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Crystal and molecular structure of the dimer of variable domains of the Bence-Jones protein ROY
J. Mol.
Biol.
(1977) 116, 73-79
Crystal and Molecular Structure of the Dimer of Variable Domains of the Bence-Jones Protein ROY P. M. COLMAN*, H. J. SCHRAM~C~~ AND J. M. Gussa %‘choo1 of Chemistry,
bMax Planck
Institut
of Sydney, @ydney 2006, Australia, and Biochemie, SO33 Martinsried hei Miinchen West Germany
University
jiir
(Received 19 April
1977)
The crystal and molecular structure of the dimcr of variable domains of the Bence-Jones protein ROY has been determined by Patterson function search procedures, using the known structure of the protein REI. The structure has been partially refined at 3.0 ii resolution to a crystallographic R-factor value of 0.33. One of the 18 residues differentiating ROY from REI is the substitution of Tyr96 for Leu96, a substitution which makes the combining site of the ROY dimer larger, Substantial movement of Tyr49 suggests t#hat point substitutions in the hypervariable segments may affect the conformation of neighbouring residues in the antigen-combining site, possibly producing differences in specificity larger than might otherwise be expected.
1. Introduction Crystal structure studies of immunoglobulin fragments have provided basic information on the three-dimensional conformation of immunoglobulins. In particular, a Bence-Jones protein (Schiffer et al., 1973), two Fab fragments (Poljak et al., 1973; Segal et al., 1974), two light-chain variable domain dimers (Epp et al., 1974; Fehlhammer et al., 1975) and more recently an Fc fragment (Deisenhofer et al., 1976a,b) have been analysed to resolutions of 3.5 A or higher. Difficultyin crystallizing whole immunoglobulins, probably due to their inherent flexibility, has required that functional inferences be drawn from work on the structure of fragments. In most cases these inferences are likely to be sound, although the analysis of a whole IgGl (Colman et al., 1976a) has led to the suggestion that the free Fab fragment may adopt a conformation similar to that found in intact molecules only after t)hey are complexed w&h antigen (Colman et al., 1976a; Huber et al., 1976). Although the notion of sequence changes in the hypervariable segments of the variable domains being directly related to a specific antigen-binding capacity is now well understood, there are few hard data on stereochemical aspects of such sequence of the changes. A convenient system for studyin, D such effects takes advantage availability of crystals of a number of light-chain variable domains which differ f ‘orn each other at a limited number of positions only. These proteins exist in solution as dimers (Solomon & McLaughlin, 1969; Schramm, 1971) and X-ray studies on two of them, REI (Epp et al., 1974) and AU (Fehlhammer et al., 1975) have shown that t,he mode of aggregation of the domains into the dimer is very similar to that found between variable domains of heavy and light chains in Fab fragments. This mode
74
P.
M.
COLMAN,
H.
J.
SCHRAMM TABLE
AND
J.
M.
CUSS
1
Amino acid difference.s in the cariable domains of the proteins ROY, REI and AU ROY
REI
AU
&3XIle Phe LYS ASP LYS Glu Ala Thr Thr ASP Phe Phe ASP Asu Leu
Ile LYS TY~ Thr Glu Am Glu Ala Ser Thr ASP TY~ Tyr Gln Ser Tyr Gln Leu Gln I10 Thr
Ser ASP Tyi LVR Asp Am Glu SW G’Y Ala His Phe Tyr ASP Tyr Trp Gln Val Glu Ile LYS
-____ 30 31 32 39 50 53 55 56 65 69 70 71 91 92 93 96 100 104 106 106 107
Gly Val ASP Phe LYS
of aggregation is functionally important since the antigen-combining site comprises portions of both domains. The REI protein has now been refined to a model accurate to approximately 0.2 A (Epp et al., 1975) and we have used both the monomer and dimer of this refined model to determine the structure of a third variable domain dimer ROY. The use of refined model co-ordinates in Patterson function analysis of related proteins has proven ultimately capable of describing the differences between the model protein and the unknown protein (Fehlhammer et al., 1975; Colman et d., 19766). Since accurate co-ordinates are not yet available for the AU protein, we preferred to start with the REI model. In Table 1, the sequence differences between REI, AU and ROY are listed. REI crystallizes in apace group P6, with a = 75.8 A, c = 98.2 A and there are two domains (M, 11,000) in the asymmetric unit, AU crystallizes with one domain per asymmetric unit in space group P6,22 with a = 82.3 A, c = 77.0 A.
