Tox4a~ Vol. 29, No . 9, pp. 1031-1084, 1991 . Printed in Oreat Briuio.

0041-0101/91 53 .00 + .00 Persmoo Prat pk

REVIEW ARTICLE STRUCTURE AND STRUCTURE-FUNCTION RELATIONSHIPS OF SEA ANEMONE PROTEINS THAT INTERACT WITH THE SODIUM CHANNEL RAYMOND S . NORTON

School of Biochemistry, University of New South Wales, Kensington 2033, Australia (Received 8 Novanber 1990; accepted 4 February 1991)

R. S. NORTON . Review article-Structure and structure-function relationships of sea anemone proteins that interact with the sodium channel. Toxicon 29, 1051-1084, 1991 .-Sea anemones produce a series of toxic polypeptides and proteins with molecular weights in the range 3000-5000 that act by binding to specific receptor sites on the voltage-gated sodium channel of excitable tissue . This article reviews our current knowledge of the molecular basis for activity of these molecules, with particular emphasis on recent results on their receptor binding properties, the role of individual residues in activity and receptor binding, and their three-dimensional structures as determined by nuclear magnetic resonance spectroscopy . A region of these molecues that constitutes at least part of the receptor binding domain is proposed. INTRODUCTION SEA ANEMONES, in common with other members of the phylum Cnidaria (Ccelenterata), possess numerous tentacles containing specialized stinging cells or cnidocytes. These stinging cells are equipped with organelles known as nematocysts that contain small threads which are forcefully evened when stimulated mechanically or chemically (WATSON and HESSnvGER, 1989). Anemones use this venom apparatus in the capture of prey (crustaceans, small fish), as well as for defence against predators and in intraspecific aggression (AYRE, 1982). It is not surprising to find, therefore, that sea anemones contain a variety of interesting biologically active compounds, including some potent toxins (BERF~S, 1982). Peptides and proteins figure prominently amongst the various classes of sea anemone toxins isolated and characterized to date . BERESS (1982) has noted that these range in molecular weight from 3000 to 300,000. Since then, even smaller peptides have been identified, such as the tetrapeptide Antho-RF amide and pentapeptides Antho-RW amide I and II from Anthopleura elegantissima (GRAFF and GRIMMELIICFiIJiJZ,EN, 1988), but it is unlikely that these evolved as toxins, even though they have potent biological activities as neurotransmitters or neuromodulators . The most thoroughly characterized proteins from sea anemones are the 5000 dalton toxins that act by binding to the voltage-gated sodium channel and the membrane-active cytolysins with molecular weights in the range 15-20,000. The latter have been reviewed recently (KEM, 1988) and the known amino acid

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1052

R. S. NORTON

sequences compared (St~sox et al., 1990). The sodium channel binding polypeptides have been reviewed by Biat>3ss (1982) and KEM (1988), but since then the three~imensional structures of four of these molecules have been determined by nuclear magnetic resonance (NMR) spectroscopy and some interesting new findings have been made about the roles of individual amino-acid residues in their biological activity . This review focusses on these sodium channel binding proteins, and in particular on their structures and structure-function relationships. AMINO ACID SEQUENCES

The first representatives of the sodium channel binding proteins were isolated in the early and mid-1970s by BEIZESS and co-workers at the University of Kiel (BERESS et al., 1975; ALSiav et al., 1976) and by NORTON and co-workers at the University of Hawaii (NORTOx et al., 1976). It is now recognized that this group of toxins consists of three classes of polypeptides, two made up of molecules containing 46-49 amino acid residues and one of shorter polypeptides containing 27-31 residues. "Long" polypeptides from the genera Anthopleura and Anemonia (family Actinüdae) have been classified as Type 1 (KEM, 1988; Kia~t et al., 1989), and those from the Indopacific genera Heteractis (formerly Radianthys (DUxx, 1981)) and Stichodactyla (formerly Stoichactis), members ofthe family Stichodactylidae, as Type 2. These two classes of polypeptides are similar with respect to the locations of the six half-cystines (and presumably therefore the disulfide bonds), as well as several other residues thought to play a role in biological activity or maintenance of the tertiary structure . However, as shown in Fig . 1, while there is extensive sequence homology ( >_ 60%)within each class, there is only about 30% homology between the two classes. This is reflected in a lack of immunological cross-reactivity between the two groups and differences in their binding sites on the sodium channel, as discussed below. Recently, another long polypeptide, calitoxin (CLX), was isolated and characterized from the anemone Calliactis parasitica, which occurs in the Mediterranean and along the European coast of the Atlantic Ocean (CARIELIA et al., 1989) . Calitoxin contains 46 amino acid residues (including three disulfide bridges) and exerts similar presynaptic effects on crustacean nerve muscle preparations to those of the Anemonia sulcata toxins. However, its amino acid sequence displays a number of significant differences from those of both Type 1 and Type 2 toxins; for example, it lacks the functionally important Arg-14 (having instead a His residue at position 15), the half-cystine residue normally at position 29 is at position 26, and the C-terminal sequence is His-Glu-Ala in contrast to the basic C-terminal sequences found in the Type 1, and, even more markedly, the Type 2 toxins (Fig. 1). Provided that 2-D NMR studies currently underway (NORTON, R. S. and CARIELLO, L., unpublished results) confirm the chemically determined sequence (c.f. SCHWEITZ et al., 1985; WEMMER et al., 1986), these results suggest that calitoxin may be a representative of yet a third class of long sea anemone polypeptides. Long polypeptides from other species of anemone are also being investigated (BERESS et al., 1976; BERESS and Zwlcx, 1980; KEM, 1988) . Toxin II from Bolocera tuediae (BTTX II) has very little sequence homology with the Type 1 or 2 polypeptides (BERESS, 1988); only three residues, Cys-4, Cys-6 and Trp-31, are conserved throughout this collection of sequences, with Gly-1 in BTTX II being identical to Gly-1 in the Type 1 polypeptides and Asp-8 of BTTX II being conserved in Type 2. This is in marked contrast to the 27 conserved residues within the Type 1 family and 23 in Type 2, of which 11 are conserved across both families (Fig. 1). It seems that the effects of BTTX II on mam-

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Anemone Protein Structure and Function

105 5

malian skeletal muscle are similar to those of ATX II (As II), although it has been suggested that BTTX II may influence activation as well as inactivation of the sodium channel (TESSERAUX et al., 1989). It remains to be seen if BTTX II binds to the same receptor site as Type 1 or Type 2 polypeptides . Currently, there appears to be some correlation between the type of long polypeptide isolated from a given species of anemone and the taxonomic family to which that anemone belongs. As there are several other families of anemones that have not yet been investigated in any detail, the possibility exists that still more classes of interesting polypeptides will emerge . Relatively few "short" polypeptides have been isolated . Their sequences are summarized in Fig. 2. ATX III contains three disulfide bonds and PaTX four, and neither contains free cysteine residues. There is limited, but clear, homology between these two polypeptides; their sequences can be aligned such that 16 residues are identical. Their relationship to the Type 1 long polypeptides (Fig. 1) is very limited, with only one tripeptide sequence being identical and some similarities evident in the disposition of the half-cystines (NlsxtnA et al., 1985). The relationship to the Type 2 polypeptides is even more remote . BIOLOGICAL ACTIVITIES

These proteins were first isolated as cardiac stimulants (NoItTOx et al., 1976) and neurotoxins (BERESS et al ., 1975), and these two activities remain the primary focus of attention. The mechanism underlying these activities involves binding of the toxins to specific sites) on the voltage-gated sodium channels) of excitable tissue . The major effect of this interaction is to delay the inactivation process which converts the activated, open channel to a closed but not readily activatable state (BERGMAN et al., 1976; Root et al., 1976; ULBRICHT and SCHMIDTMAYER, 1981). This mechanism of action is discussed further in the following section. Table 1 summarizes some of the pharmacological properties of the sea anemone polypeptides. As has been noted previously ($CHWEITZ et al., 1981 ; KEM, 1988 ; KEM et al., 1990), there is a crude inverse correlation between the activity of a given polypeptide in crustacea and its activity in mice. The most potent toxin in crabs, Sh I, is practically inactive in mice (when administered i.p.), whereas two of the most potent toxins in mice, Ax II and Hm III, are among the least active in crabs. The LD P values in crabs vary by a factor of about 60, those in mice by some 2000-fold. This inverse correlation is by no means perfect, as evidenced by As V which is highly active in both crabs and mice, and the comparison between Hp II and Hp III, where the former is 1 .5-fold less active in crabs, and eight-fold less active in mice. Comparisons are futher compromised by the use of different species of crab. The crab Ln,~ for Sh I, for example, ranges from 0.5 to 3 ug/kg depending on the species used (PENNIIVGTON, M. W., KEM, W. R. and Datum, B. M., to be published). Furthermore, Af I and As II, which differ in only four locations in the sequence (Fig. 1), have mouse LD s p values that are essentially identical, but crab LD SpS that differ by 50-70-fold, presumably as a result of the use of a particularly resilient crab species for the Af I assay. Correlation of the Kp values for binding to rat brain synaptosomes with mouse Ln~ values is compromised by the fact that the binding sites for the Type 1 and Type 2 toxins are distinct (see below) and different radiolabelled ligands have been used in competitive binding assays . The rat heart Ec~ values in Table 1 are median effective concentrations for increasing sodium flux into cardiac tissue . The poor correlation between these values and the

