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Three-dimensional arrangement of conserved amino acid residues in a superfamily of specific ligand-binding proteins
Three-dimensional arrangement of conserved amino acid residues in a superfamily of specific ligand-binding proteins A. C. T. North University of Leeds, Astbury Department of Biophysics, Leeds LS2 9JT, UK
(Received 12 November 1988) Beta-lactoglobulin has been found to be a member of a super family of proteins that bind specific lioands and which share common features in their amino acid sequences. Here we show that these features are grouped spatially on the surface of the proteins and suggest that they may be concerned with binding to cell-surface receptors. Keywords: Beta-lactoglobulin;ligand binding; protein homology
We wish to draw attention to the remarkable spatial distribution of homologous and conserved amino acid residues in the recently identified super family of ligandbinding proteins exemplified by beta-lactoglobulin. Although beta-lactoglobulin (BLG) is the major component of the whey fraction of milk from cattle and other ruminants, its function has been unclear. The threedimensional structure of the bovine protein has recently been determined by high-resolution crystallographic studies t-4 of several different crystal forms which have shown it to be very closely similar to that of serum retinolbinding protein (RBP) (see Ref. 2). This similarity of structure provided a rational explanation for the observation that BLG could bind retinol and other hydrophobic molecules. In the crystal structure of RBP, retinol was seen to be bound in a deep pocket formed within the calyx-shaped fold of the protein chain and model-building studies showed that a retinol molecule could be accommodated in a closely corresponding position in BLG. These observations suggested that the physiological function of BLG might be to transport retinol, or perhaps other hydrophobic molecules, from the cow to its calf and evidence was subsequently obtained for the presence of a BLG receptor in defined segments of the gut of very young calves 2. The assumption that retinol is bound in an analogous fashion in BLG and RBP is questioned by the interpretation by Monaco et al. 4 of crystallographic data from a presumed complex of retinol and BLG, in which the internal cavity appeared to be empty, while electron density appropriately shaped for retinol occurred in a depression on the exterior surface of the protein. It would indeed be most surprising if BLG and RBP bound retinol in such different ways and possibly the complex seen by Monaco et al. represented an artefactual mode of binding due to the difficulty of obtaining a 'productive' complex in aqueous solution between the soluble protein and the highly hydrophobic ligand. 0141-8130/89/010056-o3503.00 © 1989Butterworth & Co. (Publishers)Ltd 56 Int. J. Biol. Macromol., 1989, Vol. 11, February
Two further crystal structures of related proteins have recently been published. They are of the insecticyanin from tobacco hornworm 5 and a closely similar insect bilin-binding protein 6'7, both of which bind the chromophore biliverdin. As with BLG, the full physiological function of these proteins is not yet clear, though they seem to be involved in insect camouflage. While these two proteins differ in amino acid sequence, they share with each other a similar calyx-shaped betastrand framework to that of BLG and RBP s-s. The major differences in conformation between all four proteins occur in surface loops. The biliverdin ligand is found in the interior pocket of both insecticyanin and bilinbinding protein, although the orientation in which it is bound differs somewhat in the two cases. Godovac-Zimmermann 9 has discussed the existence of further members of this ligand-binding protein superfamily, as deduced from homology in amino acid sequences. This list has been supplemented by Pevsner et al.l° and recent additions to it are the two different chains of the lobster protein crustacyanin 11, which binds the pigment astaxanthin. Table 1 lists the proteins that are now believed to belong to the superfamily, together with their putative functions and ligands, where these are known. Sawyer s, Pervaiz and Brew 12, GodovacZimmermann 9 and Pevsner et al. t° have all drawn attention to the fact that, while the overall sequence homology between members of the family is low, there are some important features of high homology that strongly suggest a common chain fold in three dimensions: a sequence -h-a-x-x-u-h-x-Gly-x-Trp-y-x-h-h- (a is usually acidic, u often basic, y aromatic, h hydrophobic) occurs near residue 20 (BLG numbering), a sequence -h-h-x-ThrAsp-Tyr-x-x-y-h- occurs near residue 100 and a disulphide bridge is formed between Cys 66 and another cysteine near to the carboxyl end of the chain. Pevsner et aL to place the two sequences specified within regions