2. Materials and Methods (a) Preparation
and crystallization
of protein
Bence-Jones protein ROY was supplied by Professor N. Hilschmann (Giittingen). The sequence is known (Hilschmann, 1967). 90 mg of the protein was digested with 0.8 mg pepsin (Sigma) in 18 ml of 0.1 M-ammonium acetate at pH 3.5. The digestion was carried out at 37°C for 40 min. The pH was adjusted to 6.0 and the light precipitate so formed was spun off. The supernatant was concentrated by pressure dialysis and passed over a Sephadex (Pharmacia) G50 column (1.5 cm x 100 cm). The column had been equilibrated with 0.5 M-acetic acid and the flow-rate was 19 ml/h. The main elution band appeared after about 5 h and upon lyophilization yielded 7.8 mg protein. This protein was then dissolved in 0.4 ml of 0.1 &I-Tris buffer at pH 8.0 and set concentrations of up in tubes for dialysis (Zeppezauer et al., 1968) against increasing
STRUCTURE
OF
THE
PROTEIN
ROY
75
ammonium sulphate. A light amorphous precipitate formed at approximately 1.6 Mammonium sulphate concentration, and some weeks later crystals were seen to have grown. The crystal morphology is a hexagonal rod and X-ray diffraction data show the space group to be P6,22 (or enantiomorph) with a = 81.8 A, c = 79.1 A. (b) Data collection A small crystal 0.6 mm x 0.3 mm x 0.2 mm was used for the measurement of intensity data. These data were measured on an Enraf-Xonius CAD -4/F automatic diffractometer using Ni-filtered CuKa radiation. The crystal-t’o-detector distance was 173 mm. Unit ~11 dimensions were refined using 28 values for automatically centred reflections (0 < IsO). Each reflection was measured using an w-scan in 96 steps. The width of t,htl peak area corresponding to the central 64 steps was (1.1 + 0.14 tan0) degrees. ‘l’h(, scanning time per reflection was 150 s. The backgrounds B, and Bz were the sum of the counts for the 16 steps at the extremes of the scan and the peak P was the sum of the counts for the central 64 steps. The intensity (I) is given by I = V[P - 2(B, + B,)], where 1’ is a factor to account for the different scan speeds. The standard deviation in I is defined by o(l) = [B(P +4(B, + Hz))]*. All, the data were collected from a single crystal and at the conclusion the 3 reference reflections measured every 1000 s of exposure time had decayed to 800,,, of their initial intensity. Lorrntz (L), polarization (p) and decomposition (U) corrections were applied to the intensity data. No absorption corrections were applied. The resulting observed structure and the statist,ical standard deviations by o( F,) = factors are given by F, = [(DI)/(Lp)]” D/(2Lf1jE’j)~(l). The crystals diffracted poorly and only 45(!. of the reflections for 1.5” < H < 15” satisfied the criterion that I > 20(I). (c) Rotation
functions
(Rossmann
& Blow,
1962)
Two models of the REI protein were used, the first being the monomer and the second the dimer. In each case the atomic co-ordinates were placed in a cube 70 A on the edge, and an intensity data set was calculated with an overall temperature factor of 20 A”. At all side-chains where ROY and REI differ, side-chain and main-chain atoms were omitted from the calculatjion. The excision of main-chain atoms was excessively cautious, Prior to this calculation the REI protein was pre-oriented in the box in such a way that its dimer axis of symmetry, a local dyad axis, was parallel to the 02 axis of the box. This pre-orientation of the model (achieved by an Eulerian angle rotation of (12, - 90, 0)” about OZ, OS’ and 02”) facilitated interpretation of the rotation function results since the Eulerian angle system employed there involves rot,ation about’ 02, 01” and 02”. Both of the model intensity data sets were rotated against the ROY crystal data using the method and programs of Crowther (1972). All data between 10 and 4.5 A were used and the search involved all Pat,terson vectors within a 26 A radius of the origin. The Patterson function origins were not removed. (d ) Trandution
functions
The model (dimer or monomer) was oriented in the ROY cell according to the resulbs of the rotation functions and it was initially established in the cell as close as possible to the position determined for the model REI protein in the AU crystals (Fehlhammer et al., 1975). Translation functions (Tollin, 1966; Crowther & Blow, 1967) were calculated using t,hat shell of data between 15 and 5 d. Searches using the monomeric model of REI must- bo done in the full space group of ROY, viz. P6,2 2 in order to define the z co-ordinate of the model. The translation functions with the dimeric model, however, presuppose alignm(‘nf of the tlimer dyad with the ROY crystal dyad and were calculated in PG1. (e) Rejinement For the purpose of refinement the monomer was treated the appropriately oriented and translated REI monomer, CD positions at residues where REI and ROY differ, were
as a rigid body. All atoms of excepting those beyond the used. Initially a special rigid
70
P.
M.
COLMAN,
H.
J.
SCHRAMM
AND
J.
M.
GUSS
body refinement program (Scheringer, 1963) was written and used. In the flnal stages of refinement we used a general refinement program written by A. D. Rae in which the rigid body constraint is treated somewhat differently (Rae, 1976). Here, only data between 6.5 A and 3 .k resolution were used, and, in particular, structure amplitudes which were less than 3.5 times the standard deviation of the measurement were omitted. This left 780 reflections which in the refinement were weighted according to 1/(2(F) + 0.0009 P).
