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Eklund, H. & BrandCn, C.4. (1976) Int. Congr. Biochem. Abstr. 10,206 Eklund, H., Nordstrom, B., Zeppezauer, E., Soderlund, G., Ohlsson, I., Boiwe, T., Soderberg, B.-O., Tapia, O., Brandkn, C.-I. & Akeson, A. (1976a) J. Mol. Biol. 102,27-59 Eklund, H., Brandh, C.-I. & Jomvall, H. (19766) J. Mol. Biol. 102,61-73 Krebs, H. A. & Perkins, J. R. (1970) Biochem. J. 118,635-644 Jones, G. M. T. & Harris, J. I. (1972) FEBSLett. 22, 185-189 Jomvall, H. (1970) Eur. J. Biochem. 16,4149 Jomvall, H. (1977) Eur. J. Biochem. 72,425442 Okuda, K. & Takigawa, N. (1970) Biochim. Biophys. Acta 220, 141-148 Rossmann, M. G., Liljas, A..Brandtn, C.-I. & Banaszak, L. J. (1975) Enzyrnes3rdEd. 11,61-102 Schwartz, M., & Jornvall, H. (1976) Eur. J. Biochem. 68, 159-168 Wills, C. & Phelps, J. (1975) Arch. Biochem. Biophys. 167, 627-637

Agonists, Antagonists and Models for Glucocorticoid-Receptor Interactions PHILIP A. BELL Tenovus Institute for Cancer Research, The Welsh National School of Medicine, The Heath, CardiifCF4 4XX, U.K. Although any extensive analysis of the mechanisms of steroid-receptor interaction must await the purification of these receptors, much useful information can be obtained from physicochemical studies with crude preparations or with intact cells. Such techniques have been of particular value in the study of glucocorticoid-receptor interactions. Glucocorticoid target tissues, such as thymus and liver, are characterized by d o s e related physiological responses to a wide range of steroid agonists, including both naturally occurring compounds, such as cortisol and corticosterone, as well as synthetic steroids, such as dexamethasone and triamcinolone acetonide. The responses of these optimal agonists can be diminished, either partially or completely, by a further group of steroids which include progesterone and cortexolone (1 1-deoxycortisol) (Munck & Brinck-Johnsen, 1968; Samuels & Tomkins, 1970). This latter group of steroids have accordingly been classified as antagonists, or at least as partial agonists/antagonists. It is likely that a continuous spectrum of activities exists between the two extremes of optimal agonist and optimal antagonist; nevertheless the existence of these two extremes implies that any model for glucocorticoid action must provide at some point for both ‘active’ and ‘inactive’ states. Glucocorticoid-binding proteins of high affinity and limited capacity have been detected in most target tissues and appear to possess very similar properties. Their identification as receptors rests on the evidence that their dose-saturation responses parallel those of the biological responses, that the inhibition of agonist binding by antagonists parallels the inhibition of the biological response, and on the finding that the concentration of these binding proteins is often decreased in steroid-resistant cell lines. They are readily distinguished from the major plasma binding protein, corticosteroid-binding globulin, by their high affinity for 9a-fluorinated corticosteroids. Little in the way of mechanistic insight has been provided by the physical characterization of these receptors; in common with most cytoplasmic steroid-receptor complexes the glucocorticoid-receptor complexes prepared by incubation of target-tissue cytosol with steroid agonists at 04°C display sedimentation coefficients of about 4 s and 8 s at high and low ionic strengths respectively, when analysed by equilibrium centrifugation in sucrose density gradients (Bell et al., 1975; Beato & Feigelson, 1972; Baxter &Tomkins, 1971). Under conditions approaching physiological ionic strength, I have observed that the glucocorticoid-receptor complex from rat thymus cytosol sediments with an szo, of 6s;this may represent a dimer of the 4 s form, similar to that reported for the oestrogen VOl. 5