Spatial distribution of conserved amino acid residues: A. C. T. North Table 1 Properties and functions of the ligand-binding protein family Protein Beta-lactoglobulin Serum retinolbinding protein Purpurin Alpha-l-acidglycoprotein Androgen-dependent secretory protein Endometrial alpha-2-globulin Alpha-1-microglobulin (Protein HC) Apo-lipoprotein D Major urinary protein (alpha-2u-globulin) Bowman's gland odorantbinding protein Insecticyanin (Bilin-binding protein) Crustacyanin
Size 2 x 162
Source
Ligands
Properties
Milk whey
Retinol and other hydrophobic molecules Retinol in complex with transthyretin Retinol, heparin Progesterone
Vitamin transport to gut receptor Retinol transport to the eye Cell survival Mediates inflammatory response Binds to sperm membrane Synthesized in early pregnancy Mediation of neutrophil chemotaxis Lipid transport
182
Serum
196 187
Retina Hepatic cells, serum, urine Epididymal luminal fluid Amniotic fluid
165 ~ 21 kDa 183
? ? IgA and a yellowbrown retinoid Lecithin, cholesterol Pheromones?
160
Cerebrospinal fluid, serum Gland and gut secretions Hepatic cells, serum, urine Nasal mucus
189
Haemolymph
Biliverdin IX
170 + 177
Carapace
Astaxanthin
169 162
Odorants
Signalling? Presentation of odorants to receptor? Camouflage Coloration, photo-reception
o
U
F i b r e 1 Stereo diagrams of the chain fold of beta-lactoglobulin (a) showing the alpha-carbon backbone, the invariant and homologous side chains specified in the text and the position of bound retinol in our model; (b) showing a ribbon representation of the backbone, invariant side chains (with atoms marked) and homologous side chains (with skeletal representation)
respectively 17 and 15 residues long that show a degree of homology higher than the remainder. The disulphide bridge referred to occurs in most but not all of the proteins listed in Table I and it is possible that there are in fact two subsets of the family, with alternative arrangements of the bridge. There is only one amino acid that is invariant throughout, namely Trp at position 19,
Gly 17 being present in all but one of the proteins, where it is replaced by Asp. The -Thr-Asp-Tyr- triplet occurs in most examples, but variants occur in all three positions; it is particularly interesting that all three differ in horse BLG-II, which is otherwise generally very similar to horse BLG-I and the BLGs from other species. What functions can be ascribed to the regions of high
Int. J. Biol. Macromol., 1989, Vol. 11, February
57
Spatial distribution o f conserved amino acid residues: A. C. T. North
homology? Huber et al.7 suggest that the -Thr 97-Asp 98(BLG numbers) side chains are essential for the formation of a tight turn but, as has been said, these residues are not absolutely invariant. Fioure 1 shows the spatial position of the near-invariant and homologous side chains listed above, together with Arg 124 which also is nearinvariant ~3, in the positions in which they occur in the acid p H form of cow B L G 3. What is remarkable is that they form a closely-spaced group. Trp 19 faces the interior pocket where it is close to the retinol in our model. Two other side chains face inwards and one is a spacer between strands, but the remainder occur nearby on the outside of the protein. This implies that their importance lies neither in a structural role nor in common features of internal ligand binding. The proteins of this family appear to fulfil a wide range of biological roles; in all cases where these have been identified, they involve the transport of hydrophobic ligands through aqueous or hostile environments. Many of the proteins are clearly required to transport their ligand to a cell receptor or from a transmitter. We suggest that the common surface features of the proteins in this family are concerned with interacting with common features on the cells to which they bind. The other, variable, surface side chains will allow discrimination between different cell receptors, so as to ensure that the ligand is delivered to, or picked up from, its correct site. The invariant Trp residue must have an important role, such as 'signalling' to the receptor whether or not the protein is carrying a ligand. Such a property is required because a 'full' protein arriving at a receptor must be able, by binding more strongly, to displace an 'empty' protein
58
Int. J. Biol. Macromol., 1989, Vol. 11, February
that has discharged its load. Recent evidence x4 supports such behaviour in the case of RBP.
