWKI DGS~AO
150
RVTMSN$TLPAVECP
NGVLNSW~DODSKDSTVSMSSTLTLTK~EYERH
160
170
180
190
I
EGESVKCSVOIJIS
NP VOELLI~NC
NSYTCEATN
KTSTSPIVKSFNRNEC
200
210
l,O
220
(b) Figure 6. The separation (in d) of corresponding alpha-carbon atoms of the superimposed domains of (a) VH and VL, and (b) CHl and CL. The upper sequence is that of the heavy chain and the lower sequence is of the light chain. Every 10th letter in each sequence is underlined. The numbers below the sequences refer to the residue number in th(e light chain. Where the structural alignment has matched identical residue types, the corresponding letters are shown in bold face. Caps occur when there are no corresponding residues for comparison. The horizontal broken lines indicate the r.m.s. separation.
Y. Xatow et al.
602
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 MePC603 are compared in Figure 6(a) and (b). The sequence identity derived from the structural alignment 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 Asn155 (CHl) 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 Asnl60 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 An analogous poorly defined trace of moiety. density is found in the electron density map of the Fab of 5539 (Suh et aZ., 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 2 M) 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 int,eractions 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 molecule, TyrlOOL, Glu35H, another water AsnlOlH, 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
Figure 7. Part of the combining site residues of McPC603, showing the key residues that, surround the 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 Llo2 are from the light chain, the remaining residues being from the heavy chain. Potential hydrogen bonds are illustrated with thin cont,inuous 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 ver8u8 2 X lo* mol-’ in an aqueous environment without ammonium sulfate the sulfate (Rudikoff et al., 1972). Assuming eoncent’ration 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-I. This can be compared with the value of lo6 mol-l for the association constant of the sulfate ion and a sulfate binding protein quoted by Pflugrath & Quiocho (1985). Here the sulfate has a total of seven hydrogen bonds from the protein and is sufficiently different from the binding observed in McPC603 to readily account for the difference in the binding constants.
The Fabs whose structures have been elucidated all display strong lateral interactions between homologous domains in the light and heavy chains, and wesk longitudinal interactions between the variable and constant domains of the same chain. This weak longitudinal interaction results in differences between the relative dispositions of the
Phosphocholine
Binding
Immunoglobulin
variable and constant pairs in each Fab. The Fab of McPC603 and New have an elbow bend near 133”(Navia et al., 1979); the elbow bend of 5539 is 145” (Suh et aZ., unpublished results), while Kol Fab is essentially straight (Marquart et al., 1980). The bend in McPC603 results in the close approach of CHl 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, 5539, 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 CHl probably allows these Fabs to be bent in what appears to be an extreme position, as found in McPC603, 5359 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 VLjCL 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 residues in the light chain and 11 residues in the heavy chain are from hypervariable regions (Table 4). In McPC603 Fab, approximately 45% of the VL/VH atomic contacts are due to hypervariablel hypervariable interactions, while less than onethird of the contacts is contributed by framework/ framework interactions (Table 4). This large contribution of hypervariable/hypervariable 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 viable antibody structures. Alternatively, CLjCHl 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 VLjVH 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 Ll of McPC603 could not be totally ascertained, so that the
complete McPC603
structure of the is not available.
combining site of The present data
represent
the
collection
limit
of data
with
our
available equipment and we do sot plan any further refinement
of the
structure.
The
complex
with
phosphocholine has been refined independently and elsewhere (Padlan et al., will be reported unpublished results). We a,re grateful to Drs Stuart Rudikoff and Michael Potter for the gift of McPC603. We also acknowledge the
Fab McPC603
helpful advice and comments Stuart Rudikoff.
603
of Drs Elvin
Kaba,t and
