Sulfhydryl groups and disulfide bridges in subunit c of Panulirus interruptus hemocyanin

Sulfhydryl groups and disulfide bridges in subunit c of Panulirus interruptus hemocyanin

126 Biochimica et Biophysica Acta, 998 (1989) 126-130 Elsevier BBAPRO 33452 Sulfhydryl groups and disulfide bridges in subunit c of Panulirus inter...

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126

Biochimica et Biophysica Acta, 998 (1989) 126-130 Elsevier

BBAPRO 33452

Sulfhydryl groups and disulfide bridges in subunit c of Panulirus interruptus hemocyanin Ben N e u t e b o o m , Peter A. Jekel, R o b e r t M . W . H o f s t r a a n d J a a p J. B e i n t e m a Biochemisch Laboratoriung Rijksuniversiteit Groningen, Groningen (The Netherlands)

(Received 24 March 1989)

Key words: Hemocyanin; Disulfide bridge; Cooperativity; (P. interruptus)

Subunit c of Panulirus interruptus bemocyanin contains, after reduction, three cysteine residues. Determination of suifhydryls in the native subunit revealed one free SH group. The cysteine-containing peptides were obtained from a pepsin digest. A disulfide bridge was established between Cys3 and CysssT, while Cys~ bears a free sulfhydryl group. These results are in agreement with expectations based on the three-dimensional structure of subunit a. The position of the disulfide bridge in subunit c is unique among arthropedan hemoeyanins, and is possibly responsible for the absence of eooperativity in subunit c homo-hexamers.

Introduction Hemocyanins are copper-containing oxygen carriers occurring freely dissolved in the hemolymph of many molluscs and arthropods. Arthropod hemocyanins consist of hexamers or multihexamers with subunits of about 75 kDa, each of which contains one binuclear copper site [1]. In the spiny lobster, Panulirus interruptus, hemocyanin molecules are present as hexamers. Four different subunits have been distinguished in this species, called a, b, b' and c. Subunit c is the minor component, and accounts for about 16% of the hemocyanin. The amino acid sequences of subunits a and b have been elucidated completely [2,3]. The three-dimensional structure of P. interruptus hemocyanin has been determined by X-ray diffraction at a resolution of 0.32 nm, with crystals consisting of a mixture of a and b in roughly equal amounts [4]. From previous studies it was clear that subunit c is very different from the a and b subunits, both in structure [5,6] and in functional properties, the most striking difference being the absence of cooperativity in homohexamers of subunit c [7,8]. In two hemocyanin molecules, the disulfide bridges and free sulfhydryls have been determined. In P. interruptus hemocyanin subunit a, the disulfide bridges were deduced from the X-ray structure [4]. Subunit a con-

Correspondence: J.J. Beintema, Biochemisch Laboratorium, Rijksuniversiteit Gronin~en, Nijenborgh 16, 9747 AG Groningen, The Netherlands.

tains three disulfide bridges and no free sulfhydryls. In subunit d of Eurypelma californicum hemocyanin, the presence of two disulfide bridges and three free sulfhydryls has been proven chemically [9]. 97% of the amino acid sequence of P. interruptus subunit c has been determined so far [10]. Sequence difference with subunits a and b is 41~. Three cysteine residues have been established after reduction and carboxymethylation, of which only one is in the same position as the cysteine residue in subunit a. Amino acid analysis of the undetermined stretch showed no carboxymethylcysteine. There are no cysteines in positions corresponding with those in Eurypelma subunit d. Since no X-ray structure of subunit c has been determined, an attempt was made to position disulfide bridges a n d / o r sulfhydryls chemically. Materials and Methods Hemolymph of P. interruptus was obtained from Pacific Bio-Marine Laboratories (Venice CA, U.S.A.). Hemocyanin subunit c was isolated as previously described [11]. The number of free sulfhydryl groups was determined both by labelling with 5,5'-dithiobis(2-nitrobenzoic) acid (DTNB; Sigma, St. Louis, MO, U.S.A.) as described by Butterworth et al. [12], in the absence of denaturing agents, and by carboxymethylation (also in the absence of denaturing agents) with subsequent amino acid analysis on a Kontron Liquimat III analyser. 32 mg (400 nmol) subunit c was cleaved into small fragments by successive dig~3tions and cleavages. First,

