Crystal structures of two intensely sweet proteins

"FIBS 13 - January 1988 E. H. (1986) IRCS Med. Sci. 14, 701-702 20 Widdicombe, J. H. (1986) Am. J. Physiol. 251, R818-R822 21 Cotton, C. U., Stutts, M. J., Knowles, M. R., Gatzy, J. T. and Boucher, R. C. (1987)J. Clin. Invest. 79, 80-85 22 Welsh, M. J. and Liedtke, C. M. (1986) Nature 322, 467-470 23 Frizzell, R. A., Rechkemmer, G. and Shoemaker, R. L. (1986) Science 233, 558-560 24 Pedersen, P. S., Larsen, E. H and Brandt,

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N. J. (1987)Med. Sci. Res. 15, 151-152 25 Yankaskas, J. R., Knowles, M. R., Gatzy, J. T. and Boucher, R. C. (1985)Lancet i, 954956 26 McPherson, M. A., Dodge, J. A. and Goodchild, M. C. (1983)Clin. Chim. Acta 135,181188 27 Berridge, M. J. (1987) in Calcium Regulation and Bone Metabolism : Basic and ClinicalAspects, Vol. 9 (Cohn, D. V., Martin, T. J. and

Meunier,P. J., eds), pp. 8-15, Elsevier

Crystal structures of two intensely sweet proteins Sung-Hou Kim,Abraham de Vos and Craig Ogata Three-dimen~,o, ud ~ o f two intensely sweet proteins (thaumatin and monellin) have been determined by X-ray crystallographic m e ~ d s . Despite their resemblance in taste, the two proteins have no s i ~ t similarities in ~ i r crystal structures or amino acid sequences; and yet the two proteins are immunologically cross-reactive.

The sweetest natural molecules known to man are two plant proteins, thaumatin and monellin. They were isolated from the fruits of two African plants, katemfe (Thaumato~tt'us dan/e/Ti Benth) l and serendipity berries (Dioscoreophyllum acmmins// Diels) 2,3, respectively. Thaumatin is a 207 residue single chain protein, and monellin is made up of two polypeptide chains, one 44 and the other 50 amino acids long. Both proteins contain many basic residues, but neither has carbohydrates or modified amino acids. These two unusual proteins possess the interesting property of having a very high specificity for the sweet taste receptors. They are approximately 100 000 times sweeter than sugar on a molar basis and several thousand times sweeter on a weight basis4. Several interesting observations have been made about the two proteins: (1) native conformations are important for the sweet taste; (2) thaumatin contains 16 cysteines forming eight disulfide bonds, but monellin has only one cysteine with no disulfide bond; (3) although both proteins are intensely sweet, there are no statistically signifi~ cant sequence homologies and only five homologous tripeptide sequences between the moleculesS; (4) despite the S-H. Kim, A. de Vos and C Ogata are at the Department of Chemistry, Un~ersity of Ca~fomia, Berkeley, CA 94720, USA. C. Ogata is currently at the Department of Biochemistry and Biophysics, College of Physicians and Sw'geons, Cohanbia University, New York, N Y 10032, USA.

absence of sequence homology, antibodies rai~d against thaumatin competed for monellin as well as many other sweet compounds 6,7, but chemically modified non-sweet monellin did not7; (5) a reciprocal experiment showed that antibodies raised against monellin also cross-reacted with thaumatin s. Most sweet-tasting molecules are small compounds with a relatively low specificity for the sweet taste receptor. The two unique exceptions are thaumatin and monellin, which can be perceived as sweet at a concentration as low as 10-s M, indicating a specificity for the receptor comparable to that found in hormone-receptor stu~ii~a,

Three-dimensional.structures The crystal structures of thaumatin and monellin have been determined at 3 A resolution by X-ray crystallographic methods 9,10. The schematic drawings of the structures are shown in Fig. 1. The thaumatin structure can be considered as composed of three structural 'domains'. The largest domain is a flattened ~-barrel formed by 11 anti-parallel [5-strands, each strand being six residues long on average. Attached to this domain are two 'finger' domains with many loops closed by disulfide bonds. Monellin on the other hand is very compact without large, flexible loops. It has one five stranded, anti-parallel [l-sheet made of two strands from the B chain and the remaining three from the A chain. Each strand is ten residues long on average. The ~-sheet is twisted in the same