1056

R. S . NORTON

TABLE l . PHARMACOLOGICAL PROPERTIES OF SEA ANEMONE POLYPEPTiDES AND PROTEINS AFFECTING THE VOLTAGEGATED S()VIUM CHANNEL ~so (PS/kg) Toxin Type 1 Af I Af II As I (ATX n" As II (ATX II)" As V (ATX V) Ax I (AP-A) Ax II (AP-B) Ty~e 2 Hm I (RTX I) Hm II (RTX II) Hm III (RTX III) Hm TV (RTX IV~ Hm V (RTX V) Hp I (Rp I) Hp II (Rp II~I Hp III (Rp III~,II Hp IV (Rp IV)II Sg I Sh I Sbort ply~e~tides As III (ATX III)" PaTX

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Unless otherwise indicated in footnotes, LDSp and KD values were obtained from the following references : Af, SUNAHARA et al. (1987); As, Ax and Sg, SCHWEITZ et al . (1981); ScHwverrz (1984); Hm I-IV, ZYKOVA et al. (1988) and ODINOxOV et al. (1989) (except for LDw in crabs; see footnote) ; Hp I-IV, Scxwerrz et al. (1985) ; Sh I, KEM et al. (1989) ; PENNINGTON cl al. (19906) ; PaTX, L~CAWA et al. (1979) . Ec,~ data were obtained from REr1Atro et al. (1986), $ITNAHARA et al. (1987) and KFx et al. (1989) . Low values are based on intrahaemocoelic injection in crabs and intraperitoneal (i .p.) or intracisternal (i .c .) injection in mice. Crab Lom values for As, Ax and Sg wero estimated by halving the ~Iw values reported by ScHwErlz et al. (1981) . KD values for Hp I-IV taken from ScxvvEtlz et al. (1985) wrreapond to Ko.s values obtained from competitive binding experiments with Androctomls australis toxin II . The nomenclature for these toxins is based on the genus and species names, followed by a Roman numeral to di$èrentiate amongst multiple toxins from a single species. Original or commonly used names of toxins are indicated in parentheses . Genus and species names are as follows : Af, Anthopleurafuscoviridis; As, Anemonia sulcata, Ax, Anthopleura xanthogrammica; Hm, Neteroctis macrodacrylus; Hp, Heteractis paumotenais; Sg, Stichodactyla gigantea; Sh, Stichodactyla lerlianthus. PaTX is from Parasicyonis actinostoloides. " ~,ro values (hg/kg i .m .) in shore crab (Carcinus mamas) are: ATX I, 2; ATX II, 2 ; ATX III, 6. LD w values (Ng/kg i .v.) in mice are ATX I, 7700; ATX II, 310 (ALSEN, 1983). tThero are discrepancies among the ~>o values in mice reported for some of the Heteractis macrodactylus toxins . ZYKOVA Snd KOZLOVSYAYA (1989b) report a value of 3600 tEnucx, 1986), are also clear in defining its limits . These data further suggest that there is a ß-bulge-like distortion of the sheet in the vicinity of residue 19, and a Type II ß-reverse turn involving residues 28-31 . No evidence of regular helical structure is found in any of these polypeptides . Although the tertiary structures of three of these polypeptides have been determined using NMR data and a preliminary description of a fourth is available (see next section), it is instructive to compare their secondary structures in greater detail . In Fig. 5, the deviations from random coil polypeptide values of the backbone amide proton chemical shifts are plotted for two Type 1 and three Type 2 polypeptides . These NH chemical shifts are particularly sensitive to hydrogen bonding (WiTI'HRICH, 1986), and the close similarities in the patterns for all five molecules further emphasize the structural similarities within this series . Thus, although strand 1 of the sheet in AP-A is not shown as including residues 5 and 6 (Fig . 4), the NH chemical shifts for Cys-6 and Ser-19 experience the same significant perturbations found in the other molecules, and Cys-6 in AP-A is also slowly exchanging, indicating that the local structures are highly conserved. In a similar vein, while the amide of Cys-44 is shown as being hydrogen bonded to the carbonyl of Cys-34 in ATX la but not in the other molecules, the fact that it is slowly exchanging and experiences a large downfield shift in all cases suggests that it participates in similar interactions in all of these polypeptides (a point which is discussed further in relation to the tertiary structure) . Comparing the ß-sheets in Hp II and III, PFASE et al. (1989) noted that the N-terminal strand of the ß-sheet in Hp III is translated significantly (up to 1 .SA) relative to its neighbouring strand, such that C°H of residue 3 is further away from C°H of residue 21 and C°H (5) is closer to C°H (18) . In ATX la, the N-terminal strand is probably translated less than in Hp III, while in Sh I the positions is more similar to that in Hp II (comparing Fig. 8 of WEMMEIt et al. (1986) with Fig. 4 of Foci et al. (1989)). Finally, we note that in Hp II and III only one slowly exchanging amide proton (Val-22) is identified as a hydrogen bond donor linking the first and second strands of the sheet, possibly indicating that the N-terminal region is more flexible in these two molecules. It is of interest to compare the secondary structure elements of AP-A determined by NMR with those predicted using Chou-Fasman rules. ISHIZAKI et al. (1979) predicted ß-sheet for residues 2-6 and 45-49, and ß-nuns at positions 20-23, 25-28, 29-32, 7-10, 40-43 and 34-37, while NABIULLIN et al . (1982) predicted ß-sheet for residues 16-24 and 38-45, ß-turns at positions 10-13, 25-28 and 29-32, and a-helix for residues 2-9 and possibly 33-41 . Although these two reports agree in predicting that ß-sheet predominates over a-helix in AP-A, neither provides an accurate description of its secondary structure. This may be due in part to the extensive disulfide cross-linking in the molecule. Spectroscopic data for AP-A indicated that it had a compact structure containing ß-bends and some ß-sheet (Isxtznxi et al., 1979), although an earlier laser Raman study of ATX II was interpreted as indicating a "disordered" structure (PRESCOTT et al., 1976)' . in an eflbrt to detect structural elements other than the ß-sheet that might be conserved

1062

R . S . NORTON

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FIG. 4. SCIn~lATIC REPREE~NTATION OF REGIONS OF ß-8E>1~Er IN TYPE 1 AND 2 FOLYPEPTIDPS . In each case the sheet contains four strands arranged anti-parallel . Residues are numbered as in Fig. l . A filled circle adjacent to a residue indicates that its backbone amide proton is slowly exchanging as a result of a hydrogen bond to a backbone carbonyl in the shcet, while an open circle indicates that the NH is slowly exchanging but its hydrogen-bonding partner is not defined by the NMR data used to determine the secondary structure. The filled square adjacent to W31 in each case indicates that its NH participate in a hydrogen bond that stabilizes a type II ß turn. The arrows indicate the direction of the polypeptide chain. References : AP-A, GOOLEY and NORTON (1986), MAaaurr and NoR~roN (1989); ATX Ia, WIDLtER et al. (1988) ; Sh I, FoGx et al. (1989) ; Hp II, WESD~R et al. (1986), KurdAR et al. (1990); Hp III, PEASE et al. (1989) . The positions of hydrogen bonds are as inferred from analysis of the NMR data at the level of sequence-specific assignments and secondary structure ; for some H-bonds the identity of the acceptor is clarified upon determination of the wmplete tertiary structure . Note that the backbone amide hydrogen of Cys-6 in AP-A is slowly exchanging as in ATX Ia and Sh I, but residues 5 and 6 are not shown as part of the sheet structure in AP-A because of the absence of NOES between C°`H (5) and C'H (18) and NH (4) and NH (20), which are observed in ATX Ia and Sh I .