0167-4838/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

127 the protein was denatured by diatysis against 8 M urea in 100 mM ammonium acetate buffer (pH 4.0), dialyzed against 2 M urea, dissol,,~ in the same buffer, and digested with 3% (w/w) endoy,roteinase GIu-C (Boehringer-Mannheim, F.R.G.) at 37 ° C for 16 h. Secondly, it was cleaved with CNBr (Pierce, Rockford, IL, U.S.A.) in 70% formic acid at room temperature in the dark for 24 h. Since no noticeable cleavage had occurred, it was finally digested with pepsin (Boehringer-Mannheim, F.R.G.) as described by Ryle [13]. Incubation was continued at room temperature for 2 h. Insoluble particles were removed by centrifugation. Performic acid oxidation of isolated peptides was performed as described by Hirs [14]. The reaction was stopped by lyophilization. Thermolysin (Calbiochem, San Diego, CA, U.S.A.) was used for subdigestions. Peptic peptides were separated on a Sephadex G-50F column (1.0 × 200 cm) in 30% acetic acid. The eluate was collected in 1.6 ml fractions at a flow rate of 6.0 ml/h. The fractions were monitored at 280 nm and pooled into nine pools. Peptides were further purified by reversed-phase high-performance liquid chromatography, successively in the trifluoroacetic acid system and in the ammonium acefate system [2]. Peptides were screened for the presence of cysteine by amino acid analysis. Peptides containing cysteine were sequenced either manually, with the DABITC reagent according to Chang [15], or automatically in a gas-phase sequenator (Applied Biosystems model 470A).

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0.8

0.6

0.4

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Results and Discussion

Reaction with DTNB showed that unreduced subunit c contained 1.4-1.8 thiol groups. With iodoacetic acid, 1.0 thiol group was modified. Since the amino acid sequence determined so far contains after reduction three cystein~s [10], two cysteines must be present as a disulfide bridge and one as a free suffhydryl. Digestion with endoproteinase GIu-C did not result in the desired cleavage into small fragments. After CNBr cleavage, most methionines still were unconverted, as was checked with amino acid analysis, indicating that chain cleavage had hardly occurred. Sufficient cleavage was obtained with pepsin. Separation on Sephadex G-50F in 30% acetic acid, yielded nine pools as indicated in Fig. 1. Amino acid analysis showed that pools 1, 2, 3, 4, 5 and 8 contained half-cystine. Table I shows the cysteine content of each pool. For further purification these po0~s were subjected to reversed-phase HPLC using the tfifluoroacetic acid system. Pool 1 revealed one main peak, the other pools yielded a complex pattern of peaks (Fig. 1). Pools 1, 2 and 3 each had a cysteine-containing peak with identical elution time. The cysteine-containing peaks of pools 4 and 5 also had identical elution times. These latter peaks were further purified by reversed-phase HPLC using the am-

80

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Cys 99

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elution t i m e {mind

cys3_cysSS7

Fig. 1. Isolation of cysteine- and cystine-containing peptides. Initial fractionation of a peptic digest of P. interruptus hemocyanin subunit c was on Sephadex G-50F. The cysteine- (or cystine)-containing pools were further fractionated on reversed-phase HPLC using the trifluoroacetic acid system. Only the fractionafions of pools 1 and 4 are shown. Peptides with disulfide bridge Cys3-Cysss7 were isolated from pools 4 and 5. Pools 1 and 2 contained peptides with Cys99, and small quantities of disulfide bridge Cys99-Cys557, the latter being the result of disulfide exchange.

monium acetate system. Several peptides were isolated, which on sequencing with the DABITC method revealed two sequences, starting with positions Ala I and Arg 54s (Fig. 2). These positions precede the cysteines in positions 3 and 557. The peptides had various C-termini because of incomplete and unspecific cleavages. The amino acid analysis of the major disulfide peptide is shown in Table II). 17~is l~,eptide was oxidized with performic acid. HPLC separ~:tion resulted in two pure, cysteic-acid-contain~g pep6des that were originally connected by disu~ide boud Cys3-Cysss7 (Table II, coltrams b and c).