Note added in proof The recently determined three-dimensional structure of P2 myelin protein 1s shows it to be another member of the superfamily, with a lipid ligand bound in the central cavity.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Sawyer,L., Papiz, M. Z., North, A. C. T. and Eliopoulos, E. E. Biochem. Soc. glans. 1985, 13, 265 Papiz, M. Z., Sawyer, L., Eliopoulos, E. E., North, A. C. T., Findlay, J. B. C., Sivaprasadarao, A., Jones, T. A., Newcomer, M. E. and Kraulis, P. J. Nature (London) 1986, 324, 383 North, A. C. T. and Yewdall, S. J. (unpublished work) Monaco, H. L., Zanotti, G., Spadon, P., Bolognesi,M., Sawyer, L. and Eliopoulos, E. E. J. Molec. Biol. 1987, 197, 695 Holden,H. M., Rypniewski,W. R., Law, J. H. and Rayment, I. EMBO J. 1987, 6, 1565 Huber, R., Schneider, M., Epp, O., Mayr, I., Messerschmitt,A., Pflugrath, J. and Kayser, H. J. Molec. Biol. 1987, 195, 423 Huber, R., Schneider, M., Mayr, I., Muller, R., Deutzmann, R., Suter, F., Zuber, H., Falk, H. and Kayser, H. J. Molec. Biol. 1987, 198, 499 Sawyer,L. Nature (London) 1987, 327, 659 Godovac-Zimmermann,J. Trends Biochem. Sci. 1988, 13, 64 Pevsner,J., Reed, R. R., Feinstein, P. G. and Snyder, S. H. Science 1988, 241,336 Findlay,J. B. C. (personal communication) Pervaiz,S. and Brew, K. Science 1985, 228, 335 Yewdall,S. J. (personal communication) Sivaprasadarao,A. and Findley, J. B. C. Biochem. J. 1988, 255, 561 Jones,T. A., Bergfors,T., Sedzik, J. and Unge, T. Zeitschrift f. Kvist. 1988, 185, 120
(Received 12 November 1988) Beta-lactoglobulin has been found to be a member of a super family of proteins that bind specific lioands and which share common features in their amino acid sequences. Here we show that these features are grouped spatially on the surface of the proteins and suggest that they may be concerned with binding to cell-surface receptors. Keywords: Beta-lactoglobulin;ligand binding; protein homology
We wish to draw attention to the remarkable spatial distribution of homologous and conserved amino acid residues in the recently identified super family of ligandbinding proteins exemplified by beta-lactoglobulin. Although beta-lactoglobulin (BLG) is the major component of the whey fraction of milk from cattle and other ruminants, its function has been unclear. The threedimensional structure of the bovine protein has recently been determined by high-resolution crystallographic studies t-4 of several different crystal forms which have shown it to be very closely similar to that of serum retinolbinding protein (RBP) (see Ref. 2). This similarity of structure provided a rational explanation for the observation that BLG could bind retinol and other hydrophobic molecules. In the crystal structure of RBP, retinol was seen to be bound in a deep pocket formed within the calyx-shaped fold of the protein chain and model-building studies showed that a retinol molecule could be accommodated in a closely corresponding position in BLG. These observations suggested that the physiological function of BLG might be to transport retinol, or perhaps other hydrophobic molecules, from the cow to its calf and evidence was subsequently obtained for the presence of a BLG receptor in defined segments of the gut of very young calves 2. The assumption that retinol is bound in an analogous fashion in BLG and RBP is questioned by the interpretation by Monaco et al. 4 of crystallographic data from a presumed complex of retinol and BLG, in which the internal cavity appeared to be empty, while electron density appropriately shaped for retinol occurred in a depression on the exterior surface of the protein. It would indeed be most surprising if BLG and RBP bound retinol in such different ways and possibly the complex seen by Monaco et al. represented an artefactual mode of binding due to the difficulty of obtaining a 'productive' complex in aqueous solution between the soluble protein and the highly hydrophobic ligand. 0141-8130/89/010056-o3503.00 © 1989Butterworth & Co. (Publishers)Ltd 56 Int. J. Biol. Macromol., 1989, Vol. 11, February