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throughout this series of molecules, several comparisons of'H NMR chemical shifts have been made . (FocH et al., 1989 ; P~ et al., 1989; M~ssvrr and NORTON, 1990). Comparison of the NH and C°H chemical shifts of AP-A, ATX Ia, Hp II and Sh I revealed that there are eight spin systems in which both the NH and G~H chemical shifts

1064

R. S. NORTON

have a range 5 1031 P.P .m ., viz. Cys-4, Asp/Ser-8, Gly-10, Arg-14, Pro/Thr/Ser-17, Thr-21, Gly-30 and Arg/Lys-46. For Cys-6, the NH and C°H ranges are 0.31 and 0.19 p.p.m ., respectively, while for Pro-11 the C~H range is only 0.05 p.p .m . Only three of these ten residues, viz. Cys-4, Thr-21 and Arg-46, occur within the ß-sheet, while Cys-6 occurs at its edge . The remaining six represent residues that have similar magnetic environments in all four molecules. Considering the extent of homology among these four proteins, it is likely that these residues also participate in common structural features. In the case of Gly-30 this structure is a reverse turn, as described above. Clearly, this type of comparison does not detect all amino acid residues that participate in structures common to the four polypeptides, partly because the aromatic residues, which have a significant effect on chemical shifts from surrounding residues (WtrTxRicx, 1986), are not conserved throughout the series . It does, however, highlight some that would not have been inferred from inspection of the ordered secondary structure and the disulfide bonds. The data in Fig. 5 also highlight some local structural features which are not yet accounted for. For example, the amide resonance of residue 26 is shifted upfield in Type 1 but not Type 2 polypeptides, suggesting a local structural difference . The amide of residue 16 is shifted upfield in all molecules, while that of residue 15 is also shifted in Type 2 but not Type 1 . The amide of residue 36 is shifted downfield in all cases, although it is not part of the ß-sheet hydrogen bonding network. Finally, the amides of residues 41 and 42 are shifted upfield in all five polypeptides (although the shifts in ATX I are only slight), even though the Ala-42 amide proton in the Type 2 polypeptides is slowly exchanging and might therefore be expected to be hydrogen bonded .

Tertiary structure The throe-dimensional structures of three long polypeptides have been determined from NMR data (Toxn~ et al., 1988 ; WiDa~tt et al., 1989 ; Foci et al., 1990) and a preliminary description has been given for a fourth (KUMAR et al., 1990). These structures are based on distance information derived from NOE's and dihedral angles from spin-spin coupling constants, both of which are used as input for distance-geometry algorithms that generate a family of structures for each molecule which are consistent with these data . The resulting structures are usually refined further using either restrained energy min;m ;~ation or restrained molecular dynamics simulations, where the restraints represent the input NMR data . So far, X-ray crystallography has not succeeded in generating a structure for any of the anemone polypeptides, although diffraction data for AP-A were reported some time ago by S~urx et al. (1984) . It is possible that by application of molecular réplacement techniques, the NMR-based structures may be used to assist in solving the crystal structure. Before considering the structures themselves, it is important to emphasize that NMR-based methods generate afamily of closely-related structures for a given molecule, each of which is of low energy and satisfies the NMR data (Wi}~rf~ucx, 1986 ; NORTON, 1990). The degree of overlap between these individual structures can be evaluated using the root mean square difference (RMSD) between them . In one of the most precisely defined protein structures determined to date, that of the a-amylase inhibitor tendamistat (BILLSi'ER et al., 1989), the pairwise RMSD values between individual structures were 0.85A for the backbone atoms and 1.52A for all heavy atoms. For the anemone polypeptides, the RMSD values for the defined regions (see below) of the backbone were as follows: (mean f S.D.) : Sh I, 1 .3±0 .2 ~; ATX Ia, 1 .410 .3 ~; AP-A, 2.3 10 .5 A. In

Anemone Protein Structure and Function

1065

Fta. 6. BACICHONS A1~OLS OF Sh I t+oA ~ ~ax~r sesr a~txucruttas. Structures are fitted so as to minimize RMSDs among the backbone atoms N, C' and C for the well-defined regions of the molecule (from Ala-2 N to Asp-8 N and Pro-17 C to Lys-48, using the numbering system of Fig. 1.) . The top and bottom halves of the figure show different views of the same structures . Modified from Foax et al. (1990) .

general, the greater the number of NMR constraints (mainly NOEs), the more precisely defined will be the final structures . The RMSD values should not be regarded, however, as defining the "resolution" of the NMR-based structures . Stereo views of the structures of Sh I and ATX Ia are shown in Figs 6 and 7, respectively . Clearly, the overall topology of the two molecules is very similar; each consists of a core of highly twisted, four-stranded, antiparallel ß-sheet connected by two well-defined loops and one poorly defined loop encompassing residues 8-17 . Of the three disulfide bonds, two connect Cys-4 and Cys-6 in strand 1 of the sheet to Cys-44 in strand 3 and Cys-34 in strand 4, respectively, while the third connects Cys-45 in strand 3 to Cys-27 in the well-defined loop at the base of the structures as depicted in Figs 6 and 7. A number of hydrophobic side~hains located in the vicinity of the disulfide bonds contribute to a hydrophobic core in the molecules . Detailed descriptions of the structtues of Sh I and ATX Ia are given by Foci et al. (1990) and WII)A~R et al. (1989), respectively.

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FIG. 7.

SUPERM).4ITION OF THE POLYPEPTIDE BACKBONE IN T~ EIGHT ENEROY-REFINED DISMAN sTRUCruRFS of ATX Ia . The structures were superimposed to minimise the RMSD values for the backbone atoms N, C' and C of residues 1-7 and 19-47. Reproduced with permission from WIDMER et al. (1989) .

A distinctive feature of the structures of all four anemone polypeptides is the poorly defined loop encompassing residues 8-17 . This is manifested in each family of structures by significant variation in this region of the molecule from one structure to the next (see Figs 6 and 7). This variation is a reflection of the lack of long-range NMR constraints (NOES) between residues in this loop and those in the rest of the molecule . The most likely explanation for this lack of restraints is that the loop is flexible ; we are currently carrying out NMR spin relaxation measurements to characterize the rates of internal motions within this region . An understanding of the dynamics of the loop will be important in a complete description of the structural basis for activity of these molecules, as at least some of the residues essential for activity are located within this loop (see below). The chemical shifts of backbone protons from this loop are for the most part similar to random coil vâlues (Fig. 4; also see below), further emphasizing the probable lack of ordered structure. There is some evidence from NMR for transient local interactions, possibly hydrogen bonds, within the loop, even though these interactions are not as strong or as long-lived as, for example, those in the ß-sheet (GotrLn et al., 1991). That these interactions are local is underscored by the fact that they are also found in isolated peptide fragments corresponding to the loop in AP-A. PEA38 et al. (1989) have noted that in Hp II the aromatic ring of Phe-18 shows NOES to the methyls of Val-13, indicative of some ordered structure in the loop of this molecule. However, Hp II seems to be the exception in this respect, as these interactions are not observed in Hp III or any other Type 1 or Type 2 toxins investigated so far. As we now have available the structures of two Type 1 and two Type 2 polypeptides, it is of interest to compare them with a view to identifying structural features that might distinguish the two classes. Figure 8 shows a comparison of thé structures of Sh I, ATX Ia and AP-A . It is evident that all three molecules have the same overall fold, as also

Fca. 8. CO~wRL90H OF liü? SI7tUC1TJRE4 of Sh I wrrx ATX Ia wrro AP-A . The structure of Sh I is the energy-minimized average of the eight best refined structures (Foax et al., 1990), while those for ATX Ia and AP-A wrrespond to A1 and DMI, respectively, of Wroema et al. (1989) and Toxnw et a1 . (1988) . Note that the Arg-14 containing loop is poorly defined in all throe proteins, as shown in Figs 6 and 7, and the individual structures shown here are not representative of the time-averaged behaviour of this ttgion of the proteins in solution.

Fto. 9. Av~two8 srxvctvx$ of Sh I wtrx sosm roads xeceeroa snvnnva xssmtrFS nvntcwr~. The energy minimized average structure of Sh I is as described by Foox et al. (1990); backbone a-carbons are shown. Theme residues for which dotted van tier Weak surfaces are shown may play a role in receptor binding, as discussed in the text .

