Summary Insulin is a member of a family of hormones, growth factors and neuropeptides which are found in both vertebrates and invertebrates. A common ‘insulin fold’ is probably adopted by all family members. Although the specificities of receptor binding are different, there is a possibility of co-evolution of polypeptides and their receptors.
Introd uction Tn the years since 1955when the amino acid sequence of insulin was determined by Sanger and co-workers(l?’) our knowledge of insulin and related proteins has increased dramatically. We now have sequences of more than 80 insulins and insulin-related proteins as well as site-directed mutants and chemically modified insulins. There are structures in several oligomeric states defined in the crystals by X-ray analysic, and in solution by 2-dimensional nmr. The relationship between sequence variation and the role of parts of the structure in processing, stora e, transport and receptor binding have been explored(3 . The insulin receptor has been identified, sequenced, cloned and expressed. However, although we have extensive knowledge of insulins and their receptors, the nature of the interaction between insulin and its receptor and the events that transduce the signal are less well understood. Early proposals for a receptor-binding region on the insulin molecule centred around the first 3-dimensional structure(4) as defined in crystals of 2-zinc insulin hexamers. Receptor interaction was postulated to involve a surface region of the insulin molecule, which partly overlaps with the hydrophobic surface involved in dimer formation. However, evidence from assays of binding and activity of modified and mutated insulins, and from structural variations in the monomer in different environments, has suggested the possibility that the monomer may adopt a slightly different conformation at the receptor than it does in the dimer. Here we review the crystal and NMR structures, studies of insulin sequences, mutagenesis and chemical modification, and studies of the receptor itself. These contribute to our present understanding of the molecular mechanism of receptor binding.
The First Crystal Structure More than 20 years ago, determination of the X-ray crystal structure of 2-zinc porcine insulin(’) provided the first direct experimental evidence for the insulin fold. In this structure, insulin protomers form a hexamer composed of three identical dimers assembled around a three-fold axis (Fig. 1). The original structure has undergone subsequent refinement. but the essential components are unchanged(‘). In the dimer the two monomers are not completely identical, but both have their A and B chains folded to form compact globular structures with a number of common features (Fig. 2). These include: 1. an A chain with two helices (A2-A7 and A13-A19), joined by an extended loop(A8-Al2); 2. a B chain with an extended N-terminus, followed by a helix (B9-B19), followed by a sharp turn (B20-B23) and an extended C-terminal section; 3, three disulphide bridges (A6-All, A7-B7, A20-Bl9); and 4. a hydrophobic core. In the dimcr, an antiparallel {j-sheet is formed between the C-termini of the B chains of adjacent monomers and the B chain helices pack together. This leads to the burying of hydrophobic sidechains. Additional, but less strong, hydrophobic contacts are made between dimers in hexamer formation. Early Models for Receptor Binding An early suggestion that some of the elements involved in dimer formation wcre involved in receptor interaction was supported by the relatively high conservation of putative binding residues across the inyulin family. The biological activity of analogues with chemical modifications in the dimer-forming surface and the
?
d
Fig. 1. Assembly of an insulin hexamer. Pairs of insulin molecules come together about 2-fold axes in the plane of the diagram to form three dimers, which then assemble around a three-fold axis perpendicular to the plane of the diagram, along with two zinc ions, to form the ‘2-zinc’ insulin hexamer.
Fig. 2. The insulin monomer. A ribbon diagram through the main-chains of the insulin molecule. The A-chain is shaded, and the sulphur atoms of the disulphidebonds are represented as dotted spheres.
crystal structures of some modified in~ulins(~) were consistent with this view. Studies on sequence variant and modified insulins have also indicated that, some residues, which are not part of the diiner foi-ming surface may be iiivolved in receptor binding. For example, a low-activity mutant human insulin has been identified where A3 valine is replaced with leucine; substitution of B10 histidine by aspartic acid (as found in some patients with hyperproinsulinaemia) has been shown to make insulin more active; and the presence of histidine at A8, as found in some bird insulins, can also be associated with high activity. The spatial location of the residues on the insulin monomer, both 'dimerforming' and further removed (e.g. A3, BlO) (Fig. 3 ) gives rise to the idea of an 'extended binding region' of residues which have all been shown to be important in receptor interaction, but not all of which are solvent accessible in the protomer as found in the crystal structure. More X-ray Studies Since the initial structure determination, the threedimensional structure of insulin has been studied intensively. Crystal structures have been solved for insulins in different aggregation states (monomer, dimer, hexamer), for insulin crystallised in differing solvent conditions which alter the monomer conformation, for sequence variant insulins from different species, for chemically modified insulins and for des(B26-B30)insulin (DPI). It has become clear that, although most of the common features listed above are
Fig. 3. A receptor's view of the insulin molecule'? Residues which are labelled have been shown to be important in receptor binding.
