Structure of Bence—Jones protein, Pav: an initial report

J. Mol. Biol. (1977) 116, 619-625

Structure of Bence-Jones Protein, Pav: an Initial Report A BenceJones protein, Pav, was isolated from the urine of a myeloma patient. Crystals were grown by five different methods yielding different morphologies with slightly changed cell parameters, but the space group was the same (P2,2,2,) in every case and X-ray patterns appeared to be identical. The cell parameters are: a = 93.6(4) to 95*1(4) A, b = 92.7(3) A and c = 72*8(2) A. The crystal density and solvent content are approximately 1.128 g/cm3 and 0.64, respectively. Chemical evidence suggest that the two subunits of Pav are identical in chemical sequence. The crystal structure may prove useful in defining allosteric effects among antibody domains.

Bence-Jones proteins are immunoglobulin light chains found in the urine of some multiple myeloma patients. The three-dimensional structure of a dimeric light chain Meg, has been determined by Edmundson and his colleagues (Schiffer et al., 1973; Edmundson et al., 1974u,b). They found that the two identical chains have different three-dimensional structures. The dimer looks like an Fab fragment (Poljak et al., 1973,1974; Amzel et al., 1974; Segal et al., 1974; Davies et al., 1975a,b) since one of its light chains assumes the conformation of a heavy chain. While the overall differences in the chain conformations are intriguing, those between the variable regions are especially so because in Bence-Jones dimers lacking constant regions, such as REI (Epp et al., 1974,1975) and Rhe (Wang et al., 1975) the respective variable regions are either not significantly different in conformation or are related by a crystallographic S-fold axis of symmetry. Why then does the mere presence of the constant region engender such a difference in the conformation of one of the light chains! This question has not been previously raised nor has the question of whether this is the result of allosteric effects among antibody domains. Before defininmg and answering these and other related questions?, it is necessary to see how widely the structural feature in Meg occurs in other light-chain dimers. As part of our project to determine the crystal structures of M-components and of immunoglobulin fragments we crystallized a Bence--Jones protein, Pav, whose structure should provide greater insight into these questions. Pav and Meg are dimers of h-type light chains, but their crystals differ in space group, cell volume and solvent content. We report here the procedures of crystallization, the results of chemical characterization and the preliminary crystal data of Pav. (a) Isolation and crystallization

of Pav

Crystals of Pav can be grown under a variety of conditions. The procedures growing each kind of crystal shown in Figure 1 are as follows. (a) The urine first exhaustively dialyzed against distilled water. Then the distilled water replaced by a polyethylene glycol (PEG-4000) solution (5%) containing 0.1 M-NaCl

for was was and

t Such &s the possibility that light chains and probably also heavy chains can be present in at least 2 different conformations (Schlessinger et. al., 1976) and that the change of conformation is also responsible for the transmission of antigen-binding signal to the F, portion. 619

620

B.

Fra. 1. Crystals

C. WANG

ET AL.

of Pav grown under different

conditions

(see text).

O-02% NaN, at 6°C. Crystals grew to a maximum of 3 mm within three days inside the dialysis bag. (b) The protein was first precipitated from a urine specimen by adding ammonium sulfate. The precipitate was then dissolved, and repreoipitated with ammonium sulfate. The material was then dialyzed against distilled water at 6C. After one week crystals of the size shown were found in the dialysis bag. (c) A procedure similar to that for (b) was used except that in the last step 0.1% NH,HCO, at pH 7.9 was substituted for the distilled water. (d) When the procedure described for (b) failed to give crystals the protein solution was allowed to evaporate on a microculture slide at room temperature inside a plastic chamber of dimensions 3 cm x 12 cm x 12 cm. Crystals formed in three days. A procedure similar to that in (d) was followed except that the protein solution was first dialyzed in a 0.05 M-glycine buffer (pH 9) for two days. However, when the glycine buffer was replaced by phosphate/citrate or phosphate buffer (pH 5 to 8) crystals did not form using this method. Despite the variety of conditions under which Pav formed crystals, it did not do so in solutions of high salt concentration. (b) Characterization of Pav Immunological tests according to the methods of Grabar & Williams (1953) and Scheidegger (1955) indicate that Pav is a lambda-type Benoe-Jones protein. Molecular

LETTERS

TO

THE

TABLE

EDITOR

621

1

Amino acid analysis of Pav crystals Amino

acid

Cysteic acid? Aspartio acid ThreonineS Serinet Glutamic acid Proline Glycine Alanine Valine Methionine Isoluecine Leucine Tyrosine Phenylalanine Tryptophan § Lysine Histidine Arginine

nmol

Residues per 46,600T

31.6 94.6 127.4 198.8 130.2 97.8 102.4 127.0 99.6 0 29.7 96.4 55.6 26.4 24.8 66.8 12.0 30.7

10.3 30.7 41.4 64.6 42.3 31.8 33.2 41.2 32.3 0 9.6 31.0 18.1 8.2 8.0 21.7 3.9 10.0

All analyses were corrected for physical loss by normalization to glutamic acid. The average nanomoles are given to an accuracy of &3% based on the average of 6 samples for all amino acids except threonine, serine and tryptophan which were measured in duplicate. t Determined by oxidation with performic acid according to the procedure of Moore (1963). $ Values determined by extrapolation of the results of 24 and 48 h HCI hydrolyses to zero time. $jDetermined by hydrolysis in p-toluenesulfonic acid according to the procedure of Liu & Chang (1971). 7 Determined by a least-squares method according to Katz (1968).