References Amit, A. G., Mariuzza, R. A., Phillips, S. E. V. & Poljak, R. J. (1985). Nature (London), 313, 156-158. Amzel, L. M. & Poljak, R. J. (1979). Annu. Rev. B&hem. 48, 961-997. Amzel, L. M., Poljak, R. J., Saul, F., Varga, J. M. & Richards, F. F. (1974). Proc. Nat. Acad. Sci., U.S.A. 71; 1427-1430. Arndt, U. W. & Wonacott, A. J. (1977). The Rotation in Method Crystallography, North-Holland, Amsterdam. Auffray, C., Nageotte, R., Sikarov, J.-L., Heidmann, 0. & Rougeon, F. (1981). Gene, 13, 365-374. Bernstein, F. C., Koetzle, T. F., Williams, G. (J. B., Meyer, E. F., Brice, M. D., Rogers, J. R., Kennard, 0.; Shimanouchi, T. & Tasumi, M. (1977). J. Mol. Biol. 112, 535-542. Blow, D. M. & Crick, F. H. C. (1959). Acta Crystallogr. 12, 794-802. Busing, W. R., Martin, K. 0. & Levy, H. A. (1962). ORFLS, a Fortran Crystallographic Least-Squares Program. Report ORNL-TM-305, Oak Ridge National Laboratory, Oak Ridge, TN. Cohen, G. H., Silverton, E. W. & Davies, D. R. (1981). J. Mol. Biol. 148, 449-479. Colman, P. M., Gough, K. H., Lilley, G. G., Blagrove, R. J., Webster, R. G. & Laver, W. G. (1981). jr. Mol. Biol. 152, 609-614. Davies, D. R. & Metzger, H. (1983). Annu. Rev. Immunol. 1, 87-117. Diamond, R. (1981). In Biomolecular Structure, Function, Conformation and Evolution (Srinivasan, R. : ed. ) , vol. 1, pp. 567-590, Pergamon Press, Oxford. Dickerson, R. E., Kendrew, J. C. & Strandberg, B. E. (1961). Acta Crystallogr. 14, 1188-1195. Gibson, A. L., Herron, J. N., Ballard, D. W., Voss, E. W. Jr, He, X. M., Patrick, V. A. & Edmundson, A. B. (1985). Mol. Immunol. 22, 499-502. Hamilton, W. C., Rollet, J. S. & Sparks, R. A. (1965). Acta Crystallogr. 18, 129-130. Harrison, S. C. (1968). J. AppZ. Crystallogr. 1, 84-90. Hendrickson, W. A. & Konnert, J. A. (19811). In Biomolecular Structure, Function, Conformation and Evolution (Srinivasan, R., ed.), vol. 1, pp. 43-47, Pergamon Press, Oxford. Hill, T. L. (1985). Co-operatiwity Theory in Biochemistry, Springer-Verlag, New York, Berlin, Heidelberg and Tokyo. Huber, R. & Steigemann, W. (1974). FEBS Lettecrs, 48, 235-237. Kabat, E. A., Wu, T. T., Bilofsky, H., Reid-Miller, M. & Perry, H. (1983). Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, Public Health Service, Nabtional Institutes of Health, Washington. Kohnert, J. H. (1976). Acta Crystallogr. sect. A, 32, 614617. Luzzati, V. (1952). Acta Crystallogr. 5, 802-810. Mariuzza, R. A., Jankovic, D. L., Boulot, G., Amit, A. G.: Saludjian, P., LeGuern, A., Mazie, J. C. & Poljak, R. J. (1983). J. Mol. Biol. 170, 1055-1058. Marquart, M., Deisenhofer, J., Huber, R. & Palm, W. (1980). J. Mol. Biol. 141, 369-391. Matthews, B. W. (1966). Acta CrystaZZogr. 20, 82-86.
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Matthews, B. W., Kopfenstein, C. E. $ Colman, P. ;\I. (1972). ./. Phys. E. Sci. Instr. 5, 353-359. Navia, M. A., Segal, D. M., Padlan, E. A., Davies, D. R., Rao, D. S., Rudikoff, S. & Potter, M. (1979). Proc. Nat. Acad. Xci., U.S.A. 76, 4071-4074. Needleman, S. B. & Wunsch, C. D. (1970). J. MoZ. Biol. 48, 443-453. n’ovotny, J. & Haber, E. (1985). Proc. Nat. Acad. Sci., U.S.A. 82, 4592-4596. Piovotny, J., Bruccoleri, R., h’ewell, J., Murphey, D.: Haber, E. & Karplus, M. (1983). J. Biol. Chem. 258, 14433-14437. Padlan, E. A. (1977). Quart. Rev. Biophys. 10, 35-65. Padlan, E. A. & Davies, D. R. (1975). Proc. Nat. Acad. Sci., U.S.A. 72, 819-823. Padlan, E. A., Segal, D. M., Spande, T. F., Davies, D. R., Rudikoff, S. & Potter, M. (1973). Nature New BioE. 245, 165-167. Pflugrath, J. W. & Quiocho, F. A. (1985). Nature {London), 314, 257-260. Richards, F. M. (1968). J. Mol. BioZ. 37, 225-230. Robinson, E. A. & Appella, E. (1979). J. BioZ. Chem. 254, 11418-11430. Robinson, E. $. & Appella, E. (1980). Proc. Nat. Acad. Sci., U.S.A. 77, 4909-4913. Rossmann, M. G., Leslie, A. G. W., Abdel-Meguid, S. & Tsukihara, T. (1979). J. AppZ. Crystallogr. 12, 570581. Edited
Rudikoff, S. & Potter, M. (1974). Biochemistry, 13, %t334038. Rudikoff, S., Potter, M., Segal, D. M., Padlan, E, A. & Davies, D. R. (1972). Proc. Xa.t. Acad. Sci., U.S.A. 66. 3689-3692. Rudikoff, S., Satow, Y., Padlan, E. A., Davies, D. R,. c?r Potter, M. (1981). Mol. Immunol. 18! 705-711, Rudikoff, S., Giusti, A. M., Cook, W. D. & Scharff. M. I). [1982). Proc. Nat. Acad. Sci., U.S.A. 79, 1979-1983. Saul: F., Amzel, L. M. & Poljak, R. J. (1978). J. Riol. Chem. 253, 585-597. Segai, D. M., Padlan, E. A., Cohen, G. H.. Rudikoff: S.. Potter, M. & Davies, D. R.. (1974). Proc. *Vat. Acad. Sci., U.S.A. 71, 4298-4302. Sielecki, A. R..; Hendrickson, W. 9., Broughton, C. G.. Delbaere, L. T. J.: Brayer, 6;. D. & James, M. X. G. (1979). J. Mol. Biol. 134; 781-804. Silverton, E. W.; Padlan, E. A., Davies, D. R., SmitbGill, S. & Potter, M. (1984). J. Mol. BioZ. 180, 761765. Svasti, J. & M&stein, C. (1972). Biochem. J. 128. 427-444. Tsernoglou, D.. Petsko, 6. A.; McQueen, J. E. & Hermans, J. (1977). Science, 197, 1378-1381. Tucker, P. W., Siightom, J. R. & Blattner, F. R. (1981). hoc. Nat. Acad. Sci., U.S.A. 78, 7684-7688. Winkler, F. K., Schutt, C. E. & Harrison, S. 6. (1979). Acta Crystallogr. sect. A, 35, 901-911.
by R. Huber