retained, there is considerable conformational variation in the insulin monomer (Fig. 4). [For a detailed review, see reference 81. A recently determined X-ray crystal structure of human relaxin(') shows an insulin fold, differing only in that the truncated C-terminal region of the B chain is an extension of the B chain helix rather than an insulin-like turn. In the insulin molecule the most extreme variation is in the N-terminus of the B chain, which may exist either in an extended conformation or as an extension of the B chain helix. However, this is thought to be a property of the hexarner('") and therefore not directly relevant to receptor binding. Superimposing insulin structures defined in different crystal forms, it is seen that the major secondary structural elements are capable of individual rigid body movements without major changes to the structure. These movements are accommodated by alterations in side chain conformations and are generally determined by packing interactions in the crystal. Relatively small changes in one part of the molecule may be transmitted by this mechanism to distant regions of the molecule. The insulin monomer is, therefore, rather flexible('''. Crystallographic studies on proinsulin(12)show that the A and B chains of the insulin moiety retain a similar structure to that in other crystal structures, while the C-
\
A
B
Fig. 4. Superimpositionof some insulin crystal structures. Achains are shown in dotted, and B-chains in continuous 1ines.The structures used were: the two molecules from 2-zinc insulin, two molecules from 4-zinc insulin. one molecule from the monoclinic insulin hexamer and one molecule from des-
pentapeptide insulin. peptide is not well-defined, because of either static disorder or mobility. Molecular dynamic simulations(13) starting from several different crystallographically defined protomer structures reinforce these concepts, highlighting relative movements of the helices and flexibility of the chain termini. Computer modelling showed that the other members of the family can adopt an insulin-like fold for the regions corresponding to the A and B chains of insulin("-17), although some are single chain molecules with B and A chains linked by a C-peptide, and A or B chain extensions may be present (Fig. 5).
Nuclear Magnetic Resonance Insulin would appear to be a natural candidate for the recently developed 2-dimensional NMR methods for large molecules, but problems in obtaining a spectrum of the molecule in a single known aggregation state have impeded a complete sequential assignment. The presence of multiple aggregation states in solution is one cause of line broadening in the spectra; but dilution to a point where the solution contains only monomers leads to weak signals and consequent long data collection times. However, com lete assignments have now been made for insulin(ls2') and IGFlC2"),and spectra measured for various related molecules. The solution structures of insulin are broadly similar to the crystal structure, and the IGFl structure is similar to the model based on the insulin fold, although greater flexibility is reported in the helix extremities and chain
r:
Fig. 5. Computer models of members of the insulin family. The models werc built on computer graphics systems using the coordinatcs of the insulin crystal structures as a starting point. The regions corresponding to the insulin A chains are shown as heavy lines. (a) IGFl, (b) relaxin, ( c ) bombyxin-2, (d) molluscan insulin-like peptide-1.
termini. Again, relative movements of the structural elements are a salient feature. In both IGFl and proinsulin the C-peptides are apparently mobile.