3. Results (a) Patterson function solution Using Crowther’s (1972) convention of Eulerian angles, the REI monomer showed maximum overlap in the ROY Patterson function at (dc,/3, y) = (4, 90, 85)“. The peak height was 1.25 times the highest noise peak. The dimer search was more convincing with a peak at (0, 90, 80)” of 1.40 times the highest noise peak. This result is, in terms of signal-to-noise ratio, qualitatively very similar to that found in the AU structure determination (Fehlhammer et aE., 1975). In keeping with the AU structure, we find here that the molecular dyad axis parallels the a crystallographic axis and not the dyad axis which intersects the 3, crystallographic symmetry element (Fehlhammer et al., 1975). Note that the monomer search does not show the exact preference for aligning the molecular and crystal dyad which one expects, and finds, in the dimer search. The translation functions were expected to yield a solution near (z y z) = (0 0 0) (see Materials and Methods). The monomer searches were very unconvincing. The translation functions for the b*-dyad and the a-dyad showed peaks consistent with expectation but with signal-to-noise ratios of 0.76 and 0.96, respectively. A sum function (Colman et al., 19766) over all the symmetry elements may have better resolved this solution although in this case an unambiguous result was obtained with the translation function of the dimer model. The results for the 6,, 31 and 2, axes had signal-to-noise ratios of 1.50, 0.69 and 1.79, respectively, and not only indicated x = 0, y = 0 for the location of the model origin but also confirmed the 6-fold symmetry element as 6,. (b) Rejinement and difference synthesis In the initial stage of the refinement (done according to Scheringer, 1963), five cycles led to an R-factor (Zl\ #‘,,,I - (B’cslc~\/z(~P,,s~)for the strongest 1000 data between 6.5 A and 3.0 ,& resolution of 0.44. We then proceeded with six cycles of Rae’s program, refining the overall temperature factor and scale factor on alternate cycles and the six positional parameters of the rigid group in every cycle. This refinement terminated with R = 0.336 for the 780 strongest data in the resolution range 6.5 to TABLE
Sin 0/h dependence Resolution range (A) R Number of reflections
2 of R-factor
6.5-50
50-4.0
4.0-3.0
0.355
0.334
0.334
32
210
471
STRUCTURE
OF
THE
PROTEIN
ROY
77
TABLE 3 List of some contacts across the intramolecular and intermolecular dyad axes in ROY and REI C’ontactt Gln38 Tyr36 Tyr49
NOEl-Gln238 OEE-Gln289 CG-Leu294
12 O-409 N 12 0-Ser409 10 O-412 N Atom
not.ation
as in Epp
OG
NOE2 NOE2 CD1
ROY (.A)
REIf (4
3.2 3.x 4.8
3.1, 2.9 3.2, 3.8 3.9, 3.8
2.2
24, 2.9 3.5, 36 2.8, 2.9
2.X 24
et al. (1976).
t Residues on the 2nd monomer in the dimer have 200 added to their sequence those on the neighbouring dimer have 400 added to their sequence number. $ Tao values here for tho 2 monomers in the asymmet,ric unit,.
number
whilst
3.0 if. The sin 0/h dependence of R at the end of refinement is shown in Table 2. The largest shifts in the positional parameters over these final six cycles were approximately 1” in orientation and 0.8 A in translation. The final overall temperature factor is 15.6 A”. The estimated standard deviations in the angular and translational parameters of the rigid group are 0.12” and O-03 A. A number of contact bond-lengths across both the intramolecular dyad and the intermolecular dyad were calculated. These are listed in Table 3 and compared to the
FIG. 1. Difference electron density showing: (a) shift of Tyr49 by a combined rotation of approx. 45” around C-C!4 and approx. 70” around P-P. The C” atom is directly above Co, the atom at the far left; and (b) Asp92. Negative contours are shown by broken lines. The first contour is at & 0.07 e/A” and the contour interval is 0.07 e/A3. Bonds in the section of the map shown here are solid. Other bonds are shown as dotted lines.
78
I’.
M.
COLMAN,
H.
J.
SCHRAMM
AND
J.
M.
GUSS
values obtained from the REI structure. The intramolecular dyad appears to bc unalt,ered. There may be some slight changes across the dyad relating two dimers. The figures in Table 3 indicate a slight crowding around this dyad, a crowding which, if real, could well be accommodated by relaxing the rigid body const’raint of tjhc refinement. A conventional crystallographic difference Fourier map with 756 coefficients Fca,J exp (&& was then calculated using the data between 6.5 and 3 A. (Fobs Peak heights in this map ranged between f 0.35 e/A”. The two largest positive features corresponded to the Leu96 side-chain, excluded from Fcalc, and Tyr49. The change relative latter residue was included in Fcalc but it undergoes a conformational to REI (see Fig. 1). The third largest positive feature lies between SerlO 0” and Ser12 Oy and corresponds to the water molecule designated 431 by Epp et al. (1975) in REI. Other interpretable features include density for the omitted residues Ser30, Ile31, Asp50, Asp92, Va1104, and AsplOB. The density and model fit at Tyr49 and Asp92 are shown in Figure 1.