Two further crystal structures of related proteins have recently been published. They are of the insecticyanin from tobacco hornworm 5 and a closely similar insect bilin-binding protein 6'7, both of which bind the chromophore biliverdin. As with BLG, the full physiological function of these proteins is not yet clear, though they seem to be involved in insect camouflage. While these two proteins differ in amino acid sequence, they share with each other a similar calyx-shaped betastrand framework to that of BLG and RBP s-s. The major differences in conformation between all four proteins occur in surface loops. The biliverdin ligand is found in the interior pocket of both insecticyanin and bilinbinding protein, although the orientation in which it is bound differs somewhat in the two cases. Godovac-Zimmermann 9 has discussed the existence of further members of this ligand-binding protein superfamily, as deduced from homology in amino acid sequences. This list has been supplemented by Pevsner et al.l° and recent additions to it are the two different chains of the lobster protein crustacyanin 11, which binds the pigment astaxanthin. Table 1 lists the proteins that are now believed to belong to the superfamily, together with their putative functions and ligands, where these are known. Sawyer s, Pervaiz and Brew 12, GodovacZimmermann 9 and Pevsner et al. t° have all drawn attention to the fact that, while the overall sequence homology between members of the family is low, there are some important features of high homology that strongly suggest a common chain fold in three dimensions: a sequence -h-a-x-x-u-h-x-Gly-x-Trp-y-x-h-h- (a is usually acidic, u often basic, y aromatic, h hydrophobic) occurs near residue 20 (BLG numbering), a sequence -h-h-x-ThrAsp-Tyr-x-x-y-h- occurs near residue 100 and a disulphide bridge is formed between Cys 66 and another cysteine near to the carboxyl end of the chain. Pevsner et aL to place the two sequences specified within regions
Spatial distribution of conserved amino acid residues: A. C. T. North Table 1 Properties and functions of the ligand-binding protein family Protein Beta-lactoglobulin Serum retinolbinding protein Purpurin Alpha-l-acidglycoprotein Androgen-dependent secretory protein Endometrial alpha-2-globulin Alpha-1-microglobulin (Protein HC) Apo-lipoprotein D Major urinary protein (alpha-2u-globulin) Bowman's gland odorantbinding protein Insecticyanin (Bilin-binding protein) Crustacyanin
Size 2 x 162
Source
Ligands
Properties
Milk whey
Retinol and other hydrophobic molecules Retinol in complex with transthyretin Retinol, heparin Progesterone
Vitamin transport to gut receptor Retinol transport to the eye Cell survival Mediates inflammatory response Binds to sperm membrane Synthesized in early pregnancy Mediation of neutrophil chemotaxis Lipid transport
182
Serum
196 187
Retina Hepatic cells, serum, urine Epididymal luminal fluid Amniotic fluid
165 ~ 21 kDa 183
? ? IgA and a yellowbrown retinoid Lecithin, cholesterol Pheromones?