Sequences of Members of the Insulin Family Insulins and related peptides("] have been identified in several phyla of the animal kingdom. Most have been found in the vertebrates, but examples have also been identified in proto-chordates, insects and molluscs. This distribution may reflect the relative extent to which these groups have been studied rather than the actual distribution of the molecules in nature. The family comprises the insulins, the insulin-like growth factors (IGFs)(zz~z3j, the relaxins(24)),the bombyxins(") and the molluscan insulin-like peptides (MIPS)('@. Apart from the IGFs, all contain separate A and B chains. The C peptides of the IGFs are not cleaved. They are single-chain peptides organised in a manner analogous to proinsulin, with the C peptide linking the B chain C-terminus to the A chain Nterminus. A C-terminal extension to the A chain forms the D region and, in some forms of IGF, there is an additional C-terminal E region which may be removed in post-translational processing. Within the family the
PTTHs (bombyxins) dogfish relaxin shark relaxin skate relaxin human relaxin porcine and whale relaxins rat relaxin
lengths of the A and B chains are rather variable. In particular. the MIPs havc a long N-terminal extension to the H chain containing a cysteine which may form an additional disdphide bridge with the cysteine found at A4 in these molecules. The sequences of the insulin family have been clustered and a tree (Fig. 6), similar to a phylogenetic tree, constructed by cluster analysis techniques(27). Since bombyxins and MIPs each represent multiple sequences from the same species, this is not a true phylogenetic tree in the sense that it parallels the evolutionary relationship between species, but is rather a ‘molecular phylogeny’. The tree represents evolutionary divergence up to the point where the insulins have diverged from the IGFs. After the IGFs, the bombyxins are next most closely related to the insulins. The next are the relaxins which fall into two groups, mammalian and fish, which diverge before the bombyxins diverge from the lGFs and insulins. Surprisingly the insuliiis and IGFs are less divergent than the relaxins of different species and most remarkably, the MIPS, which are all derived from a single species, show even more divergence. In considering the common features of the sequences, it is useful to define a ‘common framework’ for
Fig. 6. Relationships in the insulin fdmily. Comparison of the sequences of the members of the famly allows them to be arranged in drder of similarity to form a ‘molecular phylogeny’.
the molecule. Sequence data suggest that structural elements conserved throughout the family include the disulphide bridges, the three helices and their spatial relationship, A and B chain turns and the buried hydrophobic residues. We exclude the extended regions of the B chain, and chain extensions or links found in some non-insulin members of the family, An analysis of sequence variability is given in Figure 7; within the common framework all sequences can be aligned without insertions or deletions. Of the structurally important residues in insulin, only thc six halfcystines and the glycine at B8 are rigidly conserved in all known sequences. The glycine at B20 is conserved in most insulins, being substituted only in hystricomorph insulins which do not dimerise. It is conserved in all relaxins and all but but one IGF (rat IGF2). B23 glycine is conserved in all known insulins and IGFs, but is substituted in the relaxins, MTPs and bombyxin-IV. Lack of glycines at B20 and B23 in MIPs makes the presence of an insulin-like B chain turn in these molecules rather unlikely. Across the family, other residues generally conserved are those buried in the insulin monomer (A2, A16, B11, B15) and residues at the N- and C-termini of the A chain. Of the residues involved in forming the insulin dimer, B12 is conserved
25
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Fig. 7. Sequence variability at each residue in the insulin molecule. The histogram represents the reciprocal of the percentage conservation for each residue in the consensus sequence for the insulin family. The data are scaled with A4 arbitrarily taken as zero, so that negative values represent more conserved residues and positive values more variable residues. The consensus sequence is shown on the X axis, and the numbers associated with each residue are the sequence numbers in the A and B chains. A database of 80 sequences of members of the insulin family was used.