4. Discussion Given the sequence data it is not surprising to find ROY more nearly isomorphous with AU rather than REI. REI differs from ROY and AU at 18 and 16 positions, respectively, but ROY and AU differ at only 13 positions. With respect to the cavity surrounded by the hypervariable segment, the most interesting changes occur at residue 96 where Trp in AU makes the cavity smaller than REI whilst Leu in ROY makes it larger. A change in conformation of Tyr49 in AU relative to REI has also been reported (Fehlhammer et al., 1975), although the direction of this change is not given. In so far as this change in conformation was attributed in AU to replacement of Tyr96 for Trp96 it seems likely on steric grounds that the movement of Tyr49 is away from Trp296 (i.e. Trp96 on the other chain of the dimer). In REI the phenol groups of Tyr49 and Tyr296 interact very weakly. They are separated by 4.1 A. The movement of Tyr49 in ROY is back towards residue 296, i.e. back into the cavity between the hypervariable loops. Whatever the direct cause of this movement, it is clear that amino acid substitutions in the hypervariable regions may have consequences not only for a change in the character of that particular part of t’he antigen-combining site but also for neighbouring positions in the site. The factor which governs the crystallization of ROY and AU in P6, 2 2 and REI a very subtle one as it does not yield to the crude refinement in P6, is apparently reported here. Whether or not LyslO7 in ROY and AU might alter the interdimer contact slightly must await a definition of its conformation. It is remarkable that this analysis performed on a single poorly diffracting specimen has yielded so much detail in the difference Fourier synthesis. It emphasizes again the power of the Patterson search method when very accurate atomic co-ordinates for the model structure are available. A rigid body refinement of the model so reduces the number of parameters that even with a marginal quality, medium resolution data set, such as was used here, differences between the model and unknown molecules are easily detectable. We thank Professor N. Hilschmann for the gift of the Bence-Jones protein ROY, and Dr A. D. Rae for help in using his least-squares refinement program. One of us (P.M.C.) is a Queen Elizabeth II Fellow.
STRUCTURE
OF
THE
PROTEIN
ROY
79
REFERENCES Colman, P. M., Deisenhofer, J. D., Huber, R. & Palm, W. (1976a). J. Mol. Biol. 100, 2.57-282. Colman, P. M., Fehlhammer, H. & Bartols, K. (1976b). Crystallographic Computing Techniques (Ahmed, F. R., ed.), pp. 248-258, Munksgaard, Copenhagen. Crowther, R. A. (1972). In The MoZecuZur Replacement Method (Rossmann, M. G., ed.), pp. 173-178, Gordon and Breach, New York. Crowther, R. A. & Blow, D. M. (1967). Acta Crystullogr. 23, 544-548. Deisenhofer, J., Colman, P. M., Huber, R., Haupt, H. & Schwick, G. (1976a). Hoppe8eyzer’s
2.
1’hysiok
Chem.
357,
435-445.
Deismhofer, J., Colman, P. M., Epp, 0. & Huber, R. (1976b). Hoppe-Seyler’e 2. Physiol. C/tern. 357, 1421-1434. Epp. O., Colman, P. M., Fehlhammer, H., Bode, W., Schiffer, M., Huber, R. 85 Palm, W. (1974). Eur. J. Biochem. 45, 513-524. Epp, O., Lattman, E. E., Schiffer, M., Hubrr, R. & Palm, W. (1975). Biochemistry, 14, 4943-4952. Fchlhammcr, H.. Schiffer, M., Epp, O., Colman, P. M., Lattman, E. E., Schwager, I’., St:xigemann, W. & Schramm, H. J. (1975). Biophys. Struct. Mechanism, 1, 139-146. Hilschmann, N. (1967). Hoppe-Seyler’s 2. Physiok Chem. 348, 1077-1080. Hllbrlr, R., Deisenhofer, J., Colman, P. M., Matsushima, M. & Palm, W. (1976). Nature (I,ondon), 264, 415-420. R. & Saul, F. (1973). Poljak, R., Amzcl, L. M., Avey, H. P., Chen, B. I,., Phizackerley, T’ror. -Vat. Acad. Sci., U.S.A. 70, 3305-3310. Rae, A. D. (1976). A& Crystallogr. sect. A, 32, 895.-897. Rossmann. M. G. & Blox~, D. M. (1962). Actu Crystullogr. 15, 24-31. &gal, I). M., Padlan, E. A., Cohen, G. H., Rudikoff, S., Potter, M. & Davies, D. R. (1974). Proc. Nat. Acad. &v’., 1J.S.A. 71, 4298-4302. Schpringer, C. A. (1963). A& Cryslallogr. 16, 546-550. Schiffcr, M., Girling, R. L., El,v, K. R. & Edmundson, A. B. (1973). Biochemistry, 12, 4620~4631. Schmmm, H. J. (1971). Hoppe-Seyler’s 2. Physiol. Chem. 352, 1134-1138. Solomon, A. & McLaughlin, C. L. (1969). J. BioZ. Cl/em. 244, 3393-3404. Tollin, P. (1966). Acta Crystallogr. 21, 613-614. 126, Zcppcx+ucr, M., Ekland, H. & Zrppezauer, E. S. (19fi8). Arch. Biochem. Biophya. 564-573.
