The double domain structure of rhodanese

J. Mol. Biol. (1975) 98, 63%643

The D o u b l e D o m a i n Structure o f Rhodanese A 3 A electron density map of bovine liver rhodanese shows, in conjunction with gel electrophoresis experiments, that rhodanese consists of a single polypeptide chain with molecular weight of 32,000. The map reveals a very clear double domain structure of the molecule. The two domains are of equal size and have very s~mflar conformations. They are related by a pseudo dyad. Nevertheless, at a few places, distinct structural differences exist between the two domains. A four-stranded parallel ~-structure occurs in each domain, with one of the helices ~mnlng antiparallel to the ~-sheet. The relationship of this super secondary structure with similar structures in other proteins is discussed briefly.

The sulphurtransferase rhodancse (E.C. 2.8.1.1) has widespread occurrence in living systems, as it has been found in bacteria, plants and animals. Although no n a ~ r a l substrates have been detected so far for this enzyme, its general occurrence, the relatively large amounts in organs such as the bovine liver, and the presence of sulphur as an element in so m a n y biological compounds point to an important function for rhodanese in living systems. An X-ray crystallographic study of rhodanese from bovine liver is under way in our laboratory (Drenth & Stair, 1971; Stair, 1973; Smit ~ al., 1974). The last published result was an electron density map of this enzyme at 3.9 A resolution, which, quite surprisingly, allowed the complete tracing of the polypcptide backbone. The molecule appeared to consist of two separate globular regions, which could be explained as different subunits, in agreement with th%rgsults of Westley and co-workers (Green & Westley, 1961; Westley & Nakamoto, 1962; Volini e~ al., 1967), who had provided evidence for rhodanese being a ~imer consisting of two identical subunits with a molecular weight of about 18,500. However, we observed two remarkable features in the map. In the first place, the chain-folding in the two subunits appeared to be very similar, but certainly not identical, and even in the structurally most similar regions in both subunits little indication for amino acid sequence identity could be found. Second, the amino terminus of one subunit and the carboxyl terminus of the other were in contact, giving rise to a continuous ribbon of density, with only a slight depression at the position where we defined the "breakpoint". These two observations led us to speculate cautiously about the possibility of g single-chain molecule with a molecular weight of 37,000, evolved from a monomer of about half t h a t molecular weight after gene duplication. We now wish to present conclusive evidence for this hypothesis based on a preliminary 3 A resolution electron density map and polyacrylamide gel electrophoresis experiments performed with dissolved crystals. Moreover, since at this resolution the polypeptide chain-folding could be established with more certainty, we want to describe briefly the main features of the structure and to make a few remarks on a possible relationship to nucleotide binding proteins.

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(a) The 3 ~ elec~ro~density map The 3.9 A resolution map, which we presented earlier (Smit, 1973; Smitet al., 1974) was based on a p-chloromercury(II)bcnzene sulphonate and a K2Pt(CN)4 derivative and the use of the multiple isomorphous replacement and anomalous scattering method (Matthews, 1966). An uranyl acetate derivative was added to this set, which increased the mean figure of merit from 0.79 to 0.86. l~urthermore, the resolution of the native and the p-chloromercury(II)benzene sulphonate derivative data were increased to 3.0 .~. A "best" Fourier map (Blow & Crick, 1959) was calculated based on multiple isomorphous replacement and anomalous scattering phases out to 3.9/~ resolution, and single isomorphous replacement and anomalous scattering phases between 3.9 A and 3.0/~ resolution. The presentation of the experimental details will be given in a later paper. The map was interpreted and a polyalauine skeleton model was built in a Richards optical comparator (Richards, 1968) with the use of Kendrew-Watson skeleton model parts, scale 1 A = 2 cm. The map is at present being studied, using the preliminary amino acid sequence information available (R. L. Heinrikson, personal communication). The structural information obtained from this study will be described later. The model is in virtually all respects in agreement with the interpretation of the previous 3.9 A map. Only one correction had to be made in the main chain conformation as proposed on the basis of that map; the previously assigned C-terminal part of the "green" chain, including 11 residues, had to be inserted elsewhere in this chain, without changing its direction, however. The most interesting observations in the new map are the continuity of the polypeptide chain between the "yellow" and the "green" subunit, and the confirmation of many side chain differences, as judged from their appearance in the map, between structurally identical parts in both chains. These observations can be explained by assuming that the gene coding for an ancestor protein of half the present size has doubled, after which the two copies have evolved independently, leading to the present two domain structure for the enzyme. The polyalanine model consists of 266 residues (Fig. 1). This number may have to be increased slightly when a higher resolution map becomes available. It is however not compatible with the molecular weight of 37,000 that has been determined for the rhodanese "dimer" (SSrbo, 1953; Wang & Volini, 1968; Blumenthal & Heinrikson, 1971). For this reason and in order to check the "single chain hypothesis" we performed a number of gel electrophoresis experiments. (b) Polyacrylamide gel electrophoresis experiments Rhodanese crystals were thoroughly washed in order to ensure that only crystalline material would be used in the experiments, and subsequently transferred to a denaturing medium. After denaturation, the solution was dialysed overnight against a 0.1 ~/o sodium dodecyl sulphate solution in Trls-acetate buffer (pH 8.0) and submitted to electrophoresis in 1 0 ~ (w/v) polyacrylamide gels (Weber & Osborn, 1969). In the first series of experiments, the denaturing medium consisted of 0.02 M-Tris.acetate buffer (pH 8.0), containing 2~/o (w/v) sodium dodccyl sulphate, 2o//o(w/v) ~-mercaptoethanol and 1~/o (w/v) EDTA. In this case no material with a molecular weight higher than 11,000 was observed (Plate I(a)). As this result could be due to proteolytic cleavage of highly susceptible peptide bonds, a second series of experiments was

