rosfr UWl
Y 'm_m_u_n_O_'O_9Y_T
Structure
r t
COOH
cooh cooh
Fig, 3. ConselVed sequence patterns in X-ray and predicted carboxy-terminal helices in a subgroup of cytokine structures. (a) Helix Dis the probable receptor-binding structure. Boxed, upper case residues indicate amino acids that are surface exposed in the GRH structure, lower case residues mark amino acids that pack in the bundle interior34 Bold letters distinguish positions of conselVed chemical character (@ is hydrophobic, +1- is basiclacidic, # is polar). Above the sequence alignment, asterisks mark specific residues in helix Dwhose mutation to Ala strongly affected binding to the GRH receptor53, 70 Residue numbers center on the most strongly neutralizing position (number 0) and count up (+) and down (-) from that amino acid. (b) The exposed surface ofhelix D. In lieu of a helical net, amino acidside chains are represented as spheres in helical progression 47 ,42 These spheres are numbered as in (a), showing that the boxed residues form an extended patch on the surface of helix D. Darker shaded spheres distinguish the neutralizing residues, other amino acids depicted as light shaded spheres are in a position to influence binding.
Implications for protein design Most of the protein fold of a helical cytokine would serve an essential function as a structural scaffold for presenting the recognition helix to the receptor. Bioactivity of the cytokine may be maintained by judicious alteration of the structure that does not disturb the receptor-binding epitope. For example, Landgraft et al. 66 have engineered a more stable, semi-synthetic version of IL-2 by incrementing the amphiphilicity of one of the core helices of the 4-a helix bundle. Conceivably, this approach could extend to the wholesale grafting of a receptorbinding helix to an engineered, host protein fold. At the other extreme, 'minimal' cytokines that retain a fair level of bioactivity could be equivalent to an amphiphilic helical peptide. (This is a functional strategy that is apparently employed by a class of small hormone-like proteins67 .) Bioactive helices that are stable in solution could be linked to form higher aggregates that exist as 2-a helix hairpins, 2-a/2-a or 4-a bundles68 , novel molecules that may be particularly effective in generating cytokine-neutralizing anti bod ies69 . The (convergent) similarity of certain key cytokine residues within the aligned recognition helices of Fig. 3(b) suggests a parallel similarity of binding sites in the set of cognate receptors. Progressive replacement of helix residues (for example in the conversion of PRL to a GRH-like cytokine 70 ) may probe individual amino acid contributions to specific binding.
Conclusion, insights and future directions The parallel analyses of receptor and cytokine structures bear several conclusions. (1) Haemopoietic receptors share a common binding fold that can be classified by sequence similarity into two divergent classes. (2) Cytokine structures are generally rich in a-helices; ad-
ditionally, subsets of helical cytokines (for example GRH/ PRL, EPO and IL-6/MGF/G-CSF) may adopt a similar tertiary fold. (3) The shared binding domains of haemopoietic receptors are proposed to feature 'rigid' troughs complementary in topography to a preferred cytokine (sub)structure; in complicity, experimental evidence implicates a prominent a-helix in diverse cytokine receptorbinding epitopes. (4) High-affinity binding may favor the use of ancillary receptors that recognize dissimilar cytokine epitopes and perhaps also participate in signal transduction. The teleological framework for receptor-cytokine interactions suggests an intrinsic degeneracy within the haemopoietic regulatory system. The structurally related binding receptors, ligands with convergent recognition elements and accessory membrane proteins, are proposed to form a dynamic pool of interacting molecules; echoing Sporn and Roberts 71 , the collective synergistic and opposing actions of subsets of these proteins could then integrate into discrete mitogenic signals for cell growth and differentiation. The wide range of pleiotropic and redundant activities of haemopoietic cytokines l - 5 may just be the most obvious manifestation of this intriguing functional strategy. A detailed analysis of class 1-2 receptor folds has recently revealed that the shared =200 amino acid binding segments are composed of two structurally similar (13strand rich) =100 residue modules; a binding crevice for cytokines is predicted to form between linked l3-barrel folds 22 . The receptor subunits are in turn predicted to be related to more economical modules called fibronectin type III domains22 . This link between primitive adhesive structures and sophisticated binding receptors is analogous to the structural kinship between primitive Ig-like adhesive molecules and antibody frameworks 72 . Accord-
353
rosfr Ullt ingly, an understanding of the evolutionary emergence of distinct receptor types (with emphasis on the phylogeny of class 1 molecules) may shed some light on the parallel development of blood cell types and, in particular, on the evolution of a regulatory network controlling the haemopoietic cell hierarchy. This work was sustained in large part by a postdoctoral fellowship from the Alfred P. Sloan Foundation. The support of Tina S. Bazan is also gratefully acknowledged.