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does Hp II (not shown) . Comparing Sh I and ATX Ia, we find that the backbone structures for residues 2-32 and 45--47 are very similar (Fig. 8). ATX Ia lacks the bend at residue 44, but otherwise the entire third strand of the ß-sheet is the same in the two molecules . One difference between them is that ATX Ia lacks the Gly-25 NH to Ile-41 C = O hydrogen bond. This is consistent with the fact that NH of residue 25 in ATX Ia does not exchange slowly with solvent (Wm~R et al., 1988), possibly because of the subtitution of a Phe in ATX Ia for Gly-25 . This effectively prohibits formation of a y-turn at residue 25 and gives rise to a structural feature that appears similar to a ß-turn in ATX Ia. The course of the backbone, howe~rer, is similar in the two molecules. The backbone structure of the other Type 2 toxin, Hp II, is less similar to Sh I than that of ATX 1 a. In Hp II, Gly-25 is replaced by a Trp and the loop at residues 25-30 commences with a gentle bend rather than a defined turn . The published structure of Hp II is a preliminary description, not yet thoroughly documented, but other differences are evident, for instance the ß-sheet is less twisted than in Sh I, and the structure is clearly different around residues 4-7 and 17-19. Significant differences among the three molecules are found in the Ala-35-I1e-40 loop, which is in closer contact with the ß-sheet core of the molecule in Sh I than in ATX Ia or Hp II. These residues also encompass several tight turns in Sh I, whereas they are mostly in an extended conformation in the other two molecules. The lack of a tight turn at residues 392 in ATX Ia and Hp II is consistent with the fast exchange of the NH proton at residue 42 in both molecules. Hp II has a proline at position 40, but this would not exclude the presence of a type I turn . The Pro-39 containing loop of AP-A appears to have a less ordered structure in AP-A than the corresponding loop in ATX Ia (Mnssuz-r and NORTON, 1990). In Sh I (NORTON et al., 1989), ATX Ia (Wmrmt et al., 1988), AP-A (TORDA and NORTON, 1987), and Hp III (PEASE et al., 1989) the NH protons of residues 32, 34 and 44 exchange slowly with solvent. For Sh I, this is explained by a ß-bulge, which also appears in Hp II. The structure of AP-A is not yet determined in sufiïcient detail to determine whether a ß-bulge is present, but an NOE between the NH protons of residues 34 and 35 shows that the regular ß-sheet cannot continue beyond the NH proton of residue 34 (Mnssvrr and NORTON, 1990). No ß-bulge was found in ATX Ia, but this may be a consequence of the way in which the structure was calculated (see Foax et al., 1990). It is surprising that the tertiary structure of Sh T appears somewhat closer to that of ATX Ia than to Hp II, when Sh I has 69% homology with the latter and only 33% with the former. Thus, it is not possible at this juncture to define a set of structural features characteristic of Type 2, as opposed to Type 1, sea anemone toxins . A further indication of the extensive overall structural homology within this series of molecules comes from a comparison of the deviation of chemical shifts from random coil polypeptide valuQs at each position in the sequence. Such a comparison is shown in Fig. 5 for the backbone amide protons and was discussed in relation to the secondary structure. In addition, PEnsE et al. (1989) have compared NH and G~H chemical shifts for Hp 1I, Hp III and ATX Ia. Leaving aside differences that can be accounted for by ring current shifts caused by non~onserved aromatic residues (e.g. . Phe-18 in Hp II, Tyr-21 in Hp III, 23-26, 37-38 and 43), the overall patterns for all five molecules are very similar. The environment of Trp-31 is quite similar in Sh I and ATX Ia. In both cases this residue appears to be substantially shielded from solvent by the side chains of residues 22, 28 and 47, the backbone at residue 46 and the 27-45 disulfide bond. It is interesting to note that while Trp-23 and Trp-31 are less than 30% exposed to solvent in ATX Ia

Anemone Protein Structure and Function

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et al., 1989), Trp-23 in ATX Ia and AP-A, as well as Trp-31 in ATX Ia, AP-A and Sh I, are all accessible to flavin dye as determined by photo-CIDNP experiments (NORTON et al., 1986; NORTON et al., 1989). KUMAR et al. (1990) do not provide any details on the conformation of side chains in Hp II, but a comparison between Sh l (Foci et al., 1989) and Hp II (WEMMER et al., 1986) shows a remarkable similarity in the chemical shifts of, among others, residues 21, 22, 28, 30-32, 38, 39 and 457. This suggests that the surroundings of the partially buried Trp-31 and Tyr-38 aromatic rings are similar in the two molecules. Since both aromatic residues are absolutely conserved among Type 2 toxins (Fig. 1), it is likely that the surrounding structure is conserved as well. The tight turn at residues 392 in Sh I is strongly hydrophobic, and the side chains of Ile-40 and Ile-41 as well as the nearby Tyr-37 and Leu-24 form an exposed hydrophobic region on the molecular surface. A similar hydrophobic region, encompassing Val-24, Phe-25, Ile-40, Ile-41 and possibly Val-2 was found in ATX Ia by Wro~t et al. (1989) . Indeed, both the second strand of the ß-sheet and the residues linking this strand to the ß-turn preceding the fourth strand are mostly non-polar in the Type 1 polypeptides, although not in Type 2. Sh I contains seven Asp and Glu, as well as seven Lys and Arg residues. Although the characteristics of the surface of Sh I cannot yet be determined with great confidence, inspection of the surface electrostatic potential using the molecular graphics program MIDAS shows that the structure contains a region of strong negative charge associated with Asp-7, Asp-8 and Glu-9, as well as a region of strong positive charge associated with Arg-46 and Lys-479. This distribution of surface charge may prove to be important in orienting the molecules as they bind to the sodium channel. Discussion of the charged residues of these toxins leads to one final comment on the tertiary structures determined so far, that they are of relatively low resolution . Methods for further refining structures based on NMR data are being developed ($LJMMEItS et al., 1990), and their application to these proteins is likely to lead to a better understanding of the molecular basis for their biological activities, and in particular of their ionic interactions. Coupled with this will be studies of the dynamics of these proteins in solution, to establish the rates and amplitudes of internal motions, especially in regions such as the Arg-14 containing loop. NMR spectroscopy is particularly well suited to this task (W~rTFnucx, 1986; ToRnA and NoRTOx, 1989 ; NoRTOx, 1990). (Wro~tt

Conjormational heterogeneity NMR spectroscopy is a powerful means of detecting and characterizing chemical and conformational heterogeneity of proteins in solution . In the case of ATX I, splitting of a few resonances was observed in our initial studies (GOOLEY er al., 1984a), but was subsequently shown to be due to the presence of both the Ala-3 and Pro-3 isotoxins (Wro~t et al., 1988 ; GOOLEY et al., 1988). By contrast, AP-A and ATX II show splitting of a large number of resonances (Goor.~r et al., 19846) which is due to conformational rather than chemical heterogeneity (Goor mr et al., 1988). On the basis of the largest chemical shift differences between the two conformers being found near Pro-39 in AP-A and the absence of this heterogeneity in ATX I, which lacks Pro-39, it was suggested by Gooi.s~r et al. (19846) that ci~trans isomerism of the Gly-38 to Pro-39 peptide bond was responsible for the heterogeneity . In the structure of AP-A, Pro-39 is located at the outermost tip of the loop joining strands 3 and 4 of the ß-sheet, where the preceding

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R. S. NORTON

peptide bond presumably has enough freedom to switch between the two forms. Nevertheless, the energy bamer to interconversion must be quite high, as the two forms give distinct NMR spectra even at extremes of pH and temperature. If is intriguing that Hp II and III, which have a Pro at position 40, do not display this kind of heterogeneity. This may reflect differences between these loops in Type 1 vs Type 2 polypeptides, as discussed above, and/or greater restrictions on the conformation of the 390 peptide bond .

COMPARISON WITH RELATED STRUCTURES DRLSCOLL et al. (1989a,b) have determined the three-dimensional structure in solution of the antihypertensive and antiviral protein BDS I, using NMR data and a hybrid distance geometry-dynamical simulated annealing procedure (DxtscoLL et al ., 1989a,b) . This 43 residue polypeptide isolated from A. sulcata, contains a three-stranded antiparallel ß-sheet to which the three disulfide bonds are connected, and the locations of the disulfide bonds are similar to those found in the long anemone proteins . Clearly, there are structural similarities between the two groups of proteins, although there are also major differences in the structures of the connecting loops (for a detailed comparison, see WmMER et al. (1989)). The sequence homology between BDS I and the A. sulcata long polypeptides (ca 30%)and the structural similarities in the ß-sheet core suggest a common evolutionary ancestry . Indeed, we have shown recently that BDS II is able to displace labelled AP-A from the rat brain sodium channel, suggesting that it competes for the same binding site (LLEWELLYN and NORTON, 1991). On isolated guinea pig atria, BDS II elicits a weak negative inotropic response . The absence from BDS I and II of the Asp residues at positions 7 and 9, and the large flexible loop incorporating Arg-14, both of which are thought to be functionally important in the long proteins (see below), undoubtedly contribute to their lack of positive inotropic activity . From a functional standpoint, a more interesting comparison can be made between the long anemone toxins and the a-scorpion toxins, which appear to bind to the same site on the sodium channel (at least for the Type 1 anemone toxins) and elicit similar biological responses, even though there is no sequence homology between the two classes. FONTECILLA-CAMPS and co-workers (1988) have determined the structure of the a-toxin Androctonus australis Hector II (AaH II) by X-ray crystallography, at a resolution of 1 .8 ~. This 64-residue protein contains a three-stranded antiparallel ß-sheet (residues 2~, 32-37 and 45-51) and an a-helix (residues 19-28) running roughly parallel to the sheet. Two disulphide bonds link the helix to the sheet, a third is nearby and the fourth extends to the C-terminal region . The overall molecular structure is very similar to that of the weakly toxic ß-scorpion toxin Centruriodes sculpturatus variant 3, determined previously by the same group (ALMASSY et al., 1983), although loops extending from the core of secondary structure differ in the two molecules, as does the C-terminal region . In particular ; the "conserved hydrophobic" surface recognized in variant 3 (FONTECILLACAMPS et al., 1981) is also present in AaH II . This surface contains a number of aliphatic and aromatic residues which may participate in hydrophobic interactions with the sodium channel. It also encompasses a group of residues which are conserved throughout the scorpion toxins and are therefore thought to be essential for activity . It was suggested that these conserved residues, especially those with positive charges, confer some degree of specificity on the toxin's interaction with the channel, more so than the hydrophobic interactions, which contribute mainly to the free energy of binding. Further support for