28 Doughney, C., Pedersen, P. S., McPherson, M. A. and Dormer, R. L. (1987) in Pediatric Pulmonology Suppl. 1, 119 (Abst. 45) 29 Scambler, P. J., McPherson, M. A., Bates, G., Bradbury, N. A., Dormer, R. L. and Williamson, R. (1987) Hum. Genet. 76, 278-282 30 McPherson, M. A., Dormer, R. L., Bradbury, N. A., Shod, D. K. and Goodchild, M. C. in The Cellular and Molecular Basis of Cystic Fibrosis (Mastella, G. and Quinton, P. M., eds),

San FranciscoPress(in press)

handed sense as observed in [3-sheets of many other proteins. The only a-helix nestles in the gently twisted concave side of the I~-sheet. These 3/~ structures provide explanations for the unusual stability of the proteins. Thaumatin's thermal stability is probably due to the extensive network of eight disulfide bonds stabilizing otherwise flexible loops in the structure. The fact that the two chains of monellin can only be separated by very strong denaturation can be explained by the extensive hydrogen-bonding between the two chains. This strong/nterchain ~-sheet is further stabilized by hydrophobic interactions between one side of the ahelix and the concave side of the [I-sheet. Structural comparison Despite the fact that both proteins share the unusual properties of being sweet and immunologically cross-reactive, it is clear that there is no significant homology either in amino acid sequence or in the three-dimensional backbone structures. The structural features responsible for the sweet taste of these proteins and for the immunological cross-reactivity must thus be found at a local level. An examination of the amino acid sequences 5,11.12 of both proteins show that there are five pairs of homologous tdpeptide sequences in the two proteins. Even though the tripeptide homologies are statistically not significant, the immunological cross-reactivity implies the existence of common chemical and structural features, and the possibility exists that a joint site made of several of these short homologous regions may act as an antigenic determinant. For these reasons, the homologous tripeptides have been the target of many biochemical studies and speculations. The five tripeptides in thaumatin located at residues 94-96, 100-102, 101103, 118-120 and 128-130 (labelled 1 to 5 in Fig. lb), have homologous sequences at residues B28-30, B6-8, A22-24, A29-31 and A21-23 in monellin (also labelled 1 to 5 in Fig. l a) respectively. Conformations of three of the five (~) 1988.ElsexierPublicationsCambridge 0376- 51~67/88/51)2.(1l

TIBS 1 3 - January 1988

14

A

N

(a) Monellin

(b) Thaumatin

Fig. I. (a) Schematic drawing of the backbone structure of monellin. The tubed sections indicate regions with tripeptide sequence homologies with thaumatin. Each hc,mologous tripeptide pair ar,o identified by the same number as in (b). The dashed lines indicate disordered residues that could not be seen in the electron density maps. (b) The backbone structure of thaumatin. Each [l-strand is shown as a long dab with arrow indicating the polarity of the peptide chain from amino- to tarboxy-terminus. The numbered'sleeves' are the regions of sequence homology with monellin.

pairs, regions 1,4, and 3 of monellin, are found to be topologically similar to their counterparts in thaumatin at the current resolution of 3 ,~, i.e. the former two are located in looped regions and the third in a 13-strand. Of these, region 3 was not considered to be a cross-reacting antigenie site because only the side chain of one out of the three residues points toward the exterior of the protein. This leaves only regions I and 4 of monellin, similar to looping regions 1 and 4 of thaumatin, respectively. Conclusions

Three conclusions can be drawn. First, despite the fact that both proteins are intensely sweet, there is no similarity in the overall backbone structures of the proteins. Second, at the local level, monellin and thaumatin have only two small regions (both tripeptides), located in exposed, looping regions, sharing the same sequence and topology. According to the conventional notion, each of these regions is too small by itself to be the complete antigenic determinant. Third, since the locations of these two regions are far apart, we can rule out the possibility that the two tripeptides jointly form a single antigenic site. The only remaining possibility is that several smaller regions (shorter than three residues), with or without one of the two above tripep-

tides, jointly form the common antigenic site. What is the molecular basis of sweet taste?