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.

' 68,000

' 43,000

4

4

25,000

12,400

I','

(a)

(b)

PLATE ~. Polyacrylamide cleetrophoresis of dissolved rhodanese crystals in the presence of sodium dodecyl sulphate. A small n u m b e r of crystals was dissolved in 0-02 ~ - T r i s - a c e t a t e buffer (pH 8.0), subsequently d e n a t u r e d a n d subjected to electrophoresis as described in the text. (a) Result w i t h o u t the addition of pretense inhibitors. (b) Protease inhibitors (10 mM-iodoacetamide a n d 13 mM-phenylmethylsulphonyl fluoride) added to the dissolving buffer a n d the denaturing medium. To the right of the gels the b a n d positions of the s t a n d a r d proteins are indicated. The s t a n d a r d proteins were bovine serum a l b u m i n (Mr ~ 68,000), ovalbumin (Mr = 43,000), chymotrypsinogen (Mr ~ 25,000) and cytoehrome c (Mr ~ 12,400).

[ f~ing ~. 038

LETTERS TO THE EDITOR

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Fro. 1. A stereo picture of the Ca atoms of rhodanese viewed along the pseudo dyad.

carried out in which protease inhlbitors (10 mM-iodoacetamide, and 13 mM-phenylmethylsulphonyl fluoride) were added to the denaturation medium while ~-mercaptoethanol was omitted. After electrophoresis a single, distinct band corresponding to a molecular weight of 32,000 (4-2500) was observed (Plate I(b)) t. These experiments show that rhodanese is a single-chain protein with a molecular weight of about 32,000. This value is in better agreement with the 266 residue model than the earlier value of about 37,000 for the dlmer and confirms the results obtained recently by Trumpower et al. (1974), who reported a molecular weight of 32,000, and by Ellis & Woodward (1975) and Russell et al. (1975), who determined a molecular weight of 35,000. (e) Description of the molecule In the electron density map the polypeptide chain is represented by about the same density throughout its length. The direction of the polypeptide chain, determined from the C~ positions in the helices, is as assumed previously (Smit, 1973). From now on we will call the previous "yellow chain" domain A and the "green chain" domain B. These domains each comprise about 125 residues. The connecting chain has a length of about 16 residues (Fig. 1). The central part of each domain is a four-stranded parallel pleated sheet structure with a left-handed twist (Chothia, 1973). Its strands are spatially related in the order BACD (ABCI) is the order of the strands in the a.mlno acid sequence). Four, sometimes seemingly irregular, helices are found per domain, varying in length from 2 to 3.5 turns. A 3.5 turn helix connects the pleated sheet strands C and D and runs antiparallel to them. For a quantitative comparison of the polypeptide chain conformation in the domains, a computer program was written which minimized, by means of a non-linear least-squares procedure, the sum of the squares of the distances between equivalent atoms in domain A and domain B by varying three rotation and three translation parameters using a method similar to that used by Rao & Rossmann (1973). An t See Note added in proof, p. 643. 42

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initial set of 80 C a atom pairs, selected b y visual inspection, was used in order to calculate start parameters. These enabled us to prepare a stereo picture of domain B superimposed on domain AI \Prom this picture, 110 C a pairs in equivalent positions were found. These were used as input for the program. After each refinement cycle, the atom pairs t h a t had a distance of more t h a n three times the standard deviation were discarded. Ultimately 101 C a pairs with a root mean square distance of 1.88 A remained. Table 1 gives more details about the comparison. The position and orientation of the pseudo-twofold axis which relates domain.A and B appears to be very close to that derived, b y visual inspection, from the 3.9 A map (Stair, 1973). Figure 2 is a stereo picture of the ~-carbon chain of domain B superimposed upon that of domain A. TABLE 1

~Reaults of the comloarisor~ of the two domains of bovine liver rhodanese Rotation angles: K = 181.1~ ~ = 100.3~ ~b= 118.7~ Translation: ~ = 22.3 A ; ta = 3 1 . 9 A ; ts = - - 1 8 . 8 A . The rotation axis is a "pure" rotation axis, as the translation parallel to the axis is zero within experimental errors. Root mean square difference for 101 equivalenb Ca-pairs: 1.88 A (mean difference, 1.64 A).