Biol.
(1977) 116, 73-79
Crystal and Molecular Structure of the Dimer of Variable Domains of the Bence-Jones Protein ROY P. M. COLMAN*, H. J. SCHRAM~C~~ AND J. M. Gussa %‘choo1 of Chemistry,
bMax Planck
Institut
of Sydney, @ydney 2006, Australia, and Biochemie, SO33 Martinsried hei Miinchen West Germany
University
jiir
(Received 19 April
1977)
The crystal and molecular structure of the dimcr of variable domains of the Bence-Jones protein ROY has been determined by Patterson function search procedures, using the known structure of the protein REI. The structure has been partially refined at 3.0 ii resolution to a crystallographic R-factor value of 0.33. One of the 18 residues differentiating ROY from REI is the substitution of Tyr96 for Leu96, a substitution which makes the combining site of the ROY dimer larger, Substantial movement of Tyr49 suggests t#hat point substitutions in the hypervariable segments may affect the conformation of neighbouring residues in the antigen-combining site, possibly producing differences in specificity larger than might otherwise be expected.
1. Introduction Crystal structure studies of immunoglobulin fragments have provided basic information on the three-dimensional conformation of immunoglobulins. In particular, a Bence-Jones protein (Schiffer et al., 1973), two Fab fragments (Poljak et al., 1973; Segal et al., 1974), two light-chain variable domain dimers (Epp et al., 1974; Fehlhammer et al., 1975) and more recently an Fc fragment (Deisenhofer et al., 1976a,b) have been analysed to resolutions of 3.5 A or higher. Difficultyin crystallizing whole immunoglobulins, probably due to their inherent flexibility, has required that functional inferences be drawn from work on the structure of fragments. In most cases these inferences are likely to be sound, although the analysis of a whole IgGl (Colman et al., 1976a) has led to the suggestion that the free Fab fragment may adopt a conformation similar to that found in intact molecules only after t)hey are complexed w&h antigen (Colman et al., 1976a; Huber et al., 1976). Although the notion of sequence changes in the hypervariable segments of the variable domains being directly related to a specific antigen-binding capacity is now well understood, there are few hard data on stereochemical aspects of such sequence of the changes. A convenient system for studyin, D such effects takes advantage availability of crystals of a number of light-chain variable domains which differ f ‘orn each other at a limited number of positions only. These proteins exist in solution as dimers (Solomon & McLaughlin, 1969; Schramm, 1971) and X-ray studies on two of them, REI (Epp et al., 1974) and AU (Fehlhammer et al., 1975) have shown that t,he mode of aggregation of the domains into the dimer is very similar to that found between variable domains of heavy and light chains in Fab fragments. This mode
74
P.
M.
COLMAN,
H.
J.
SCHRAMM TABLE
AND
J.
M.
CUSS
1
Amino acid difference.s in the cariable domains of the proteins ROY, REI and AU ROY
REI
AU
&3XIle Phe LYS ASP LYS Glu Ala Thr Thr ASP Phe Phe ASP Asu Leu
Ile LYS TY~ Thr Glu Am Glu Ala Ser Thr ASP TY~ Tyr Gln Ser Tyr Gln Leu Gln I10 Thr
Ser ASP Tyi LVR Asp Am Glu SW G’Y Ala His Phe Tyr ASP Tyr Trp Gln Val Glu Ile LYS
-____ 30 31 32 39 50 53 55 56 65 69 70 71 91 92 93 96 100 104 106 106 107
Gly Val ASP Phe LYS
of aggregation is functionally important since the antigen-combining site comprises portions of both domains. The REI protein has now been refined to a model accurate to approximately 0.2 A (Epp et al., 1975) and we have used both the monomer and dimer of this refined model to determine the structure of a third variable domain dimer ROY. The use of refined model co-ordinates in Patterson function analysis of related proteins has proven ultimately capable of describing the differences between the model protein and the unknown protein (Fehlhammer et al., 1975; Colman et d., 19766). Since accurate co-ordinates are not yet available for the AU protein, we preferred to start with the REI model. In Table 1, the sequence differences between REI, AU and ROY are listed. REI crystallizes in apace group P6, with a = 75.8 A, c = 98.2 A and there are two domains (M, 11,000) in the asymmetric unit, AU crystallizes with one domain per asymmetric unit in space group P6,22 with a = 82.3 A, c = 77.0 A.