Taste is one of two chemical senses (senses initiated by chemical compounds), the other being smell. The first step in the process that results in the perception of a taste sensation is the interaction of the sweet compound with its taste receptor. This interaction produces membrane potential changes in the cells that carry the receptors, resulting in electrical signals that are carded to the brain by special taste nerves 13. Four primary taste qualities are generally recognized in man: sour, salt, bitter and sweet. In the case of sour and salt, the relationship between the structure of the stimulus and the taste sensation it elicits is relatively well understood 14, sour taste being primarily determined by the pH of the solution involved, and salty taste by the nature of the salt in the solution tasted. Bitter and sweet taste sensations, however, can be elicited by a wide range of chemicals. Presumably the precise three-dimensional structure of the molecule involved determines the quality of the sensation, a well-known example being the difference between the bitter L- and the sweet D-form of several amino acids. However, what struc-

tural elements are recognized by sweet or bitter taste receptors are not known at the present time. As for sweet (or bitter) taste activity, there is no in-vitro assay, and no taste receptor molecule has yet been isolated. However, the in-vitro immunological cross-reactivity may cast some light on taste activity. The possible relevance is suggested by the facts that (1) the antibodies of one protein cross-react to the other and vice versa6-S; (2) antibodyprotein complexes lose their ability to elicit sweet taste (van tier Wel and Kim, unpublished); (3) other sweet compounds such as aspartame compete for the same antibodies but non-sweet aspartame derivatives do not7; (4) the cross-adaptation of monellin and thaumatin in human taste experiments 15 and electrophysiologicai experiments suggest that they are recognized by the same receptor; (5) since antibody binding sites and receptor binding sites are likely to be exposed, one may be a subset of the other. At present these are the only two proteins with intense taste activity. Biochemical and immunological studies are in progress to identify the regions which may be responsible for the antibody cross-reactivity and the sweet taste receptor binding. High resolution refinement of both proteins is also in progress

TIBS 13 - January 1988 to define the precise conformation of each side chain. These: two protein structures may provide the structural basis for understanding the sweet taste receptor interaction and antibody cross-reactivity without sequence homology. They can also be the starting point for protein engineering to design protein sweeteners with better physical and chemical properties than the natural counterparts. Acknowledgements The work described in this article has been supported by the National Institutes of Health (NS15174) and the NutraSweet Co., Mount Prospect, IL.

15 References

1 van der Wel, H. and Loeve, K. (1972)Eur. J. Biochem. 31,221-225 2 Monis,J. A. and Cagan, R. H. (1972)Biochim. Biophys. Acta261,114-122 3 van der Wel, H. (1972)FEBS Lett. 21, 88-90 4 van der Wel, H. (1980) Trends Biochem. Sci. 5, 122-123 5 lyengar,R. B., Smits,P., van der Ouderaa, F., van der Wel, H., van der Browershaven, J., Ravenstein, P., Richters, G. and van Wassenaar, P. D. (1979) Eur. J. Bioehem. 96, 193-204 6 Hough,C. A. M. and Edwardson,J. A. (1978) Nature 271,381-383 7 van der Wel, H. and Bel, W. J. (1978) Chem. SensesFiavour3, 99-104 8 Kang,C. H. (1987)DoctoralThesis, University

Aluminum ion in biological systems Timothy L. Macdonald and R. Bruce Martin Aluminum ion has been proposed to be a.factor contributing to the toxicily of aquatic acidif~tion caused by acid rain, and to the etiology o f a variety of neurological and skeletal disorders in man. The biological processes and molecular mechanisms that underlie lhese palhological processes are begbming to be ~ . This review o ~ the current state of our knowledge concerning the s i ~ factors a s s ~ ~ ~ ion in biological systems. Aluminum, bound as oxides and complex aluminosilicates, is the most abundant metal in the earth's crust. However, surface water concentrations of aluminum ion (AP +) have until recently remained minimal due to the insolubility of aluminium hydroxide complexes at neutral pH (Refs 1 and 2). The acidification of surface waters through acid precipitation dramatically releases Al3+ from mineral stores. As illustrated in Fig. 1, the mole fraction of free Al3+ (relative to total aluminium ion species) varies from <10 -6 at pH = 7) to 1 at pH = 4 and thus even small shifts in pH within this narrow range profoundly affect Al3+ activityl. Living organisms, having developed at pH ranges around 7, evolved without the ability to cope with high Al3+ activity and consequently the toxicity of aquatic acidification may be largely attributable to the biological effects of aluminum ion 3. Concern over the appearance of high Al3+ concentrations in acidified waters has been enhanced by the unequivocal demonstration of an association of

T. L. Macdonald and R. B. Marlin are at tlw Department of Chemistry, University of Virginia, McCormick Road, Charlottesville, VA 22901, USA.