References 1 Metcalf, D. (1989) Nature 339, 27-30 2 Nicola, NA (1989) Annu. Rev. Biochem. 58,45-77 3 Balkwill, F.R. and Burke, F. (1989) Immunol. Today 10, 299-304 4 O'Garra, A, Umland, S., DeFrance, T. and Christiansen, J. (1988) Immunol. Today 9, 45-54 5 Paul, WE. (1989) Ce1/57, 521-524 6 Bazan, J.F. (1989) Biochem. Biophys. Res. Commun. 164, 788-796 7 Carpenter, G. (1987) Annu. Rev. Biochem. 56,881-914 8 D'Andrea, AD., Fasman, G.F. and Lodish, H.F. (1989) Cel/ 58, 1023-1024 9 Mosely, B., Beckmann, P., March, e.J et al. (1989) Ce1/59, 335-348 10 Gearing, D.P., King, JA, Gough, N.M. and Nicola, NA (1989) EMBO 1. 8, 3667-3676 11 Itoh, N., Yonehara, S., Schreurs, J. et al. (1990) Science 247,324-327 12 Goodwin, R.G., Friend, D., Ziegler, S.F. et al. (1990) Cel/ 60,941-951 13 Fukunaga, R., Ishizaka-Ikeda, E., Seto, Y. and Nagata, S. (1990) Ce1/61, 341-350 14 Saltzman, E.M., Luhowskyj, S.M. and Casnellie, H. (1989) 1. BioI. Chem. 264, 19979-19983 15 Hatakeyama, M., Mori, H., Doi, T. and Taniguchi, T (1989) Ce1/59, 837-845 16 Taga, T., Hibi, M., Hirata, Y. et a!. (1989) Ce1/58, 573-581 17 Saragovi, H. and Malek, T.R. (1990) Proc. Natl Acad. Sci. USA 87, 11-15 18 Opdenakker, G., Cabeza-Arvelaiz, Y. and Van Damme, J. (1989) Experientia 45, 513-520 19 Langer, JA and Pestka, S. (1988) Immunol. Today 9, 393-400 20 Bazan, J.F. Ce1/61, 753-754 21 Nemerson, Y. (1988) Blood 71, 1-8 22 Bazan, J.F. Proc. Natl Acad. Sci. USA (in press) . 23 Schrader, J.W, Ziltener, H.J. and Leslie, K.B. (1986) Proc. Natl Acad. Sci. USA 83, 2485-2489 24 Leutz, A, Damm, K., Sterneck, E. et al. (1989) EMBO 1. 8, 175-181 25 DeGrado, WF., Wasserman, Z.R. and Chowdhry, V. (1982) Nature 300, 379-381 26 Cohen, F.E., Kosen, P.A., Kuntz, I.D. et al. (1987) Science 234,349-352 27 Wingfield, P., Graber, P., Moonen, P., Craig, S. and Pain, R.H. (1988) Eur. 1. Biochem. 173, 65-72 28 Windsor, W, Syto, R., Nagabhushan, T.L., Trotta, P.P. and Le, HV (1988) FASEB 1. 2, A 1337 29 Chou, C-C, Witherspoon, S.M., Carter, J.M. et al. (1990) FASEB 1. 4, A2068 30 Lai, P-H., Everett, R., Wang, F-F., Arakawa, T. and Goldwasser, E. (1986)1. BioI. Chem. 261, 3116-3121 31 Lu, H.S., Boone, T.e., Souza, L.M. and Lai, P-H. (1989) Arch. Biochem. Biophys. 268,81-92 32 Williams, R.W (1985) 1. BioI. Chem. 260, 3937-3940 33 Brandhuber, B.J., Boone, T., Kenney, W.e. and McKay, D.B. (1987) Science 238, 1707-1709 . 34 Abdel-Meguid, S.S., Shieh, H-S., Smith, WW et a!' (1987)
354
Y 'rn_rn_u_n_O'_09_Y_T
Y 'm_m_u_n_O_'O_9Y_T
Structure
r t
COOH
cooh cooh
Fig, 3. ConselVed sequence patterns in X-ray and predicted carboxy-terminal helices in a subgroup of cytokine structures. (a) Helix Dis the probable receptor-binding structure. Boxed, upper case residues indicate amino acids that are surface exposed in the GRH structure, lower case residues mark amino acids that pack in the bundle interior34 Bold letters distinguish positions of conselVed chemical character (@ is hydrophobic, +1- is basiclacidic, # is polar). Above the sequence alignment, asterisks mark specific residues in helix Dwhose mutation to Ala strongly affected binding to the GRH receptor53, 70 Residue numbers center on the most strongly neutralizing position (number 0) and count up (+) and down (-) from that amino acid. (b) The exposed surface ofhelix D. In lieu of a helical net, amino acidside chains are represented as spheres in helical progression 47 ,42 These spheres are numbered as in (a), showing that the boxed residues form an extended patch on the surface of helix D. Darker shaded spheres distinguish the neutralizing residues, other amino acids depicted as light shaded spheres are in a position to influence binding.