2. Materials and Methods (a) Preparation
and crystallization
of protein
Bence-Jones protein ROY was supplied by Professor N. Hilschmann (Giittingen). The sequence is known (Hilschmann, 1967). 90 mg of the protein was digested with 0.8 mg pepsin (Sigma) in 18 ml of 0.1 M-ammonium acetate at pH 3.5. The digestion was carried out at 37°C for 40 min. The pH was adjusted to 6.0 and the light precipitate so formed was spun off. The supernatant was concentrated by pressure dialysis and passed over a Sephadex (Pharmacia) G50 column (1.5 cm x 100 cm). The column had been equilibrated with 0.5 M-acetic acid and the flow-rate was 19 ml/h. The main elution band appeared after about 5 h and upon lyophilization yielded 7.8 mg protein. This protein was then dissolved in 0.4 ml of 0.1 &I-Tris buffer at pH 8.0 and set concentrations of up in tubes for dialysis (Zeppezauer et al., 1968) against increasing
STRUCTURE
OF
THE
PROTEIN
ROY
75
ammonium sulphate. A light amorphous precipitate formed at approximately 1.6 Mammonium sulphate concentration, and some weeks later crystals were seen to have grown. The crystal morphology is a hexagonal rod and X-ray diffraction data show the space group to be P6,22 (or enantiomorph) with a = 81.8 A, c = 79.1 A. (b) Data collection A small crystal 0.6 mm x 0.3 mm x 0.2 mm was used for the measurement of intensity data. These data were measured on an Enraf-Xonius CAD -4/F automatic diffractometer using Ni-filtered CuKa radiation. The crystal-t’o-detector distance was 173 mm. Unit ~11 dimensions were refined using 28 values for automatically centred reflections (0 < IsO). Each reflection was measured using an w-scan in 96 steps. The width of t,htl peak area corresponding to the central 64 steps was (1.1 + 0.14 tan0) degrees. ‘l’h(, scanning time per reflection was 150 s. The backgrounds B, and Bz were the sum of the counts for the 16 steps at the extremes of the scan and the peak P was the sum of the counts for the central 64 steps. The intensity (I) is given by I = V[P - 2(B, + B,)], where 1’ is a factor to account for the different scan speeds. The standard deviation in I is defined by o(l) = [B(P +4(B, + Hz))]*. All, the data were collected from a single crystal and at the conclusion the 3 reference reflections measured every 1000 s of exposure time had decayed to 800,,, of their initial intensity. Lorrntz (L), polarization (p) and decomposition (U) corrections were applied to the intensity data. No absorption corrections were applied. The resulting observed structure and the statist,ical standard deviations by o( F,) = factors are given by F, = [(DI)/(Lp)]” D/(2Lf1jE’j)~(l). The crystals diffracted poorly and only 45(!. of the reflections for 1.5” < H < 15” satisfied the criterion that I > 20(I). (c) Rotation
functions
(Rossmann
& Blow,
1962)
Two models of the REI protein were used, the first being the monomer and the second the dimer. In each case the atomic co-ordinates were placed in a cube 70 A on the edge, and an intensity data set was calculated with an overall temperature factor of 20 A”. At all side-chains where ROY and REI differ, side-chain and main-chain atoms were omitted from the calculatjion. The excision of main-chain atoms was excessively cautious, Prior to this calculation the REI protein was pre-oriented in the box in such a way that its dimer axis of symmetry, a local dyad axis, was parallel to the 02 axis of the box. This pre-orientation of the model (achieved by an Eulerian angle rotation of (12, - 90, 0)” about OZ, OS’ and 02”) facilitated interpretation of the rotation function results since the Eulerian angle system employed there involves rot,ation about’ 02, 01” and 02”. Both of the model intensity data sets were rotated against the ROY crystal data using the method and programs of Crowther (1972). All data between 10 and 4.5 A were used and the search involved all Pat,terson vectors within a 26 A radius of the origin. The Patterson function origins were not removed. (d ) Trandution
functions
The model (dimer or monomer) was oriented in the ROY cell according to the resulbs of the rotation functions and it was initially established in the cell as close as possible to the position determined for the model REI protein in the AU crystals (Fehlhammer et al., 1975). Translation functions (Tollin, 1966; Crowther & Blow, 1967) were calculated using t,hat shell of data between 15 and 5 d. Searches using the monomeric model of REI must- bo done in the full space group of ROY, viz. P6,2 2 in order to define the z co-ordinate of the model. The translation functions with the dimeric model, however, presuppose alignm(‘nf of the tlimer dyad with the ROY crystal dyad and were calculated in PG1. (e) Rejinement For the purpose of refinement the monomer was treated the appropriately oriented and translated REI monomer, CD positions at residues where REI and ROY differ, were
as a rigid body. All atoms of excepting those beyond the used. Initially a special rigid
70
P.
M.
COLMAN,
H.
J.
SCHRAMM
AND
J.
M.
GUSS
body refinement program (Scheringer, 1963) was written and used. In the flnal stages of refinement we used a general refinement program written by A. D. Rae in which the rigid body constraint is treated somewhat differently (Rae, 1976). Here, only data between 6.5 A and 3 .k resolution were used, and, in particular, structure amplitudes which were less than 3.5 times the standard deviation of the measurement were omitted. This left 780 reflections which in the refinement were weighted according to 1/(2(F) + 0.0009 P).