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(Chamness & McGuire, 1972) and progesterone (Schrader et at., 1975) receptors. The 4s form of the glucocorticoid-receptor complex from rat liver cytosol has been estimated to possess a mol.wt. of approx. 70000 (Koblinsky et al., 1972). As a consequence of their high rates of dissociation, the characterization of complexes of glucocorticoid antagonists with the receptor by these non-equilibrium techniques isexceedingly difficult; Turnell et al. (1974) have reported, however, that cortexolone yields a 3.5 S complex at both high and low ionic strengths. Whether this pattern of association of protomeric units truly reflects an oligomeric structure or merely an aggregation which is an artifact, and whether the phenomenon has any significance for the mechanism of action of these steroids, remains to be established. A report by Cake et nl. (1 976) suggesting that the interaction of dexamethasone with the rat liver cytosol receptor requires the presence of a low-molecular-weight component which must be lost to permit the steroid-receptor complex to interact with DNA, when taken together with reports that partial purification of the 4s forms of the oestradiol and progesterone receptors prevents their reassociation to the 8 s form (Vonderhaar et al., 1970; Schrader & O'Malley, 1972), may be significant in this respect. Equilibrium and kinetic studies of the interactions of glucocorticoid agonists and antagonists with receptors have proved more informative. Both direct and competitive equilibrium binding studies have, in general, shown good agreement between relative binding affinity and potency for steroid agonists; for the cytoplasmic receptor of rat thymus the relative affinities are in the order: triamcinolone acetonide > dexamethasone > cortisol (Bell & Munck, 1973).Bindingstudieswith triamcinolone acetonide and dexamethasone, both in the presence and absence of steroid antagonists, have failed to reveal any evidence for co-operativity in binding; in all cases the slopes of Hill plots at 50% receptor occupancy were unity. Measurements of the inhibition of [I,2-3H2Jdexamethasone binding by other steroids have revealed that both agonists and antagonists are competitive inhibitors, but suggest that antagonists bind at a second site. Similar conclusions h a w been reached by Suthers et al. (1976) from studies of the effects ofagonists and antagonists on the dissociation of dexamethasone from the glucocorticoid receptor of rat liver. The kinetic parameters of glucocorticoid-receptor interactions have also been studied, and it is clear that the different affinities of various steroid agonists for the receptor are determined largely by their rates of dissociation (Munck et al., 1972). At low steroid concentrations the association is apparently second-order, with a rate constant of the order of 5 x 105-1 x 1061itre.min-'.mol-1 at 0 4 ° C for cortisol, dexamethasone and triamcinolone acetonide (Bell & Munck, 1973), suggesting that the interaction is entropydriven (Munck et al., 1972). More recent studies, using a wider range of steroid concentrations, have shown that association is not a simple second-order process, but instead follows Michaelis-Menten kinetics with a K,,,of the order of lOOnm (Kaine et al., 1975). The association of glucocorticoid antagonists with the receptor does appear to follow second-order kinetics; in addition, these steroids display higher rate constants of association than agonists (Rousseau et al., 1972). Since the association of both agonists and antagonists is competitive, these results suggest that the binding of steroid agonists proceeds via some intermediate state characterized by high rates of association and dissociation, so that equilibrium is reached very rapidly. The high rate of dissociation would ensure that the intermediate state escapes detection by the usual disequilibrium methods of analysis. In contrast, antagonists would bind only to the intermediate state, and with a higher affinity than agonists. This intermediate state may well correspond to the second site detected by competitive binding studies. Two models compatible with much of the binding data have been proposed. One, proposed by Pratt el al. (1975), is formally similar to the induced-fit model of Koshland & Neet (1968); the other, applied to steroid-receptor interactions by Rousseau et al. (1972), is essentially the allosteric model of Monod et al. (1965). Both are basically static two-state models which explain the behaviour of partial agonists as being due to the simultaneous presence of both states. Both are capable of providing an explanation for the low apparent second-order rate constant of association for steroid agonists 1977