128 TABLE I Amounts of cysteine detected at three stages of the isolation of cystineand cysteine-containing peptides

Gel filtration

HPLC fractionation

(nmol)

(nmol)

Pool I

70

49

2 3 4 5 6 7

40 60 300 9O 0 0

28 35 215 9O -

8

15

0

0 120

-

9 Pellet

Final isolation (nmol) ] 50Cys 99 6 Cysss7 90Cys

110 Cysss~

-

The cysteine-containing peaks of pools 1 and 2 contained a complex mixture. One peptide was present at a sufficiently high ratio and could be identified by manual sequencing. It started at position 82 (Fig. 2). The amino acid composition of this fraction fits fairly well with the composition of peptide AsnS2-Ala]°9 (Table If). Further purification with HPLC techniques failed. Therefore, peak 1-1 (Fig. 1) was treated with performic acid and digested with thermolysin. Peptides were separated by HPLC and characterized by sequence determination in a gas-phase sequenator. Most peptides were from two parts of the sequence, viz. 81-109 and 534-563. The fragments were N-terminally and Cterminally heterogeneous. All peptides from 81-109 were isolated, most of them in a yield 8-times higher

than those from the sequence 534-563. Since peptide 81/82-109 is the only peptide present in pools I and 2 in appreciable amounts, Cys 99 must have a free sulfhydryl group. It seems strange that a peptide of 28 residues elutes so early from a SepL:dex G-50 gel-filtration column. However, this phenomenon has been noticed before. Eyerle and Schartau mention that a sulfhydryl-containing peptide of 12 residues elutes together with a peptide of 60 residues [9]. Minor quantities of Cys s57 were detected in pool 1. Since a disulfide peptide has been isolated from pools 4 and 5 in which Cys 557 is linked to Cys 3, disulfide exchange must have occurred during digestions. Because disulfide exchange or interchange in acidic solutions is very slow, disulfide bridge Cys3-Cys557, which was present in the digest in a 20-fold excess over disulfide bridge Cys99-CysS57 must be the original disulfide bridge in the native structure (Fig. 2). The three-dimensional structure of the homologous subunit a lends support to the chemically determined disulfide bridge and, the sulfhydryl of subunit c. In subunit a, the residue corresponding with Lys]1 is the first residue that had its coordinates determined. This residue is at 1.2 nm distance of both positions 99 and 557. The maximum distance between Lys" and Cys 3 is 3.0 nm, so both Cys 99 and Cys s57 are within reach of Cys 3. Therefore, two disulfide bridges are allowed, viz. Cys3-Cys99 and Cys3-Cys557. Positions corresponding with residues 99 and 557 are at about 2.5 nm distance from each other. Disulfide finkage between Cys 99 and Cys 557 therefore would demand a radically different structure, which is in conflict with the generally accepted idea that homologous proteins have similar chain folding. • ,

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129 TABLE II

Amino acid composition of cysteine-containing peptides a, intact disulfide peptide with bridge Cys3-CysSST; b and c, sub-fragments of this peptide after performic acid oxidation; d, composition of (impure) HPLC fraction 1-1. The composition of peptide 82-109 derived from its sequence is indicated between brackets, e and f, peptides isolated in high yield from the thermolytic digest of oxidized fraction 1-1. a

Cysteic acid Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteine Valine Methionine Isolencine Leucine Tyrosine Phenylalanine Lysine Histidine Ar~nine Tryptophan Total Position

b

6.7 (7) 0.2 (-) 2.9 (3) 3.9 (4)

c

1.0 (1) 2.4 (2)

1.1 (1) 3.0 (3)

1.6 (2) 1.2 (1)

1.0 (1) 4.0 (4)

1.0 (1) 3.7 (4)

1.9 (2) 7.4 (8) 1.9 (2) 0.8 (1)

0.9 (1)

1.2 (1) 3.8 (4) 1.1 (1)

0.2 (-) 2.0 (2) 0.8 (1)

1.6 (2) 2.1 (2) 1.0 (1)

1.9 (2) 1.0 (1)

38 1-22 + 548-563

d

1.0 (1) 4.3 (5)

22 1-22

3.7 (4) 1.2 (1) 1.2 (1) 3.2 (3) 1.3 (-) 3.0 (-) 2.3 (3) 0.8 (1) 2.5 (2) 1.5 (3) 0.5 (-) 1.9 (2) 1.3 (-) 1.2 (1) 1.8 (1) 1.8 (2) 2.9 (3) n.d. a (1)

0.9 (1) 1.8 (2)

0.9 (1) 0.8 (1) 16 548-563

28 82-109

e

f

1.0 (1) 0.9 (1) 2.9 (3)

1.0 (1) 1.0 (1) 2.1 (2) 1.1 (1)

2.0 (2) 8 82-89

0.9 (1) 1.0(1) 6 90-95

a n.d., not determined.