Anemone Protein Structure and Function

107 1

the role of this region of the molecule in activity comes from recent chemical modification studies on the toxins AaH I, II and III (KHARRAT et al., 1989). It is possible to draw some crude analogies between the scorpion and anemone toxin structures . Both contain a core of antiparallel ß-sheet, although in the scorpion toxins the sheet lacks a fourth strand and is not highly twisted as in the anemone toxins . Also, both contain positively-charged residues that have been implicated in activity (residues 1 and 58 in the scorpion toxins, Arg-14 and one or more lysines and histidines in the anemone toxins). The anemone proteins appear not to possess an extensive hydrophobic surface, but it is noteworthy that photochemically induced dynamic nuclear polarization NMR studies on ATX I, ATX II and AP-A demonstrated that Trp-31 and the aromatic residue at position 43 (Trp or Tyr) were exposed to solvent, with Trp-23 being partially exposed (NORTON et al., 1986). Given the close proximity of the aromatic rings of Trp-23 and -31 and the conservation of their local environment, at least in the Type 1 polypeptides (GOOLEY et al., 1986), this region may constitute a small "hydrophobic patch" on the surface of the anemone toxins as described above. In any case, it is clear that a further, more detailed comparison of the anemone and scorpion toxin structures is called for. IMMUNOLOGICAL PROPERTIES

Immunological cross-reactivity has been a useful tool in distinguishing the Type 1 and Type 2 anemone polypeptides, with antibodies raised against the H. paumotensis toxins ($CI-IWEITZ et al ., 1985) and Sh i (K~t et al., 1989) failing to cross-react with Type 1 polypeptides . In addition, site-directed and monoclonal antibodies have proven to be valuable probes of the structure and mechanism of action of the sodium channel (CATTERALL, 1988 ; THOMSEN and CATTERALL, 1989). Relatively little work has been carried out at the molecular level on the interaction of antibodies with the anemone polypeptides, partly no doubt because their small size makes them less antigenic than larger proteins . This appears to be a fruitful area for further research, however, and the current situation is summarized below. EL-AYES and co-workers (1986) purified subpopulations of antibodies directed against ATX I and I1, and found that these toxins contain a minimum of one common antigenic region, with ATX II having at least one additional antigenic determinant on its surface. An attempt was made to map the common antigenic site using chemically modified derivatives of ATX II, with the result that the positively charged groups of Arg-14, Lys-35, -36 and -46 and the amino terminus appeared not to be involved, whereas two of the three carboxylates of Asp-7 and -9 and the C-terminus were. It should be noted, however, that modification of one or both of the carboxylates of Asp-7 and -9 of AP-A with the same reagent led to major conformational changes (GRUEN and NORTON, 1985), as discussed further in the following section. It is conceivable that these changes could have interfered with the structure of an antigenic region elsewhere in the molecule, thereby modifying its antibody binding affinity . An interesting finding of this work was that the common antigenic region on ATX 1I was still accessible to the antibody when bound to the rat brain synaptosomal sodium channel. This suggests that there is minimal overlap between the sodium channel binding site on ATX II and the common antigenic region, which is consistent with the result that, wherever it is, the latter does not include the basic side chains, some of which are thought to play a role in sodium channel binding (see below). The same group also investigated the antigenic properties of the short polypeptide

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ATX III (Fig. 2). In the work just described, ATX III failed to inhibit antibody binding to ATX I and II . As ATX III appears to bind to the same site (3) on the sodium channel as ATX I and II, this lack of cross-reactivity further indicated the lack of overlap between the antigenic and channel binding sites on ATX I and II. Subsequently, antibodies were raised against ATX III itself (BAHRAOUI et al., 1989). The results pointed to the existence of a minor fraction of anti-ATX III antibodies that cross-reacted with ATX I and II. The corollary, that ATX III possesses several antigenic determinants, is rather surprising, and bears further investigation. Nonetheless, more detailed analysis of the anti-ATX III subpopulation that did recognize ATX I and I1 suggested that the antigenic determinant included a guanidino group (Arg-14 in ATX I and II) and at least two carboxylate groups . It is therefore distinct from the common antigenic region in ATX l and II described above. More intriguing is the finding that anti-ATX III antibodies are able to neutralize ATX II administered intracerebroventricularly, which suggests that the determinant shared by ATX II and III partially or totally overlaps the site responsible for biological activity . The anti-ATX III antibodies did not, however, show any binding to a-scorpion toxins, which are thought to bind to the same site as ATX II. In summary, these studies indicate how antibodies can be employed to assist in identifying the active pharmacophore on the anemone polypeptides . Further work using monoclonal antibodies raised against the toxins and more detailed mapping of the various antigenic determinants will enhance the clarity of interpretation of this approach . A monoclonal antibody that recognized the pharmacophore on the Type 1 polypeptides and which was able to inhibit their binding to the sodium channel would constitute a particularly interesting candidate for study. Structural studies by NMR or X-ray crystallography on the complex between the toxins and an Fab fragment from such a monoclonal may be expected to provide valuable information on the structures of the receptorbound toxins .

AMINO ACID RESIDUES ESSENTIAL FOR ACTIVITY

Our knowledge of which residues are required for receptor binding and biological activity is based on the results of chemical modification studies and comparison of amino acid sequences. It is therefore far from complete . Furthermore, interpretation of the chemical modification studies that have been carried out is compromised by apparent disagreements in the literature concerning the effects of individual modifications. A rigorous understanding of the structure-function relationships within and between the Type 1 and Type 2 polypeptides must await the results of systematic studies with synthetic analogues (Sh I has been synthesized, as described by PENIVINC~roN et al., 19906) and mutant recombinant proteins produced by site-directed mutagenesis. In the meantime, some useful conclusions can be drawn from the available data, as summarized below. The residue numbering system of Fig. 1 will be used here . Chemical modification

The first chemical modification study was carried out on AP-A by NEWCOMB et al. (1980) . They found that the two His residues could be modified without loss of cardiac stimulatory activity . Likewise Tyr-25 was found not to be essential. Modification of one