Gompariso~ of the fl-strands

flA fiB tic flD

Residue in domain A

Residue in domain B

Mean difference

23-28 49-51 84-88 103-113

160-166 190-192 222-226 244-249

1.16 A 1.84 A 1.16 A 1.51 A

The differenoe in position between two corresponding Ca atoms is defined as: [xA -- ([C]zs -b 0 b where [C] is a rotation matrix expressed in the polar angles ~band ~, and the rotation angle K. These angles are related to the Cartesian X1, X2 and Xs axes as in Rossmann & Blow (1962), whereas the X1 axis is parallel to a, -Yaparallel to b and Xa parallel to c*. The vector $ = (tl, t2, ta), where tl represents the translation along X1, $2 along Xa and ta along Xa. The tertiary structures of the domains turn out to be very s~mflar indeed. The most significant differences are: (1) loop 30 to 37 in domain A is absent in domain B; (2) loop 57 to 64 in domain A differs in conformation from its equivalent loop 198 to 202 in domain B; and (3) loop 172 to 183 in domain B has no counterpart in domain A. The sequence 128 to 138 is unique and forms a part of the long loop 126 to 141 which connects the two domains and winds in a near half circle around the molecule (Fig. 1). This loop might be highly susceptible to proteolytic attack, which could explain earJier descriptions of rhodanese as being a dimeric molecule. The differences between the two domains are also reflected b y the fact t h a t the h e a v y atom positions in the isomorphous derivatives do not obey the pseudo d y a d (Smit, 1973; Smitet al., 1974). (d) Relationshilo with nudeotide binding l~roteins The observed parallel fl-pleated sheet structure in rhodanese and its inhibition by nucleotides (Lawrence, 1967) suggest a possible evolutionary relationship to the

LETTERS TO THE EDITOR 7@

641 7@

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RHODRNESE,

DONRIN B ON R

RHODRNESE,

DOMRIN B ON R

FIG. 2. Domain B superimposed on domain A. The single line bonds connect Ca atoms of domain B, the double bonds eonneob those of domain A. nucleotide binding domain in e.g. lactate dehydrogenase (Rossmann et al., 1974) and to flavodoxin (Watenpaugh et al., 1972; Andersen et al., 1972). Although we wish to postpone a quantitative comparison of rhodanese and the nucleotide binding proreins to a later stage, some preliminary remarks can be made based on a comparison of stereo pictures of ~r models. Lactate dehydrogenase and related dehydrogenases all contain a CBADEF arrangement of six parallel # strands. ~ flavodoxin occur five strands in an arrangement BACDE, which are structurally hotmologous to the BADEF strands in the dehydrogenases. This homology also includes, to a large extent, the (predominantly helical) loops between the # strands. In rhodanese the BACI) pleated sheet arrangement seems to be structurally related to the equivalent strands in flavodoxin and the BADE arrangement in the dehydrogenases. The return loops between the strands in rhodanese are consistently on the same side of the sheet as the equivalent loops in lactate dehydrogenase and flavodoxin. The significance of this has been pointed out by Schulz & Sehirmer (1974). However, the course of the polypeptide chain in the connecting loops in rhodanese seems to be quite different from the course of the loops in the other proteins. The ~C-~-~D structural unit in rhodanese is strongly reminiscent of similar building blocks in the nucleotide binding proteins, but occurs in a different position of the pleated sheet structure. The "parallel pleated sheet-anti parallel helix" building block occurs also in other proteins which do not bind nucleotides under physiological conditions. Examples are triose phosphate isomerase (Banner el al., 1975) and phosphogiycerate mutase (Campbell e~ al., 1974). Therefore, one could argue that the similarities in structure between rhodanese and the nucleotide building proteins are a result of the laws governing protein folding which may favour the formation of #-~-fl building blocks. However, in view of the independent observation that nucleotides inhibit rhodanese (Lawrence, 1967) the observed structural similarities obtain an extra significance.