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which, as Kaine et al. (1975)have pointed out, is too low to reflect a diffusion-limited process. If the intermediate binding state does indeed correspond to the second binding site detected by competitive equilibrium binding, then both models require expansion to allow for the simultaneous binding of agonists and antagonists. The fact that either model provides for the existence of two forms of the steroidreceptor complex suggests that the site of discrimination between agonists and antagonists is at the level of the interaction of the steroid with the cytoplasmic receptor. This implies that the discrimination should be apparent at all subsequent stages of the response. A feature of glucocorticoid target tissues, as of most steroid-responsive systems, is that the primary steroid-receptor complex formed at low temperatures displays only a low affinity towards nuclear binding sites, but this affinity is markedly increased after an activation process which is dependent on temperature and ionic strength (Munck & Wira, 1971 ;Higgins et al., 1973;Kalimi et al., 1975). This activation process is apparently first-order and yields a monomolecular product, and therefore probably represents a conformational change in the complex (Atger & Milgrom, 1976). Rousseau et al. (1973) have reported that, in hepatoma tissue-culture cells, complexes of the receptor with the antagonist progesterone, unlike those with agonists such as dexamethasone, do not leave the cytosol and bind to nuclear sites, and therefore presumably do not undergo activation. Contrary findings, however, have been reported for rat thymus cells, in which complexes of the receptor with the antagonist cortexolone d o appear to exhibit a temperature-dependent transfer to the cell nucleus (Turnell et al., 1974;Wira & Munck, 1974). Unlike the nuclear agonist-receptor complexes, however, the nuclear antagonist-receptor complex can be extracted from the nuclei with media of low ionic strength, suggesting that it is less tightly bound (Turnell et al., 1974). If these observations with rat thymus cells can be confirmed by the direct demonstration of a temperature-dependent conformational change with antagonist-receptor complexes, then models which impose a clear-cut distinction between two forms of steroid-receptor complex, such as those described previously, may prove inadequate. A multistate model which reflects more closely the dynamic nature of molecular structure has been proposed by Munck & h u n g (1977), and may well represent a closer approximation to the actual situation. In this model each steroid produces a conformational change that differs in degree from that produced by another steroid. A prediction of this model that distinguishes it from the two-state models is that partial agonist-receptor complexes should exist in a conformation intermediate between those of agonist- and antagonist-receptor complexes, rather than as a mixture of the two. I am grateful to the Tenovus Organisation for continuing financial support.