160
Cerebrospinal fluid, serum Gland and gut secretions Hepatic cells, serum, urine Nasal mucus
189
Haemolymph
Biliverdin IX
170 + 177
Carapace
Astaxanthin
169 162
Odorants
Signalling? Presentation of odorants to receptor? Camouflage Coloration, photo-reception
o
U
F i b r e 1 Stereo diagrams of the chain fold of beta-lactoglobulin (a) showing the alpha-carbon backbone, the invariant and homologous side chains specified in the text and the position of bound retinol in our model; (b) showing a ribbon representation of the backbone, invariant side chains (with atoms marked) and homologous side chains (with skeletal representation)
respectively 17 and 15 residues long that show a degree of homology higher than the remainder. The disulphide bridge referred to occurs in most but not all of the proteins listed in Table I and it is possible that there are in fact two subsets of the family, with alternative arrangements of the bridge. There is only one amino acid that is invariant throughout, namely Trp at position 19,
Gly 17 being present in all but one of the proteins, where it is replaced by Asp. The -Thr-Asp-Tyr- triplet occurs in most examples, but variants occur in all three positions; it is particularly interesting that all three differ in horse BLG-II, which is otherwise generally very similar to horse BLG-I and the BLGs from other species. What functions can be ascribed to the regions of high
Int. J. Biol. Macromol., 1989, Vol. 11, February
57
Spatial distribution o f conserved amino acid residues: A. C. T. North
homology? Huber et al.7 suggest that the -Thr 97-Asp 98(BLG numbers) side chains are essential for the formation of a tight turn but, as has been said, these residues are not absolutely invariant. Fioure 1 shows the spatial position of the near-invariant and homologous side chains listed above, together with Arg 124 which also is nearinvariant ~3, in the positions in which they occur in the acid p H form of cow B L G 3. What is remarkable is that they form a closely-spaced group. Trp 19 faces the interior pocket where it is close to the retinol in our model. Two other side chains face inwards and one is a spacer between strands, but the remainder occur nearby on the outside of the protein. This implies that their importance lies neither in a structural role nor in common features of internal ligand binding. The proteins of this family appear to fulfil a wide range of biological roles; in all cases where these have been identified, they involve the transport of hydrophobic ligands through aqueous or hostile environments. Many of the proteins are clearly required to transport their ligand to a cell receptor or from a transmitter. We suggest that the common surface features of the proteins in this family are concerned with interacting with common features on the cells to which they bind. The other, variable, surface side chains will allow discrimination between different cell receptors, so as to ensure that the ligand is delivered to, or picked up from, its correct site. The invariant Trp residue must have an important role, such as 'signalling' to the receptor whether or not the protein is carrying a ligand. Such a property is required because a 'full' protein arriving at a receptor must be able, by binding more strongly, to displace an 'empty' protein
58
Int. J. Biol. Macromol., 1989, Vol. 11, February
that has discharged its load. Recent evidence x4 supports such behaviour in the case of RBP.
Note added in proof The recently determined three-dimensional structure of P2 myelin protein 1s shows it to be another member of the superfamily, with a lipid ligand bound in the central cavity.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Sawyer,L., Papiz, M. Z., North, A. C. T. and Eliopoulos, E. E. Biochem. Soc. glans. 1985, 13, 265 Papiz, M. Z., Sawyer, L., Eliopoulos, E. E., North, A. C. T., Findlay, J. B. C., Sivaprasadarao, A., Jones, T. A., Newcomer, M. E. and Kraulis, P. J. Nature (London) 1986, 324, 383 North, A. C. T. and Yewdall, S. J. (unpublished work) Monaco, H. L., Zanotti, G., Spadon, P., Bolognesi,M., Sawyer, L. and Eliopoulos, E. E. J. Molec. Biol. 1987, 197, 695 Holden,H. M., Rypniewski,W. R., Law, J. H. and Rayment, I. EMBO J. 1987, 6, 1565 Huber, R., Schneider, M., Epp, O., Mayr, I., Messerschmitt,A., Pflugrath, J. and Kayser, H. J. Molec. Biol. 1987, 195, 423 Huber, R., Schneider, M., Mayr, I., Muller, R., Deutzmann, R., Suter, F., Zuber, H., Falk, H. and Kayser, H. J. Molec. Biol. 1987, 198, 499 Sawyer,L. Nature (London) 1987, 327, 659 Godovac-Zimmermann,J. Trends Biochem. Sci. 1988, 13, 64 Pevsner,J., Reed, R. R., Feinstein, P. G. and Snyder, S. H. Science 1988, 241,336 Findlay,J. B. C. (personal communication) Pervaiz,S. and Brew, K. Science 1985, 228, 335 Yewdall,S. J. (personal communication) Sivaprasadarao,A. and Findley, J. B. C. Biochem. J. 1988, 255, 561 Jones,T. A., Bergfors,T., Sedzik, J. and Unge, T. Zeitschrift f. Kvist. 1988, 185, 120