Anemone Protein Structure and Function

107 3

or two Trp residues did not affect activity, but modification of all three reduced activity . Reductive alkylation of the two Lys residues, which did not alter their positive charge, had no effect on activity, but modification of both with trinitrobenzene-sulfonic acid, which introduces a bulky aromatic moiety and destroys the positive charge, eliminated activity . This suggested a role for the positive charges of lysines, although the possibility of N-terminal amino group modification appears not to have been eliminated. Modification of Arg-14 with phenylglyoxal was claimed to have no efféct, but there is reason to doubt this result, as discussed below. Not surprisingly, reduction of the three disulfide bonds, followed by carboxymethylation of the resulting cysteines, completely inactivated the molecule. NEWtbMB et al. (1980) also found that reaction of two or three of the carboxylate moieties (Asp-7 and -9, and the C-terminus) with glycine ethyl ester or taurine inactivated AP-A. Subsequently, Gtttrt=tr and Nottrox (1985) showed that modifications of Asp-7 and Asp-9, but not the C-terminus, with glycine ethyl ester was sufficient to destroy activity . However, CD and NMR spectra of the modified derivative showed that its structure was drastically altered. This finding raised the question of whether the Asp carboxylates were required for activity because they interact directly with the sodium channel, or because they participate in electrostatic and/or hydrogen-bonding interactions in native AP-A that are necessary to maintain its biologically active conformation-or both. In other words, is their role "structural" or "functional' "C and'H NMR studies show that one of the two Asp residues in AP-A and ATX II has a low pK, ( S 2), and that protonation of this Asp residue at low pH alters the protein structure (Noltrox and NORTON, 1979; NORTON et al., 1980; Goot.mr et al., 1988). This indicates that at least one of the Asp residues is involved in interactions essential to the maintenance of native structure, but does not eliminate the possibility that one or both Asp residues also participates in ionic or hydrogen-bonding interactions with the sodium channel. The most thoroughly documented chemical modification study of a long anemone polypeptide reported to date is that of BARawxnv et al. (1981) on ATX II. Some of their results agree with those of NEWI~MB et al. (1980) on AP-A, some do not. Modification of all three carboxylates with glycine ethyl ester destroyed toxicity, but did not affect sodium channel binding. This implies that the carboxylate moieties do not interact directly with the channel, but do participate in a co-operative conformational change that occurs upon receptor binding and is required for expression of activity . Derivatives of ATX II with only one or two carboxylates modified were also isolated. They also appeared to be nontoxic, but unfortunately were not characterized further. Results for the amino groups were similar to those for AP-A, in that their charge seemed to be essential for toxicity and receptor binding. It was found that selective acetylation of the amino terminus reduced activity in crabs and mice, as well as binding affinity, by a factor of less than two. In contrast to the results for AP-A, modification of both His residues of ATX II by carbethoxylation caused a significant loss of activity, although iodination of His-37 had very little effect on activity or receptor binding. The most significant conflict with the results for AP-A, however, concerns Arg-14. BARHAttIN et al. reacted Arg-14 of ATX II with 1,2-cyclohexanedione in 0.2 N NaOH, and found that the product was inactive and unable to bind to the receptor . However, high pH values inactivate these proteins even in the absence of modifying agents (NoRrorr and NORTON, 1979), and although a control was carried out, this inherent loss of activity has compromised general acceptance of these results as confirming a role for Arg-14 in activity . The situation is confused by the fact that the claim by Newcoxra et al . (1980) that Arg-14 is not required for activity is also

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R. S. NORTON

compromised, in this case by the possibility that the phenylglyoxal modification may have been reversed at alkaline pH. Some clarification of the role of Arg-14 has emerged as a result of more recent work . M~xNtR er al. (19896) found that modification of Arg-14 of the Type 2 toxin Hm III with cyclohexanedione or phenylglyoxal decreased its mouse toxicity five-fold. The second Arg residue at position 46 was unreactive, consistent with its involvement in the ß-sheet, as discussed above. These results suggest that Arg-14 is important for activity, but not absolutely essential, implying that some other regions of the molecule are also required . it should be noted, however, that in our hands modifications of AP-A by cyclohexanedione under similar conditions produced extensive side-reactions which could compromise interpretation of the results (Goul.n, A . R. and NORTON, R. S., to be published). An alternative approach to characterizing the role of Arg-l4 has been pursued by GouLn et al. (1990), who prepared a derivative of AP-A in which the Arg-14 to Gly-15 peptide bond had been cleaved by trypsin. This species was devoid of cardiac stimulatory activity, but its receptor binding affinity (LLEWELLYN et al., 1991) was reduced by less than an order of magnitude. Its tertiary structure, as monitored by 'H NMR spectroscopy (Goul.n et al ., 1990), proved to be the same as that of native AP-A, but it must be emphasized that this refers to the well-defined regions of the structure (see above), and that the conformation of the Arg-14 containing loop is not defined in either AP-A or its trypsinized derivative . The fact that the derivative binds to the sodium channel with only slightly lower affinity than native AP-A indicates that other regions of the protein must interact with the channel, for although the Arg-14 sidechain and its flanking residues are free to make the same contacts with the channel as in native AP-A, it is most unlikely that the binding affinity would be unaltered as a result of trypsin treatment if these residues were the only ones responsible for binding. While Arg-14 and its immediate surroundings may not be sufficient for receptor binding (indeed, may not even make contact with the receptor), the lack of cardiac activity of the derivative demonstrates that this region of the molecule must participate in a co-operative conformational change upon receptor binding that clearly is essential for activity . Several other chemical modification studies have been carried out. An abstract by KOLKENBROCK et al. (1983) indicates that guanidination of the Lys residues of ATX II did not affect activity (in agreement with BARHANiN et al . (1981)), but that subsequent acetylation of Gly-1 abolished positive inotropic activity, even though crab toxicity was hardly affected . Acetylation of all four amino groups decreased activity in both assays, as did carbonyl modification. However, the carboxyl-modified ATX II appeared to bind to the sodium channel, in contrast to the results of BARHf+IVIN et al . (1981) . Unfortunately, these interesting findings, together with the results on Arg-14, His and Trp modification which have not been mentioned here because they are even less adequately described, have not been documented in a full publication . STENGELIN of al. (1981) modified the amino groups of ATX II by Schiff base formation with pyridoxal phosphate, followed by reduction with borohydride. Of the two monosubstituted derivatives isolated, the Gly-1 species had a crab LD,oo value about 20-fold higher than that of unmodified toxin, while the Lys-35 adduct had a 10-fold higher value. These results are compatible with those of BARHANIN et al . (1981), who found that acetylation or reaction with flourescamine of all four amino groups of ATX II led to an eight-fold increase in the crab ~n~ and a > 20-fold increase in mouse Ln~, but there is disagreement over the importance of the N-terminus, acetylation of which decreased toxicity and receptor binding by a factor of less than two according to BARHANiN et a/ .

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(1981) . This apparent disagreement may reflect the larger size of the pyridoxal phosphate moiety, coupled with the reversal of charge that accompanies its attachment to an amino group. Recently, MAHNiIt and KOZLOVSKAYA (1989) investigated the effect of acetylation of amino groups on the toxicity to mice of Hm III . Four monoacetyl and four diacetyl derivatives were isolated . The Gly-I adduct had 12-fold lower activity, while the Lys-4, Lys-47 or -48 and Lys-48 or -49 adducts had half the toxicity . Diacetylation caused a 30 to 35-fold decrease in toxicity, the N-terminal amino group being modified in all cases . MAHNIR et al. (1989a) also modified Trp-30 of Hm I1I with 2-hydroxy 5-nitrobenzyl bromide (Koshland's reagent). The resulting adduct had full activity in mice. Elsewhere, the same group reported that modification of Trp-30 of Hm iII reduces toxicity three-fold (Ontrrotcov et al., 1989). In attempting to draw definitive conclusions about the role of individual residues in activity from the above results, we are hampered by several problems : different bioassays have been used (crab and mouse toxicity, positive inotropic activity), receptor binding has usually not been measured, minor products from side-reactions have often not been separated (the older studies did not employ HPLC), and the exact identity of the major product has not always been established rigorously. It seems prudent therefore to confine ourselves to qualitative conclusions and state that the positive charges of one or more amino groups (including the N-terminus), the carboxylates of one or both of Asp-7 and -9, and perhaps the guanidinium group of Arg-14 are all required for full activity . The exact roles of each of these groups, however, remain to be clarified . While this article was under review MAHNIR et al. (1990) published a paper on the effects of carboxyl modification on the mouse toxicity of Hm 1fI (RTX III). They found that blocking of any single carboxyl group with glycine methyl ester caused no more than a two-fold decrease in toxicity, while the modification of two carboxyl groups reduced toxicity by no more than six-fold. They also reported that these carboxyl modifications did not alter the secondary structure of the protein as judged by circular dichroism spectroscopy. While their findings on the secondary structure are in accord with those of KEnt et al. (1990) and PENNiNGTON et al. (1990a) on synthetic analogues of Sh I (results Which MAHNiR Pt al. appear not to have been aware of), the effects of carboxyl modification are much less dramatic than found previously for Type I toxins (on mouse toxicity and positive inotropic activity) and the Type 2 toxin Sh I (on crab toxicity) . The origins of this apparent disagreement need to be clarified . It may be that they reflect once again the importance of confining any discussion of essential residues to a specific class of anemone toxin acting on a specific target tissue . S}~nthetic polypeptides A more systematic approach to elucidation of the structure-function relationships in these proteins is to utilize either site-directed mutagenesis of recombinant toxins or chemical synthesis . The latter has been achieved in good yield for Sh I (PENNINGTON et al., 1990a,b ; KFat et al., 1990), the synthetic material being indistinguishable from native Sh I in chemical composition, spectroscopic properties and crab toxicity . Results for the crab toxicity of a number of synthetic analogues of Sh I have been reported by KEM et al. (1990) and PENNiNGTON et al. (1990u) . Activity was abolished ( > 10°-fold decrease in toxicity) beach of the following changes : Asp-7 to Asn, Asp-8 to Asn or Glu-9 to Gln. These results are entirely consistent with those of the chemical

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modification studies described above . Somewhat surprisingly, the CD spectra of these analogues did not differ significantly from those of native Sh I, but the conformations of these analogues need to be characterized in more detail by 2-D NMR There is not necessarily any conflict, however, between the finding that replacement of sidechain carboxylates at positions 7, 8 or 9 in Sh i by sidechain amides does not affect the gross structure and the earlier results that modification of Asp-7 and Asp-9 of AP-A by glycine ethyl ester does alter the structure (GRUEN and NORTON, 1985), because in the latter case both residues were modified, and the modifying group was much larger . Furthermore, it has already been pointed out that while Sh I undergoes conformational changes associated with carboxylate protonation at low pH, these changes are neither as extensive nor as clearly linked to a single carboxylate as in AP-A (NORTON et al., 1989) . Substitution of Asp-12 by Asn and Lys-4 by N-acetyl Lys reduced toxicity by 120-fold and 80-fold, respectively (PeNNixcTON et al., 1990a) . The synthetic approach is obviously a very powerful one, and can be expected to make a significant contribution to our understanding of the molecular basis for activity of both Sh I and AP-A, which has also been synthesized recently (ICEM, W . R ., Personal communication) .