as valine in all insulins, IGFs and mammalian relaxins; B24 is conserved as phenylalanine in all insulins and IGFs. Regions of high variability occur in the regions A8-Al0, the N-terminus of the B chain and at B27. Of the helices, residues in the second A chain helix are less well conserved than in the other two. Examination of the sequence variation enables us to assess the possibility of attaining the 2Zn insulin type of fold, and models have been constructed of IGFs, relaxins, bombyxins and MIPS (Fig. 5 ) . Most residues in the extended binding region of insulin are well conserved in other members of the family. Exceptions are at A8, where the consensus residue histidine is associated with high activity in avian and mammalian insulins but not in fish: and A17, A18 and B21. So far regions determining receptor specificity have not been identified for these molecules. The Insulin Receptor The first step in the action of both insulin and IGFs is binding to a cell surface receptor. Both insulin and IGFl receptors have been sequenced, and shown to be derived from distinct but related genes(28729)); insulin and IGFl can bind to both receptors, but each binds to its own with much greater affinity. The receptors are membrane proteins, which in their mature forms are disulphide bonded a& heterotetramers. with extracellular, mcmbrane-spanning and intracellular (tyrosine kinase) domains. A diagram of our proposed model of an ap subunit of the insulin and IGFl receptors, based on sequence alignment and secondary structure prediction(”) is shown in Figure 8. The large variable regions (L1 and L2) apparently consist of four structural motifs joined by variable loops. or five a-@ The cystine residues in the S domains appear to form both inter- and intra-chain disulphide bonds, although the connectivity is unknown. Ligand binding is confined to the extracellular domain of the receptor, which contains one high affinity binding site per receptor subunit. Detailed discussion of the IGF1-IGF1 receptor system is beyond the scope of this essay, but there is evidence that binding specificity is determined by distinct regions of their receptors(31).Each of the two ligand-receptor systems could thur have co-evolved from a common ancestor. When insulin binds to the extracellular domain, tyrosine kinase activity is stimulated, leading to phosphorylation in the intracellular domain of the /?subunit, the initial step in the cellular events. The ability to construct and express mutants of the extracellular domain of the insulin receptor now allows systematic investigation of areas of sequence involved in insulin binding. Monomeric a-subunit can bind insulin only slightly less well than secreted intact extracellular domain(32). Within the subunit, both L and S regions have been proposed as the insulin binding site. Chimaeric receptors formed by coupling the extracellular domain of one receptor to the intracellular
Alpha Subunit
Ll
1
Large subunits
\
L2
1
W Fig. 8. The insulin and lGFl receptors. The schematic diagram shows just an a$’ dimer of the a2J$receptor. Binding of the ligand to the extracellular domain causes a signal to be transmitted through the cell membrane which activates autophosphorylation between subunits in the cytoplasmic domain, the first of a cascade of events in the cell.
domain of another are functional, with ligand specificity determined by the extracellular domain(33). It has been shown(”) that binding of epidermal growth factor causes a conformational change in the ectodomain of its receptor (which is similar to insulin’s, but monomeric). In the insulin receptor two conformational changes have been postulated; the first, on binding insulin to the a-subunit increases ATP binding to the B-subunit; the @-subunitthen undergoes a further conformational chan e which is important for transmembrane signallingb”’. Ligand-receptor contacts can be assumed to be rather extensive, and not necessarily confined to one portion of primary, or even secondary structure.
Discussion If we assume that insulin binds to its receptor as a monomer, then we must consider the possible effects of stability, flexibility and other properties of the monomer in receptor binding. The two interchain disulphide bridges are essential to the stability of the insulin molecule, as shown by the ease with which the chains can be separated following oxidation of the disulphides