3. Results (a) Patterson function solution Using Crowther’s (1972) convention of Eulerian angles, the REI monomer showed maximum overlap in the ROY Patterson function at (dc,/3, y) = (4, 90, 85)“. The peak height was 1.25 times the highest noise peak. The dimer search was more convincing with a peak at (0, 90, 80)” of 1.40 times the highest noise peak. This result is, in terms of signal-to-noise ratio, qualitatively very similar to that found in the AU structure determination (Fehlhammer et aE., 1975). In keeping with the AU structure, we find here that the molecular dyad axis parallels the a crystallographic axis and not the dyad axis which intersects the 3, crystallographic symmetry element (Fehlhammer et al., 1975). Note that the monomer search does not show the exact preference for aligning the molecular and crystal dyad which one expects, and finds, in the dimer search. The translation functions were expected to yield a solution near (z y z) = (0 0 0) (see Materials and Methods). The monomer searches were very unconvincing. The translation functions for the b*-dyad and the a-dyad showed peaks consistent with expectation but with signal-to-noise ratios of 0.76 and 0.96, respectively. A sum function (Colman et al., 19766) over all the symmetry elements may have better resolved this solution although in this case an unambiguous result was obtained with the translation function of the dimer model. The results for the 6,, 31 and 2, axes had signal-to-noise ratios of 1.50, 0.69 and 1.79, respectively, and not only indicated x = 0, y = 0 for the location of the model origin but also confirmed the 6-fold symmetry element as 6,. (b) Rejinement and difference synthesis In the initial stage of the refinement (done according to Scheringer, 1963), five cycles led to an R-factor (Zl\ #‘,,,I - (B’cslc~\/z(~P,,s~)for the strongest 1000 data between 6.5 A and 3.0 ,& resolution of 0.44. We then proceeded with six cycles of Rae’s program, refining the overall temperature factor and scale factor on alternate cycles and the six positional parameters of the rigid group in every cycle. This refinement terminated with R = 0.336 for the 780 strongest data in the resolution range 6.5 to TABLE
Sin 0/h dependence Resolution range (A) R Number of reflections
2 of R-factor
6.5-50
50-4.0
4.0-3.0
0.355
0.334
0.334
32
210
471
STRUCTURE
OF
THE
PROTEIN
ROY
77
TABLE 3 List of some contacts across the intramolecular and intermolecular dyad axes in ROY and REI C’ontactt Gln38 Tyr36 Tyr49
NOEl-Gln238 OEE-Gln289 CG-Leu294
12 O-409 N 12 0-Ser409 10 O-412 N Atom
not.ation
as in Epp
OG
NOE2 NOE2 CD1
ROY (.A)
REIf (4
3.2 3.x 4.8
3.1, 2.9 3.2, 3.8 3.9, 3.8
2.2
24, 2.9 3.5, 36 2.8, 2.9
2.X 24
et al. (1976).
t Residues on the 2nd monomer in the dimer have 200 added to their sequence those on the neighbouring dimer have 400 added to their sequence number. $ Tao values here for tho 2 monomers in the asymmet,ric unit,.
number
whilst
3.0 if. The sin 0/h dependence of R at the end of refinement is shown in Table 2. The largest shifts in the positional parameters over these final six cycles were approximately 1” in orientation and 0.8 A in translation. The final overall temperature factor is 15.6 A”. The estimated standard deviations in the angular and translational parameters of the rigid group are 0.12” and O-03 A. A number of contact bond-lengths across both the intramolecular dyad and the intermolecular dyad were calculated. These are listed in Table 3 and compared to the
FIG. 1. Difference electron density showing: (a) shift of Tyr49 by a combined rotation of approx. 45” around C-C!4 and approx. 70” around P-P. The C” atom is directly above Co, the atom at the far left; and (b) Asp92. Negative contours are shown by broken lines. The first contour is at & 0.07 e/A” and the contour interval is 0.07 e/A3. Bonds in the section of the map shown here are solid. Other bonds are shown as dotted lines.
78
I’.
M.
COLMAN,
H.
J.
SCHRAMM
AND
J.
M.
GUSS
values obtained from the REI structure. The intramolecular dyad appears to bc unalt,ered. There may be some slight changes across the dyad relating two dimers. The figures in Table 3 indicate a slight crowding around this dyad, a crowding which, if real, could well be accommodated by relaxing the rigid body const’raint of tjhc refinement. A conventional crystallographic difference Fourier map with 756 coefficients Fca,J exp (&& was then calculated using the data between 6.5 and 3 A. (Fobs Peak heights in this map ranged between f 0.35 e/A”. The two largest positive features corresponded to the Leu96 side-chain, excluded from Fcalc, and Tyr49. The change relative latter residue was included in Fcalc but it undergoes a conformational to REI (see Fig. 1). The third largest positive feature lies between SerlO 0” and Ser12 Oy and corresponds to the water molecule designated 431 by Epp et al. (1975) in REI. Other interpretable features include density for the omitted residues Ser30, Ile31, Asp50, Asp92, Va1104, and AsplOB. The density and model fit at Tyr49 and Asp92 are shown in Figure 1.