So far, the disulfide bridges and free sulfhydryls of only one or more hemocyanin chain, the Eurypelma subunit d, have been determined chemically [9]. This chain is reported to have two disulfide bridges and three free sulfhydryls. One disulfide bridge is in the same location as the third disulfide bridge of Panulirus subunit a, which was deduced from the X-ray structure [4]. Since all arthropodan hemocyanins sequenced so far, except P. interruptus subunit c, have cysteine residues in the corresponding positions, this disulfide bridge probably has been highly conserved. It connects two conserved regions which are in contact with the oxygen-binding domain. These regions may be involved in cooperativity [16]. I-fitherto, P. interruptus subunit c is the only arthropodan hemocyanin subunit for which absence of cooperativity has been described [7,8]. This hemocyanin subunit is also the only one with a disulfide bridge that connects the N-terminal part with the C-terminal region of the molecule. Possibly, this disulfide bridge causes a certain rigidity, which interferes with the conformational changes essential for cooperativity in oxygen binding.

Acknowledgements We thank Dr. W.G.J. Hol, Dr. E.F.J. van Bruggen and Dr. R.N. Campagne for their comments, and Dr.

R. Amons and J. de Graaf for automatic sequence analyses. This work was supported ~y the Netherlands Foundation for Chemical Kesearch (S.O.N.) with financial aid from the Netherlands Organization for Scientific Research (N.W.O.).

References 1 Van Holde, K.E. and Miller, K.I. (1982) Q. Rev. Biophys. 15, 1-129.

2 Bak, HJ. and Beintema, J.J. (1987) Eur. J. Biochem. 169, 333-348. 3 Jekel, P.A., Bak, HJ., Soeter, N.M., Vereijken, J.M. and Beintema, JJ. (1988) Eur. J. Biochem. 178, 403-412. 4 Gaykema, W.P.J., Hol, W.G.J, Vereijken, J.M., Soeter, N.M., Bak, HJ. and Beintema, LJ. (1984) Nature (Lond.) 309, 23-29. 5 Folkerts, A. and Van Eerd, J.P. (1981) in Invertebrate oxygenbinding proteins: Structure, Active Site and Function (Lamy, J. and Lamy, J., eds.), pp. 215-225, Marcel Dekker, New York. 6 Bak, H.J., Neuteboom, B., Jekel, P.A., Soeter, N.M., Vereijken, J.M. and Beintema, J.J. (1986) FEBS Lett. 204, 141-144. 7 Soeter, N.M., Beintema, J.J., Jekel, P.A., Bak, H.J., Vereijken, J.M. and Neuteboom, B. (1986) in Invertebrate Oxygen Carriers (Linzen, B., ed.), pp. 153-163, Springer-Verlag, Berlin. 8 Johnson, B.A., Bonaventura, J. and Bonaventura, C. (1987) Biochim. Biophys. Acta 916, 376-380. 9 Eyerle, F. and Schartau, W. (1985) Hoppe-Scyler's Biol. Chem. 366, 403-409. 10 Nenteboom, B. and Beintema, JJ. (1988) in Neuteboom, B., Thesis Rijksuniversiteit Groningen. 11 Neuteboom, B., Beukeveld, G.J.J. and Beintema, JJ. (1986) in Invertebrate Oxygen Carriers (Linzen, B., ed.), pp. 169-172,

130 Springer-Vedag, Berlin. 12 Butterworth, P.H.W., Baum, H. and Porter, J.W. (1967) Arch. Biochem. Biophys. 118, 716-723. 13 Ryle, A.P. (1970) Methods Enzymol. 19, 316-358. 14 Hirs, C.H.W. (1967) Methods Enzymol. 11, 197-199. 15 Chang, J.Y., Brauer, D. and Wittmann-Liebold, B. (1978) FEBS Lett. 93, 205-214.

16 Linzen, B., Soeter, N.M., Riggs, A.F., Schneider, H.J., Schartau, W., Moore, M.D., Yokota, E., Behrens, P.Q., Nakashima, H., Takagi, T., Nemoto, T., Vereijken, J.M., Bak, H.J., Beintema, J.J., Volbeda, A., Gaykema, W.P.J. and Hol, W.G.J. (1985) Science (Wash. DC) 229, 519-524.