and the difficulties associated with chain recombination reactions. Whilst these disulphides restrict monomer flexibility, considerable potential remains, and this has long been recognised as important for receptor interaction(36). Flexibility can be reduced by crosslinking the C-terminus of the B chain and the Nterminus of the A-chain, which are quite close in space. Links between the sidechain of B29 and the LY amino group of A1 cause reduced activity(37);and a peptide bond between B29 and A1 in des-B30 single chain insulin (SCI)(38)causes a total loss of activity which cannot be completely explained by the loss of charge at A l . Al-B29 crosslinked insulin is known from crystallographic evidence to be similar to the native protomer in three-dimensional structure, except for the reduced flexibility(”). The N-terminus of the B chain shows considerable variation in that it cxists in helical and several extended conformations. A recent study has em hasized the importance of LeuB6 in receptor bindinggo), probably through stabilisation of the tertiary structure, although dirwt receptor interactions are not excluded. The C-terminus of the B chain appears to play a pivotal role in the expression of insulin’s biological activity. Superimposition of crystal structures shows the C-terminus of the B chain to be relatively mobile and in DPI, which does not dimerise, it adopts a conformation (determined by crystal packing interactions) , different from that in any other insulin crystal structure. Investigation of insulin analogues by analytical HPLC has suggested that in the presence of organic solvent the B chain may unfold from B24(41).NMR studies of the insulin monomer in aqueous solution suggest that the residues up to B28 are in a similar position to that found in the crystal structure(”), although in solutions containing organic solvent it appears to be displaced(18). of a mutant insulin with B24 An NMR phenylalanine replaced by glycine, which is a full agonist, shows destabilisation of the B chain turn and a loss of contact between the C-terminal residues of the B chain and the rest of the molecule. This exposes residues such as A2 and A3 which form part of the binding region shown in Figure 3. The identification of mutant human insulins with reduced activity and substitutions at B24 and B25 (both Phe in native insulin) (for a review, see ref. 43) has reinforced interest in this part of the molecule. In DPI, the B chain from B25 to the C-terminus is missing, yet the molecule retains significant activity. Removal of B25 and B24 leads to progressively reduced activity and, where both residues are missing, the molecule is virtually inactive. However, amidation of the free carboxylate of B25 in DPI restores activity to levels similar to native insulin(44),suggesting that the C-terminal pentapeptide is not necessary for full activity, and indeed that DPI may be a good starting point from which to model the receptor-bound conformation of insulin. In studies with DPI amides, it has been shown that, where B25 is substituted with an aromatic amino acid,
particularly one with a polar sidechain. activity may be either normal or significantly enhanced(45). However, effects observed for specific substitutions in whole insulin may be significantly different in DPI amide, suggesting a modulating role for the terminal pentapeptide. B25 has been implicated as a potential primary contact between the hormone and its receptor. It has been suggested(46) that, following initial binding between the B25 sidechain and a specific pocket in the receptor, conformational changes take place in both the hormone and the receptor which lead to a secondary binding event involving other areas of the insulin monomer, leading to expression of full biological activity. Recent studies on the region B24 Phe, B25 Phe and B26 Tyr suggest(j7)that these residues all have a role in determining insulin-receptor interactions, influencing the conformation of the monomer and its ability to aggregate. But none of the native side chains is absolutely required for receptor binding, since analogues have been identified where these residues are individually either deleted or substituted by residues with non-aromatic sidechains but which retain the ability to bind the receptor, albeit with altered affinity. Conformational constraints imposed by a D amino acid at B24 can lead to enhanced affinity. The C-terminus of the insulin B chain has, therefore, an obvious role in receptor interaction. There is, however, considerable indirect evidence that this part of the molecule must undergo some conformational change on receptor binding. If it moves away from the surface of the monomer, a5 it does in non-aqueous solution, a surface similar to that in DPI will be exposed. The receptor binding region would then involve residues such as A3 which would otherwise be sterically hindered by the C-terminal residues of the B chain. It would also involve other residues such as A4. A8 A19, B9 and I310 that are not involved in dimer formation; this would explain the greater affinity of a monomer for the receptor than for another monomer. Aromatic residues in positions corresponding to B24B26 are found in all insulins (apart from some