TABLE 2

Cell parameters of Pav crystals (d) Crystal Native

a

b

0

9&O(4) 93.8(2) 93.6(4) 9&l(4) 93.9(3) 946( 1)

92.6(3) 92.6(2) 926(2) 92.9(3) 92.7(3) 93.1(2)

72.7(2) 72*9(2) 72.7(2) 72.7(2) 72.8(2) 72.8(2)

Pav

(a)t (b)t (c)t (4t Wt Pa+ + PtCI, t Sections of Fig. 1. $ The native crystal was grown by method

(a).

622

FIG. 2. Peptide map of EXtryptic map.

B. C. WANG

digest of oxidized

ET

AL.

Pav. The inset is a photograph

weight determination by gel filtration (Andrews, sulfate/polyacrylamide gel electrophoresis (Weber et of two polypeptide chains each of molecular weight peptide chains were further shown to be identical examination of a tryptic peptide map of Pav (Fig.

of the actual

1965) and by sodium dodecyl al., 1972) indicate that it consists 25,009f2500. These two polyin chemical sequence, from an 2) combined with its amino acid

LETTERS

FIQ. 3. Precession diffraction patterns

TO

THE

photographs (p = 9”, crystal-to-film of hk0 reflections.

EDITOR

distance

623

= 7.5 cm) showing

X-ray

composition (Table 1) according to the logic used by Gainey et al. (1972) for bovine liver UDPG-dehydrogenase, by Lowe & Reithel (1975) for phosphoglucose isomerase from bakers’ yeast, and by Hogue-Angelleti et al. (1976) for nerve growth factor from cobra venom. The amino acid analysis was performed according to Moore (1963) and Liu & Chang (1971). The tryptic digestion of Pav was carried out according to the procedures of Hirs (1967) and Brown & Wold (1973). The peptide map illustrated in Figure 2 was produced by electrophoresis of the tryptic digest on Whatman 3 MM paper at 25 V/cm for three hours in pyridinelacetic acid/water (1 :I0 :444), pH 35, followed by descending chromatography in butanol/acetic acid/water (3:l :I) for 18 hours. (c) Preliminary

crystal data

X-ray precession photographs which were taken at room temperature with nickelfiltered CuKa radiation indicate that all five kinds of crystals shown in Figure 1 have the same space group symmetry, P2,2,21, and all give seemingly identical intensity distributions. A precession photograph of the hk0 zone is shown in Figure 3. A search for isomorphous heavy-atom derivatives has resulted in our obtaining reproducible changes in intensity upon soaking in a solution of K,PtCl, (1 mM). Cell parameters of crystals of native Pav and of the derivative were obtained from film data and are listed in Table 2. The density of Pav crystals is 1.1275 g/cm 3. This value is obtained by extrapolating the measured densities, determined by a flotation method, to zero time since they increase slowly while the crystals are suspended in the water-saturated carbon tetrachloridelxylene mixture. We also found that the rates of density changes differ from crystal to crystal, but these differences are related to the crystal sizes rather than to the methods by which the crystals were grown (Fig. 4). Based on the assumption of a partial specific volume of O-738 ml/g (Noelkin et al., 1965) the estimated solvent content of Pav is 64% (v/v). The molecular weight based on this finding and 40

624

B.-C.

I

I.121 0

E!Z’ AL.

WANG

Crystal from

I IO

I 20

I 30

I 40

I 50

I 60

I 70

1

Time (min)

FIG 4. Crystal densities of Pav wer8u8 the time of suspension in the water-saturated mixture of carbon tetrschloride and nt-xylems. The alphabetical symbols under “crystal from” refer to the methods used for growing the crystals ((a) refers to text concerning Fig. l(a), etc.). The numbers in parentheses indicate the crystal sizes.

on the assumption that there are four molecules in a unit cell is 47000&2350 (estimated 5% error). In photographic “stills” the X-ray diffraction patterns of all Pav crystals extend beyond 2.6 A. Reflections with this resolution have also been observed with a diffractometer. The determination of the three-dimensional structure and of the chemical sequence of Pav are now under way in our laboratories. We thank Dr David Schreiner for his helpful discussions and comments. This work was supported by the Medical Research Service of the Veterans Administration, a grant from the National Institutes of Health (AM-CA18827) and a fellowship from the Winter Foundation to one of us (W. E. B.). Biocrystallography Laboratory Box 12065, VA Hospital Pittsburgh, Pa 15240, U.S.A. Department of Crystallography University of Pittsburgh Pittsburgh, Pa 15260, U.S.A.

B. C. WANG

c. s. Yoo R. Y. HW~LN M. SAX

Department of Biological Sciences Carnegie-Mellon University Pittsburgh, Pa 15213, U.S.A.

W. E. BROWN

Allegheny Pittsburgh,

M. MICHAELS

Received

General Hospital Pa 15212, U.S.A. 21 September

1976, and in revised

form

17 May

1977.

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EDITOR

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