Implications for protein design Most of the protein fold of a helical cytokine would serve an essential function as a structural scaffold for presenting the recognition helix to the receptor. Bioactivity of the cytokine may be maintained by judicious alteration of the structure that does not disturb the receptor-binding epitope. For example, Landgraft et al. 66 have engineered a more stable, semi-synthetic version of IL-2 by incrementing the amphiphilicity of one of the core helices of the 4-a helix bundle. Conceivably, this approach could extend to the wholesale grafting of a receptorbinding helix to an engineered, host protein fold. At the other extreme, 'minimal' cytokines that retain a fair level of bioactivity could be equivalent to an amphiphilic helical peptide. (This is a functional strategy that is apparently employed by a class of small hormone-like proteins67 .) Bioactive helices that are stable in solution could be linked to form higher aggregates that exist as 2-a helix hairpins, 2-a/2-a or 4-a bundles68 , novel molecules that may be particularly effective in generating cytokine-neutralizing anti bod ies69 . The (convergent) similarity of certain key cytokine residues within the aligned recognition helices of Fig. 3(b) suggests a parallel similarity of binding sites in the set of cognate receptors. Progressive replacement of helix residues (for example in the conversion of PRL to a GRH-like cytokine 70 ) may probe individual amino acid contributions to specific binding.
Conclusion, insights and future directions The parallel analyses of receptor and cytokine structures bear several conclusions. (1) Haemopoietic receptors share a common binding fold that can be classified by sequence similarity into two divergent classes. (2) Cytokine structures are generally rich in a-helices; ad-
ditionally, subsets of helical cytokines (for example GRH/ PRL, EPO and IL-6/MGF/G-CSF) may adopt a similar tertiary fold. (3) The shared binding domains of haemopoietic receptors are proposed to feature 'rigid' troughs complementary in topography to a preferred cytokine (sub)structure; in complicity, experimental evidence implicates a prominent a-helix in diverse cytokine receptorbinding epitopes. (4) High-affinity binding may favor the use of ancillary receptors that recognize dissimilar cytokine epitopes and perhaps also participate in signal transduction. The teleological framework for receptor-cytokine interactions suggests an intrinsic degeneracy within the haemopoietic regulatory system. The structurally related binding receptors, ligands with convergent recognition elements and accessory membrane proteins, are proposed to form a dynamic pool of interacting molecules; echoing Sporn and Roberts 71 , the collective synergistic and opposing actions of subsets of these proteins could then integrate into discrete mitogenic signals for cell growth and differentiation. The wide range of pleiotropic and redundant activities of haemopoietic cytokines l - 5 may just be the most obvious manifestation of this intriguing functional strategy. A detailed analysis of class 1-2 receptor folds has recently revealed that the shared =200 amino acid binding segments are composed of two structurally similar (13strand rich) =100 residue modules; a binding crevice for cytokines is predicted to form between linked l3-barrel folds 22 . The receptor subunits are in turn predicted to be related to more economical modules called fibronectin type III domains22 . This link between primitive adhesive structures and sophisticated binding receptors is analogous to the structural kinship between primitive Ig-like adhesive molecules and antibody frameworks 72 . Accord-
353
rosfr Ullt ingly, an understanding of the evolutionary emergence of distinct receptor types (with emphasis on the phylogeny of class 1 molecules) may shed some light on the parallel development of blood cell types and, in particular, on the evolution of a regulatory network controlling the haemopoietic cell hierarchy. This work was sustained in large part by a postdoctoral fellowship from the Alfred P. Sloan Foundation. The support of Tina S. Bazan is also gratefully acknowledged.