Atger, M. & Milgrom, E. (1976)J. Biol. Chem. 251,4758-4762 Baxter, J. D.& Tomkins, G. M. (1971)Proc. Natf. Acud. Sci. U.S.A. 68,932-937 Beato, M.& Feigelson, P. (1972)J. Biof. Chem. 247,7890-7896 Bell, P. A. & Munck, A. (1973)Biochem. J. 136,97-107 Bell, P. A., Rees, A. M. & Sloman, J. C. (1975)Acta Endocrinol.(Copenhagen)Supl. 199,245 Cake, M. H., Goidl, J. A., Parchman, L. G. & Litwack, G. (1976)Biochem. Biophys. Res. Comntun. 71,45-52 Chamness, G. C. & McGuire, W. L. (1972)Biochemistry 11,2466-2472 Higgins, S . J., Rousseau, G. G., Baxter, J. D. & Tomkins, G. M. (1973)J. Biol. Chem. 248, 5866-5872 Kaine, J. L., Nielsen, C. J. & Pratt, W. B. (1975)Mol. Pharmacof. 11, 578-587 Kalimi, M.,Colman, P. & Feigelson, P. (1975)J. Biol. Chem. 250, 1080-1086 Koblinsky, M., Beato, M., Kalimi, M. & Feigelson, P. (1972)J.Biol. Chem. 247,7897-7904 Koshland, D.E. & Neet, K. E. (1968)Annu. Rev. Biochem. 37, 359410 Monod, J., Wyman, J. & Changeux, J.-P. (1965)J. Mol. Biol. 12, 88-118 Munck, A. & Brinck-Johnsen, T. (1968)J. Biol. Chem. 243, 5556-5565 Munck, A. & Leung, K. (1977)in Receptors and Mechanism of Action of Steroid Hormones (Pasquahi, J. R., ed.), Marcel Dekker, Basel, in the press Munck, A. & Wira, C. (1971)Adv. Biosci. 7, 301-330 Munck, A., Wira, C., Young, D. A., Mosher, K. M., Hallahan, C. &Bell, P.A. (1972)J.Steroid Biochem. 3,567-578

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Pratt, W. B., Kaine, J. L. & Pratt, D. V. (1975) J. Biol. Chem. 250,4584-4591 Rousseau, G. G., Baxter, J. D. & Tomkins, G. M. (1972) J. Mol. Biol. 67,99-115 Rousseau, G. G., Baxter, J. D., Higgins, S. J. & Tomkins, G. M. (1 973)J. Mol. Biol. 79,539-554 Samuels, H. H. & Tomkins, G. M. (1970) J. Mol. Biol. 52, 57-74 Schrader, W. T. & O’Malley, B. W. (1972) J. Biol. Chem. 247,51-59 Schrader, W. T., Heuer, S. S. & O’Malley, B. W. (1975) Biol.Reprod. 12, 134-142 Suthers, M. B., Pressley, L. A. & Funder, J. W. (1976) Endocrinology 99, 26@-269 Turnel1,R. W.,Kaiser,N., Milholland,R. J. &Rosen,F.(1974)J. Biol. Chem.249,1133-1138 Vonderhaar, B. K., Kim, L‘. H. & Mueller, G. C. (1970) Biochim. Biophys. Acta215,125-133 Wira, C. R. & Munck, A. (1974) J. Eiol. Chem. 249, 5328-5336

STRUCTURE AND FUNCTION OF CONSECUTIVE GLYCOLYTIC ENZYMES : a Colloquium organized on behalf of the Molecular Enzymology Group by J. J. Holbrook (Bristol), R. N. Perham (Cambridge) and J. E. Fothergill (Aberdeen) An Analysis of the Three-Dimensional Structure of Chicken T r i m Phosphate Isomerase D. C. PHILLIPS, P. S. RIVERS, M. J. E. STERNBERG, J. M. THORNTON and I. A. WILSON Laboratory of Molecular Biophysics, Department of ZooIogy, South Parks Road, Oxfv-d OX1 3PS, U.K. The three-dimensional structure of chicken triose phosphate isomerase ( D - ~yceraldehyde I 3-phosphate ketol isomerase; EC 5.3.1 .l)has been determined at 0.25nm (2.5A)resolution by X-ray-crystallographic methods (Banner et a/., 1975, 1976). Triose phosphate isomerase catalyses a crucial isomerization in the Embden-Meyerhof-Parnas pathway of glycolysis. The interconversion of dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate is catalysed by the dimer with no requirement for cofactors, metals or activators. The equilibrium of the reaction in oitro lies in the direction of dihydroxyacetone phosphate (96%). However, in uioo, a constant flux through the glycolytic pathway is generally maintained by removal of glyceraldehyde 3-phosphate. The structure was determined by the method of multiple isomorphous replacement (Blow &Crick, 1959)incorporating the use of anomalous-scattering information (North, 1965). Five heavy-atom derivatives were used. Three of these were mercurials in which the mercury bound mainly to the free thiol groups of cysteine-217 on the enzyme. The other two were prepared with chloroplatinite which bound near to histidine-26 and lysine-54. The electron-density map of the dimer of 53000 daltons was interpreted in terms of the known amino acid sequence (Furth et a/., 1974), and 247 residues were placed in each subunit. Structure The triose phosphate isomerase structure contains a high percentage of secondary structure: 55 % a-helix, 23 % &sheet and 3 % 1-bends. Eight strands of 8-sheet, varying in length from four to nine residues, run parallel to each other. Each successive strand is laid down consecutively as it occurs in the sequence, a, h, c . . . h, and the final strand h makes hydrogen-bonds with both strand g and strand a to complete the cylindrical 1977