Essential residues : structural or functional?

With a knowledge, albeit incomplete, of which functional groups in these toxins contribute to their activity, it is appropriate to return to Fig. 1 and consider which residues are conserved within or between the Type 1 and Type 2 sequences. The 11 residues that are absolutely invariant throughout all 15 sequences are indicated by asterisks in Fig. l, while a further five that are conservatively substituted are denoted by daggers. It is useful to try to classify these 16 residues as being either "structural", that is essential for the maintenance of tertiary structure or as participants in a co-operative conformational change upon receptor binding, or "functional", which participate in direct interactions with the sodium channel . The structural category clearly includes the six halfcystine residues, reflecting the conserved locations of the three disulfide bonds, and the combination of Ser/Thr-19 and Gly-20, which must be necessary for the characteristic (but as yet incompletely defined) distortion of strand 2 of the ß-sheet that is evident in all toxin structures characterized to date . They are preceded by a residue which, although not conserved within or between Type I and Type 2, is always hydrophobic . Gly-10 and Pro-I I constitute another pair of "structural" residues (see also Fig . 5); they are probably associated with a reverse turn, but because they are near the start of the poorly defined loop, the nature of this turn cannot be specified at present. Finally, the ß-turn involving residues 28-31 is found in all structures, which accounts for the conservation of Trp-31 and Gly-30 (which is found in all sequences except that of Hm II, where it is replaced by Ser) .

Of the five residues that remain to be classified, only Arg-14 stands out as being clearly "functional" . With the exception of some early work that suggested it was not essential, there is general agreement that it plays some role in activity, even though the exact nature of that role may vary depending on which class of polypeptide and which bioassay are considered . The only evidence that it interacts directly with the sodium channel comes from the effect of modification by cyclohexanedione on binding (BARHANIN et al., 1981), and these results urgently require confirmation . The carboxylate-containing residue at position 9 is essential for activity, but it is not clear whether its role is structural or functional . It is conceivable that it is a mixture of both, with perhaps less emphasis on the

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former in Type 2 than Type I . The Asp residue at position 7 was not included in the list of 16 conserved residues because of its substitution by Lys in ATX I (Fig. 1). However, its conservation in every other sequence argues for a role in structure or activity. Indeed, it is likely that one of thesc Asp residues has a functional role while the other is principally structural . Of the other three conserved residues, Ile/Val-22 and Lys/Arg-46 are both in the ß-sheet, and must have roles that are at least partly structural . Lys/Arg-46 may also have a functional role, as discussed below. Finally, Ile/Val-41, which lies at the end of the loop preceding strand 3 of the ß-sheet, probably has primarily a structural role . This analysis leaves us with a dearth of residues that can be classified exclusively as "functional" . There may be several reasons for this, but the main ones are that we have taken into account both Type 1 and Type 2 polypeptides, which may not even bind to the same receptor site, and that the 15 toxins we have considered encompass a wide range of biological activities, ranging from those with negligible activity in mammals and high crustacean toxicity to those in which the reverse is true. Thus, the identity of residues essential for, say, positive inotropic activity in mammals but not crustacean toxicity is obscured in this comparison, and we are left largely with those residues essential in maintaining the common tertiary structure of these toxins . A comparison of conserved residues within the Type 1 and Type 2 sequences highlights several major differences between the two groups . In Type 2 the N-terminus is truncated and the C-terminus is extended by the addition of several lysines. Charged residues in Type 2 replace either uncharged residues or residues with opposite charge at positions 5, 8, 23, 32 and 33 in Type 1, while the reverse is true at positions 35 and 37 . These changes may be expected to cause local structural perturbations and/or changes to receptor binding. The same is true of changes such as the substitution of Pro-28 by Asn (which will affect the ß-turn), Pro-39 by Ser or Thr (affecting the loop connecting strands 3 and 4 of the ß-sheet) and Trp-43 by Ser, Asp or Glu in Type 2. From a functional standpoint, the replacement of Gly-15 and Asn-16 in Type 1 by Ser/Thr and Ala, respectively, are of interest because they are adjacent to Arg-14 in the sequence and there is evidence from'H NMR that Asn-16 is involved in local interactions within the loop (Gout.n et al., 1991). It would be premature to attempt a detailed interpretation of the differences between Types 1 and 2; the changes are simply too numerous. The picture is further complicated by the unusual sequence of ATX I, which has a number of non-conservative substitutions relative to the other Type 1 polypeptides, viz. Val-2 to Ala, Asp-7 to Lys, Ala/Ser-25 to Phe, His-32 to Asn, Lys-35 to Glu, and His-37 to Arg. What is clearly needed is a comparison of a series of analogues of one protein using a single bioassay and a relevant receptor binding assay. This will be possible using the synthetic approach (KEM et al., 1990 ; PEIVriINGTON et al., 1990a,b) or by generation of a series of mutants of a recombinant toxin expressed in cell culture. It will be imperative in such studies to monitor the structure and dynamics of mutant proteins using NMR spectroscopy, and to correlate these data with the results of bioassays and receptor binding measurements . Only when a single residue change that alters activity or binding affinity has been shown not to affect conformation will it be possible to classify that residue as functional. Of course for residues with both a structural and a functional role, the latter will be masked by the former . In the meantime, some predictions can be made about the receptor binding surface of these toxins . Given the importance of Arg-l4 and the carboxylate-containing residues at 7 and 9, it is likely that some or all of the flexible region between residues 7 and 14 makes contact with the sodium channel. Gly-10 and Pro-11 are important in maintaining the

1078

R . S . NORTON

biologically active conformation of this region, but this does not exclude the possibility that they also make contact with the sodium channel . The disulfide bond linking Cys-6 and -34 brings residues at the beginning of the Arg-14 loop into proximity with residues in strands 3 and 4 of the ß-sheet, and may account for the importance of Lys/Arg-46 or the histidines (in Type 1) . As Trp-30 is on the same side of the molecule, it is possible that it makes contact with the sodium channel in the bound state even though it is largely shielded from solvent . Figure 9 shows the average structure of Sh I with .some of these residues highlighted . It can be seen that while they do not constitute a contiguous surface, they are all on the same face of the molecule and may all be able to interact with the sodium channel . While I believe that this region constitutes at least part of the receptor binding domain, it is almost certainly not complete . For example, it does not take into account a role for the N-terminal amino group . In the case of the scorpion toxins, the sodium channel binding surface comprises approximately 13 amino acid residues (FONTECILLA-CAMPS Pt al., 1981, 1988), while in antigen-antibody complexes the number of residues on the antigen that are in direct contact with the antibody is in the range 15-20, with a surface contact area of 700-800 t1=. By analogy, we might expect 10-15 residues of the anemone polypeptides to contribute to the sodium channel binding surface, although it is possible that weaker, but still productive, binding may be achievable with fewer residues in a low molecular weight analogue.

CONCLUDING REMARKS The availability of three-dimensional structures for several long sea anemone proteins has provided a starting point for understanding how these molecules function . At the same time, the number of toxin sequences has continued to grow and further information on the identity and role of residues essential for activity has come to light as a result of recent chemical modification and proteolysis studies. These results allow us to identify a region of these proteins that may constitute part or all of their sodium channel binding domain . Much remains to be done, however, both to confirm this proposal and to identify other regions of these proteins that may be essential for activity . A major thrust of future work must be the production of series of analogues of several individual proteins, including representatives of Type 1 and Type 2 as well as mammalian-active and crustacean-active toxins . In each case, the focus should be on a single bioassay, coupled with an appropriate receptor binding assay, and the results must be correlated with structural information from techniques such as NMR spectroscopy . In this way we can expect to gain a detailed understanding of not only the common features of the mechanism of action of these toxins, but also what distinguishes a Type 1 from a Type 2 toxin or one that is mammalian active from one that is crustacean active. Further work is also likely to generate three-dimensional structures for additional long toxins, as well as short toxins such as ATX III . At the same time as information on the structure of the ligands is becoming available, progress is also being made in identifying components of the receptor site on the voltage-gated sodium channel . There are already indications that new classes of sea anemone toxins may exist that also act on the sodium channel . Understanding the molecular basis for their activity will provide a new challenge . And finally there is the possibility of therapeutic applications of these proteins if the active pharmacophore(s) can be identified and incorporated into a smaller molecule . Thus, we

Anemone Protein Structure and Function

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can look forward to a continuing output of interesting results on this fascinating class of molecules. dcknowkdgenents-I am indebted to my co-workers R~tus Foci, Auaox GoutD, Lnvnox L~ewEt .t.nv and

Bwnet:r Meaevrr for their helpful discussions and comments on this article, as well as their assistance with the preparation of figures . My own work on these proteins owes much to collaborations with Prof. Bn.t. Kent and Dr LAC71ll Ham, and has been supported by the Australian Research Council and National Health and Medical Research Council. Finally, 1 wish to thank Prof. W. Keel, D. W©te®t and K. WOrEmtcx for communicating results prior to publication.