A P + with several pathological states in man 4-6. Evidence has accumulated that A P + is the etiologic agent in an encephalopathy and a type of osteomalacia observed in patients with chronic renal failure on long-term hemodialysis 2,4-6. In addition, AP + has been impficated in the pathogenesis of the anemia and metastatic calcification noted in hemodialysis patients. Aluminum ion has also been proposed as a contributor to the pathogenesis of several neurologic disorders which appear to have an environmental etiologyT, 8. High A P + concentrations have been identified in the nuclear region of neuro4ibrillary tangle-bearing neurons of the hippocampns in brain tissue obtained from patients with Alzheimer's disease; it is uncertain whether aluminium ion has an affinity for these abnormal neural regions or possesses an etiologic relationship with the disease 9,1~. The biochemical processes and the molecular mechanisms through which Al3+ may exert its toxicity, paricularly its potent and often selective neurotoxicity, are not well understood. However, there are studies on the levels of total AP + in normal and toxic states in humans and a variety of studies on experimental animals which encourage studies at the

of California, Berkeley 9 de Vos, A. M., Hatada, M., van der Wel, H., Krabbendam. Y~., Peerdeman,A. F. and Kim, S-H. (1985) 7:~oc.NatlAcad. Sci. USA 82,14061409 10 Ogata, C., Hatada, M., Tomlinson, G., Shin, W-C. and Kim, S-H. (1987) Nature 328, 739742 11 Hudson, G. and Biemann, K. (1976)Biochem. Biophys. Res. Commun. 71,212-220 12 Frank, G. and Zuher, H. (1976)Hoppe-Seyler's Z. Physiol. Chem. 357,585-592 13 Sato, T. and Beidler, L. M. (1975)J. Gen. Physiol. 66, 735-763 14 Kurihara, Y. J. (1973)Syn. Org. Chem. (Jpn) 31,900--905 15 van der Wel, H. and Arvidson, K. (1978) Chem. SensesFlavour3, 291-297

molecular level. This review will address briefly our knowledge of the biochemical and molecular mechanisms that may underlie AP+-mediated toxicity. The chemistry of aluminum ion

¢omplexalion The behavior of AIa+ species in cells and biological fluids can be described by four different ligand-states or forms: as 'free' or mononuclear ions; as low molecular weight complexes; as reversible macromolecular complexes; and as irreversible macromolecular complexes. A brief summary of each form will precede a discussion of the nature and relevance of these components to AP+-mediated toxicity. Understanding the state of A P + in any aqueous system demands awareness of the species that AP + ('free' ions) forms at different pH values with the components of water, regardless of other ligands that may be present. In solutions more acidic than pH = 5, AP + exists as the octahedral hexahydrate, AI(H20)63+, often abbreviated as Al3+ (with the convention employed herein being 'free AP+'). As a solution becomes less acid, AI(H20)63+ undergoes successive deprotonations to yield AI(OH) 2+ and AI(OH)2 +. Neutral solutions give an AI(OH)3 precipitate that redissolves in basic solutions, due to formation of tetrahedral AI(OH)4-,, Polynuclear species of time-dependent composition may also form. At pH = %4 virtually all the soluble aluminum ion occurs as AI(OH)4-; the molar ratio of [AI(OH)4-]/[AP+] = 2.5 x 1'06 (Ref. 1). The amounts of AP + and AI(OH)4- in solution are= limited by the solubility of AI(OH)3. 'Unless a solution is supersaturated with respect to amorphous AI(OH)3, greater than nanomolar concentrafions of free AP + in neutral solutions are unattainable I. For example, 1988. Elsevier PublicationsCambn~dge 0376- 5067/88/$02.00