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Soaking and co-crystallization experiments unfortunately so far have failed to demonstrate nueleotide binding to rhodanese in the crystal, although the area corresponding to the "nucleotide binding site" in the dehydrogenases seems to be fully accessible. The a t t e m p t s are being continued. D a t a collection on native and h e a v y a t o m derivative crystals with the aim to extend the resolution to 2.5 A is in progress. As he does not accept the theory of evolution on religious grounds, one of the authors (J. H. P.) does not agree with the given explanation of the observations b y gene duplication. We are indebted to Mr J. Spoelstra for skilful technical assistance and to Mrs G. ])rent for reliable experimental work. We thank Mr L. Lijk for checking the measured model co-ordlnates. To Professor J. Drenth we are grateful for many stimulating discussions. These studies have been carried out under the auspices of the Netherlands Foundation for Chemical Research (S.O.N.) with financial aid of the Netherlands Organization for the Advancement of Pure Researoh (Z.W.O.). Laboratorium veer Struktuurchern~e Rijksuniversiteit Groningen Zernikelaau, Paddepoel Groningen, The Netherlands

J. BEROSMA W. G. J. HoT. J. N. J ~ N s o m ~ s t K. H. ~AT.~ J. H. PT.O~.GMAN J. D. G. SMr~ t

Received 20 May 1975, and in revised form 28 J u l y 1975 Present address: Abteilung Strukturbiologle, Biozentrum der Universit&t Basel, Basel, Switzerland. REFERENCES Andersen, R. D., Apgar, P. A., Burnett, R. M., Darling, G. D., Lequesne, M. E., Mayhew, S. G. & Ludwig, M. L. (1972). Prec. Na~. Acad. ~c~., U.S.A. 69, 3189-3191. Banner, D. W., Bloomer, A. C., Petsko, G. A., Phi|llps, D. C., Pogson, C. I., Wilson, I. A., Corran, P. H., Fur~h, A. J., Milman, J. D., Offord, R. E., Priddle, J. D. & Waley, S. G. (1975). iVa~ure (~mdon), 255, 609-614. Blow, D. M. & Crick, F. H. C. (1959). ~lcta Gryst~Tllogr. 8ec~. ~4, 12, 794-802. Blumenthal, K. M. & Heinrikson, R. L. (1971). J. B/eL Chem. 246, 2430-2437. Campbell, J. W., Watson, H. C. & Hodgson, G. I. (1974). iVa~wrc (Londo~), 250, 301-303. Chothia, C. (1973). J. _Mol. Biol. 75, 295-302. Drenth, J. & Smit, J. D. G. (1971). B/ochem. B/ophys. Bea. Commun. 45, 1320-1322. Ellis, L. M. & Woodward, C. K. (1975). B/ochim. B/ophys. ~lcb~, 879, 385-396. Green, J. R. & Westley, J. (1961). J. B/el. Chem. 286, 3047-3050. Lawrence, P. J. (1967). Ph.D. Thesis, University of Wisconsin. Matthews, B. W. (1966)..4ct~ C~yst~llogr. se~. A, 20, 82-86. Rao, S. T. & RossmR~u, M. G. (1973). J. MoZ. B/OZ. 76, 241-256. Richards, F. M. (1968). J. _MoL B/O[. $7, 225-230. Rossmann, M. G. & Blow, D. M. (1962). Acta C~s sect. ~4, 15, 24-31. Rossmann, M. G., Moras, D. & Olsen, K. W. (1974). Nature (Lor~on), 250, 194-199. Russell, g., Weng, L., Keim, P. S. & Heinrikson, R. L. (1976). B/ochem. B/ophys. Bes. Commun. 64, 1090-1097. Schulz, G. E. & Schirmer, R. H. (1974). iVat~re (London), 250,142-144. Stair, J. D. G. (1973). Ph.D. Thesis, University of Groningen. Smit, J. D. G., Plocgman, J. H., Kalk, K. H., Jansonius, J. N. & Drenth, J. (1974). I~rad J. Chem. 12, 287-304. SSrbo, B. H. (1953). ~lct~ Chem., Scand. 7, 1129-1136.

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Trumpower, B. L., Katki, A. & Horowitz, P. (1974). Biochem. B~ophya. Re.. Gommun. 57, 582-538. Volini, M., DeToma, F. & Westley, J. (1967). J . Biol. Chem. 242, 5220-5225. Wang, S.-F. & Volini, M. (1968). J. Biol. Chem. 248, 5465-5470. Watenpaugh, K. D., Sieker, L. C., Jensen, L. H., LeGall, J. & Dubourdieu, M. (1972). Pros. Nat. 21cad. ~e$., U.S.A. 69, 3185-3188. Weber, K. & Osborn, M. (1969). J. Biol. (Them. 244, 4406-4412. Westley, J. & Nakamoto, T. (1962). d. Biol. (Them. 237, 547-549.

Note added i n proof: I n order to cheek the possibility of a disulphide bond between the two domains, a number of experiments was carried out, where, in a third step, a large excess of fl-mereaptoethanol (with respect t o iodo acetamide) was added. Subsequent electrophoresis yielded one single band with a molecular weight of 32,000. I n addition, the electron density map contained no indication for a disulphide bond between the two domains.