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Danoor .r., P . C., Ct-oae, G. M ., Beaess, L . and GaorrExeoax, A. M . (1Î89a) A proton nuclear magnetic resonance study of the antihypertensive and andviral protein BDS-I from the sea anemone Atrcmorria sulcata: sequential and stereospecific resonance assignment and secondary structure . Biochemistry 28, 2(78-2187 . Da>scor.r ., P . C., Gaor~xaoax, A. M., Beaess, L. and CLORE, G . M . (1989b) Determination of the threedimenaional solution structure of We antihyperttnsive and antiviral protein BDS-I from the sea anemone Anemonia sulcata: a study using nuclear magnetic resonance and hybrid distance geometry-dynamcal simulated annealing. Biochemistry 28, 2188-2198. Duivw, D . F. (1981) The clownfish sea anemones : Stichodactylidae (Coelenterat~, Actirtiaria) and other sea anemones symbiotic with pomacentrid fishes . Trans. Am . Philos . Soc . 71, 3-115 . Er. Aura, M ., Bwmuorn, E . M., Gaex~r, C ., Hear, L . aad Raca~r, H. (1986) Immunachemistry of sea anemone toxins : structure-antigenidty relationships aad toxin-raxptor interactions probed by antibodies sped& for one antigenic region . Biochemistry 25, 6755-6761 . Foci, R . H ., KEet, W. R. and Noa~rorr, R. S . (1990) Solution structure of neurotoxin I from the sea anemone Stichodactyla helianthus . A nuclear magnetic resonance, distance geometry and restrained molecular dynamics study . J. Biol. Chem . 766, 13016-13028 . Focrr, R . H ., Mweeurr, B. C ., Knd, W . R . and Noa~roN, R. S . (1989) Sequence-specific 'H NMR assignments and secondary structure in the sea anemone polypeptide Stichodactyla helianthus neurotoxin I . Biochemistry 28,1826-1834 . Fowrecir.u-Cwrrrs, J . C., ALMwssv, R . J ., EALICK, S. E ., Sunnw~rrr, F . L ., Wwrr, D. D ., Fer .netwxiv, R. J . and Buac, C . E . (1981) Architecture of scorpion neurotoxins : a class of membrane-binding proteins. TIES 6, 291-296 . Foxrec,~t.u-Cw~trs, J . C ., HwaEasE~x-Rocttwr, C . and RocxwT, H . (1988) Orthorhombic crystals and threedimensional structure of the potent toxin II from the scorpion Androctonus australis Hector . Proc. Nat. Aced. Sci. USA 85, 7443-7447. FaeuN, C., Vtaxe, P., SoHwerTZ, H . and Lwznunrs~cr, M . (1984) The interaction of sea anemone and scorpion newotoxins with tetrodotoxin-resistant Na* channels in rat myoblasts . A comparison with Na* channels in other excitable and non-excitable cells. Mol. Pharmacol. 26, 70-74. GHIASUDDIN, S . M . and Sonear.uNU, D . M . (1985) Pyrethroid insecticides: potent, stereospecific enhamcers of mouse brain sodium channel activation . Pestic . Biochem. Physiol. 24, 200-206 . Goor.ev, P . R ., Beaess, L . and Noa~rox, R . S . (1984a)'H nuclear magnetic resonance spectroscopic study of the polypeptide Toxin I from Anemonia sulcata. Biochemistry 23, 2144-2152. Goor .ev, P. R., Br,urrr, J . W. and Noarox, R. S. (1984b) Conformational heterogeneity in polypeptide cardiac stimuLimts from sea anemones . FEBS Left . 174, 15-19 . GOOLEY, P . R ., BLUNT, J . W., BEREi4, L ., NOaTON, T . R . and Noa~roN, R . S . (1986) Assignment of aromatic resonances in the 'H nuclear magnetic resonance spectra of cardioactive polypeptides from sea anemones. J. Biol. Chem . 7b1, 1536-1542 . GoorEV, P . R., BLUNT, J . W., BERE43, L . and NoamN, R. S. (1988) Effects of pH and temperature on cardioactive polypeptides from sea anemones . A 'H nuclear magnetic resonance study. Biopolymers 27, 1143-1157 . GooLev, P . R . and NoaTON, R . S . (1986) Secondary structure in sea anemone polypeptides . Proton nuclear magnetic resonance study . Biochemistry 25, 2349-2356. GouLn, A . R ., Mweetrrr, H . C. and NORTON, R . S . (1990) Structure-function rolationships in the polypeptide cardiac stimulant anthopleurin-A. Effects of limited proteolysis by trypsin . Eur. J. Btochem. 189, 145-153 . COULD, A. R., Mwaevrr, B. C., t .,,$~~xN, L . E., Gosa, N. H. and NORTON, R . S . (1941) linear and cyclic peptide fragments of the polypeptide cardiac stimulant antopleurin-A. 'H NMR and biological activity (submitted for publication) . Gawr~, D. and (sauo~.uctuu~, C . J . P . (1988) Isolation of < Glu-Gly-Leu-Arg-Trp-NH z (Antho-RW amide In, a novel neuropeptide from sea anemones . FEBS Lett. 239, 137-140. Gaosa, G. J., WwaLT®r, D. C., HeaDSUrv, H . F. and Sü®ATA, S . (1985) Cardiotomic effects of anthopleurin-A lAP-A), a polypeptide from a sea anemone, in dogs with a coronary artery stemosis . Ew. J. Plearnracol. 110, 271-276 . GaueN, L . C . and NoaTON, R . S. (1985) Role of aapartate residues in the cardiac stimulatory activity of anthopleurin-A. Biochem . Intl. 11, 64-76 . Gtn, H. R. and Corm, F . (1990) Pursuing the structure and function of voltago-gated channels. TINS 13, 201-206 . Hw~r~oworr, B ., TANARA, J . C . and Bwacrn, R . L. (1986) Developmental appearance of sodium channel subtypes in rat skeletal muscle cultures. J. Neurochem . 47, 1148-I 153 . ISHIKAWA, Y ., ONODERA, K . and Twrceucrr~, A . (1979) Purification and effect of the newotoxin from the sea anemone Parasicyonis actinostoloides . J. Neurochem . 33, 69-73 . Isn~zwrc~, H ., McKwY, R . H ., NORTON, T . R ., YwsuNOau, K . T., Lee, J . and Tu, A . T . (1979) Conformational studies of peptide heart stimulant anthopleurin-A . J. Biol . Chem . 254, 9651-9656 . KAYANO, T., NODA, M., FLOCrceRZt, H ., TwKAHASH~, H . and Nut~w, S. (1988) Primary structure of rat brain sodium channel III deduced from the cDNA sequence . FEBS Gett . 228, 187-194 .

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Zvxov~, T. A . and

KOZLOVSKAYA, E . P . (19896) Amino acid sequence of neurotoxin I from the xa anemone Radianthus macrodactylus . Bioorg . Khim . 15, 1301-1306, Zvxove, T . A ., Kozwvstcwvr., E . P . and ELYAKOV, G . B. (19ß8a) Amino acid sequence of neurotoxin II from the sea anemone Radianthus macrodactyles. Bioorg . Khim . 14, 878-882. Zvxovr+, T . A ., Kozwvsxwve, E. P. and Etv~xov, G . B . (19ß8b) Amino acid sequence of neurotoxins IV and V from the sea anemone Radianthus macrodactylus. Bloorg . Khim. 14, 1489-1494.

Zncov~, T. A., Vnaoxurtov, L . M ., KOZLAV9tCAYA, E. P. and ELYAICOV, G. B . (1985) Amino acid sequence of neurotoxin III from the sea anemone Radianthus macrodactylus. Bioorg . Khirn. 11, 302-310.