References 1 Metcalf, D. (1989) Nature 339, 27-30 2 Nicola, NA (1989) Annu. Rev. Biochem. 58,45-77 3 Balkwill, F.R. and Burke, F. (1989) Immunol. Today 10, 299-304 4 O'Garra, A, Umland, S., DeFrance, T. and Christiansen, J. (1988) Immunol. Today 9, 45-54 5 Paul, WE. (1989) Ce1/57, 521-524 6 Bazan, J.F. (1989) Biochem. Biophys. Res. Commun. 164, 788-796 7 Carpenter, G. (1987) Annu. Rev. Biochem. 56,881-914 8 D'Andrea, AD., Fasman, G.F. and Lodish, H.F. (1989) Cel/ 58, 1023-1024 9 Mosely, B., Beckmann, P., March, e.J et al. (1989) Ce1/59, 335-348 10 Gearing, D.P., King, JA, Gough, N.M. and Nicola, NA (1989) EMBO 1. 8, 3667-3676 11 Itoh, N., Yonehara, S., Schreurs, J. et al. (1990) Science 247,324-327 12 Goodwin, R.G., Friend, D., Ziegler, S.F. et al. (1990) Cel/ 60,941-951 13 Fukunaga, R., Ishizaka-Ikeda, E., Seto, Y. and Nagata, S. (1990) Ce1/61, 341-350 14 Saltzman, E.M., Luhowskyj, S.M. and Casnellie, H. (1989) 1. BioI. Chem. 264, 19979-19983 15 Hatakeyama, M., Mori, H., Doi, T. and Taniguchi, T (1989) Ce1/59, 837-845 16 Taga, T., Hibi, M., Hirata, Y. et a!. (1989) Ce1/58, 573-581 17 Saragovi, H. and Malek, T.R. (1990) Proc. Natl Acad. Sci. USA 87, 11-15 18 Opdenakker, G., Cabeza-Arvelaiz, Y. and Van Damme, J. (1989) Experientia 45, 513-520 19 Langer, JA and Pestka, S. (1988) Immunol. Today 9, 393-400 20 Bazan, J.F. Ce1/61, 753-754 21 Nemerson, Y. (1988) Blood 71, 1-8 22 Bazan, J.F. Proc. Natl Acad. Sci. USA (in press) . 23 Schrader, J.W, Ziltener, H.J. and Leslie, K.B. (1986) Proc. Natl Acad. Sci. USA 83, 2485-2489 24 Leutz, A, Damm, K., Sterneck, E. et al. (1989) EMBO 1. 8, 175-181 25 DeGrado, WF., Wasserman, Z.R. and Chowdhry, V. (1982) Nature 300, 379-381 26 Cohen, F.E., Kosen, P.A., Kuntz, I.D. et al. (1987) Science 234,349-352 27 Wingfield, P., Graber, P., Moonen, P., Craig, S. and Pain, R.H. (1988) Eur. 1. Biochem. 173, 65-72 28 Windsor, W, Syto, R., Nagabhushan, T.L., Trotta, P.P. and Le, HV (1988) FASEB 1. 2, A 1337 29 Chou, C-C, Witherspoon, S.M., Carter, J.M. et al. (1990) FASEB 1. 4, A2068 30 Lai, P-H., Everett, R., Wang, F-F., Arakawa, T. and Goldwasser, E. (1986)1. BioI. Chem. 261, 3116-3121 31 Lu, H.S., Boone, T.e., Souza, L.M. and Lai, P-H. (1989) Arch. Biochem. Biophys. 268,81-92 32 Williams, R.W (1985) 1. BioI. Chem. 260, 3937-3940 33 Brandhuber, B.J., Boone, T., Kenney, W.e. and McKay, D.B. (1987) Science 238, 1707-1709 . 34 Abdel-Meguid, S.S., Shieh, H-S., Smith, WW et a!' (1987)
354
Y 'rn_rn_u_n_O'_09_Y_T