4. Discussion Given the sequence data it is not surprising to find ROY more nearly isomorphous with AU rather than REI. REI differs from ROY and AU at 18 and 16 positions, respectively, but ROY and AU differ at only 13 positions. With respect to the cavity surrounded by the hypervariable segment, the most interesting changes occur at residue 96 where Trp in AU makes the cavity smaller than REI whilst Leu in ROY makes it larger. A change in conformation of Tyr49 in AU relative to REI has also been reported (Fehlhammer et al., 1975), although the direction of this change is not given. In so far as this change in conformation was attributed in AU to replacement of Tyr96 for Trp96 it seems likely on steric grounds that the movement of Tyr49 is away from Trp296 (i.e. Trp96 on the other chain of the dimer). In REI the phenol groups of Tyr49 and Tyr296 interact very weakly. They are separated by 4.1 A. The movement of Tyr49 in ROY is back towards residue 296, i.e. back into the cavity between the hypervariable loops. Whatever the direct cause of this movement, it is clear that amino acid substitutions in the hypervariable regions may have consequences not only for a change in the character of that particular part of t’he antigen-combining site but also for neighbouring positions in the site. The factor which governs the crystallization of ROY and AU in P6, 2 2 and REI a very subtle one as it does not yield to the crude refinement in P6, is apparently reported here. Whether or not LyslO7 in ROY and AU might alter the interdimer contact slightly must await a definition of its conformation. It is remarkable that this analysis performed on a single poorly diffracting specimen has yielded so much detail in the difference Fourier synthesis. It emphasizes again the power of the Patterson search method when very accurate atomic co-ordinates for the model structure are available. A rigid body refinement of the model so reduces the number of parameters that even with a marginal quality, medium resolution data set, such as was used here, differences between the model and unknown molecules are easily detectable. We thank Professor N. Hilschmann for the gift of the Bence-Jones protein ROY, and Dr A. D. Rae for help in using his least-squares refinement program. One of us (P.M.C.) is a Queen Elizabeth II Fellow.
STRUCTURE
OF
THE
PROTEIN
ROY
79
REFERENCES Colman, P. M., Deisenhofer, J. D., Huber, R. & Palm, W. (1976a). J. Mol. Biol. 100, 2.57-282. Colman, P. M., Fehlhammer, H. & Bartols, K. (1976b). Crystallographic Computing Techniques (Ahmed, F. R., ed.), pp. 248-258, Munksgaard, Copenhagen. Crowther, R. A. (1972). In The MoZecuZur Replacement Method (Rossmann, M. G., ed.), pp. 173-178, Gordon and Breach, New York. Crowther, R. A. & Blow, D. M. (1967). Acta Crystullogr. 23, 544-548. Deisenhofer, J., Colman, P. M., Huber, R., Haupt, H. & Schwick, G. (1976a). Hoppe8eyzer’s
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Deismhofer, J., Colman, P. M., Epp, 0. & Huber, R. (1976b). Hoppe-Seyler’e 2. Physiol. C/tern. 357, 1421-1434. Epp. O., Colman, P. M., Fehlhammer, H., Bode, W., Schiffer, M., Huber, R. 85 Palm, W. (1974). Eur. J. Biochem. 45, 513-524. Epp, O., Lattman, E. E., Schiffer, M., Hubrr, R. & Palm, W. (1975). Biochemistry, 14, 4943-4952. Fchlhammcr, H.. Schiffer, M., Epp, O., Colman, P. M., Lattman, E. E., Schwager, I’., St:xigemann, W. & Schramm, H. J. (1975). Biophys. Struct. Mechanism, 1, 139-146. Hilschmann, N. (1967). Hoppe-Seyler’s 2. Physiok Chem. 348, 1077-1080. Hllbrlr, R., Deisenhofer, J., Colman, P. M., Matsushima, M. & Palm, W. (1976). Nature (I,ondon), 264, 415-420. R. & Saul, F. (1973). Poljak, R., Amzcl, L. M., Avey, H. P., Chen, B. I,., Phizackerley, T’ror. -Vat. Acad. Sci., U.S.A. 70, 3305-3310. Rae, A. D. (1976). A& Crystallogr. sect. A, 32, 895.-897. Rossmann. M. G. & Blox~, D. M. (1962). Actu Crystullogr. 15, 24-31. &gal, I). M., Padlan, E. A., Cohen, G. H., Rudikoff, S., Potter, M. & Davies, D. R. (1974). Proc. Nat. Acad. &v’., 1J.S.A. 71, 4298-4302. Schpringer, C. A. (1963). A& Cryslallogr. 16, 546-550. Schiffcr, M., Girling, R. L., El,v, K. R. & Edmundson, A. B. (1973). Biochemistry, 12, 4620~4631. Schmmm, H. J. (1971). Hoppe-Seyler’s 2. Physiol. Chem. 352, 1134-1138. Solomon, A. & McLaughlin, C. L. (1969). J. BioZ. Cl/em. 244, 3393-3404. Tollin, P. (1966). Acta Crystallogr. 21, 613-614. 126, Zcppcx+ucr, M., Ekland, H. & Zrppezauer, E. S. (19fi8). Arch. Biochem. Biophya. 564-573.