hystricomorphs where B 26 is non-aromatic) and IGFs, all of which show some affinity for the insulin receptor, but not in the relaxins, bombyxins or MIPS. There is no evidence that these latter molecules show any affinity for the insulin receptor, and their relative lack of conservation of the glycines at B20 and B23 suggest that the C-terminus of the B chain does not follow the insulin fold. It is likely, however, that the other elements of their common frameworks are similar to insulin. The receptor binding region described here involves regions occluded by the C-terminus of the B chain in the conformer stabilized in the porcine insulin dimer. This region (for example A3 Val) is relatively well conserved amongst the broader insulin family. It is interesting to speculate that the hormone and its receptor may have co-evolved. If this is the case, the receptor binding site
of the ancestral hormone probably involved the common framework, with the involvement of the B24B26 region and associated conformational changes in the C-terminus of the B chain appearing at a later stage in evolution. Acknowledgements We would like to thank G.G. Dodson and colleagues, D. Brandenburg, A. Wollmer and colleagues, M. Weiss and Liang Dong-Chai for many discussions; the York insulin group for unpublished coordinates; and thc SERC for support for J.M-R. References 1 Brown, H., Sanger, F. and Kitai, R. (1955). l h e structure of sheep and pig iiisulins. Biochem J . 60, 556-565. 2 Ryle, A. P., Sanger, F., Smith, 1,. F. and Kitai, R. (1955). The ilisulphide bondr of insulin. Riorhem. J. 60, 541-556. 3 Blundell, T. L. and Wood, S. P. (1975). Is thc evolution of insulin Darwinian or due to selectively neutral mutation? Nature 257. 197-203. 4 Blundell, T. L., Dodson, G., Hodgkin, D. and Mercola, D. (1972). Insulin: the structure in the cry5tal and its reflection in chemistry and biology. Adv. in Protein Chemistry 26, 279-402. 5 Adam, M. J., Rlundell, T. L., Dodson, E. J., Dodson, G. G., Vijayan, M., Baker, E. N., Hodgkin, D. C., Rimmer, B. and Sheat, S . (1969). Structure of rhombohedra1 2 zinc insulin crystals. Nature 224, 491-5. 6 Baker, E. K.,Blundell, T. L., Cntfield, J. F., Cntfield, S. M., Dodson, E. J., Dodson, G . G . , Hodgkin, I). C., Hubhard, R. E., Isaacs, N. W., Reynolds, C. D., Sakahe, K., Sakabe, N. and Vijayan, M. (1988). The structure of 2Zn pig insulin crystals at 1.5A rcsolution. Phil. Trans. Roy. Soc. 319, 369-456. 7 Pullen, R. A . , Lindsay, D. G., Wood, S. P., Tickle, 1. J., Blundell, T. L., Wollmer, A,, Krail. G., Brandenburg:? I)., Kahn, H., Gliemann, J. and Gammeltoft, S. (1976). Receptor-binding region of insulin. Nature 259.369-373. 8 Derewenda, U., Derewenda, Z. S., Dodson, G. G. and Hubhard, K. E. (1990). Insulin structure. In: Handbook of Experimental Pharmacology Vol 92 (cds P. Cuatrecasas and S. Jacobs) pp. 23-29. Springer-Vcrlag. 9 Eigenhrot, C., Randal, M., Quan, C., Burnier, J., O’Connell, L., Rinderknecht. E. and Kossiakoff, A. A. (1991). X-ray structure of human relaxin at 1.5A. Comparison to insulin and implications for receptor binding determinants. J . dlol. B i d . 221. 15-21. 10 Kriiger, P., Gilge, G., Cahuk, Y. and Wollmer, A. (1990). Cooperativity and intermediate states in the T + R structural transformation of insulin. Bid. Chem. Hoppr-Seyler 371, 669-673. 11 Chothia, C . , Lesk, A. M., Dodson, G. G. and Hodgkin, D. C. (1983). Transmission of conformational change in insulin. Nature 302. 500-5. 12 Blundell, T. L., Cleasby, A., Murray-Rust, J. et al. Unpublished. 13 Caves, L. S. D., Nguyen, D. T. and Hubhard, R. E. (1990). Coiiformational variability of Insulin: a molecular dynamic5 analysis. In: Molecular Dynarrzics: .4pplicafions in Molecular Biology (ed J.M. Goodfellon) pp 27-68. Macmillan, London. 14 Jhoti, H., McLeod, A. N., Blundell, T. L., lshizaki, H., Nagasawa, H. and Suzuki, A. (1987). Prothoracicotrophic hormone har an insulin-like tertiary structure. FEBS T-.P~LJ. 219, 419-425. 15 Blundell, T. L., Bedarkar, S., Rinderknecht, E. and Humbel, K. E. (1978). Insulin-likc growth factor: a model for tertiary structure accounting for immunoreactivity and receptor binding. Proc. NatlAcad. Sci. USA 75. 180-184. 16 McLeod, A. N. and ,Murray-Rnst, J. tJnpublishcd work. 17 Redarkar, S., Turnell, W. G., Blundell, T. L. and Schwahe, C. (1977). Rclaxin has conformational homology with insulin. Nature 270, 449-451. 18 Kline, A. D. and Justice, K. M., Jr (1990). Coniplcte Sequence-Specific ‘H NMR As5ignments for Human Insulin. Biochemistry 29, 2906-13. 19 Hua, Q. and Weiss, M. A. (191). Comparative 2D NMR studies of human insulin and des-pentapeptide insulin: sequential resonaiicc assignment and implications for protciii dynamics and receptor recognition. Biochemistry 30, 5505-5515. 20 Cooke, R. M . , Harvey, T. S. and Campbell, 1. D. (1991). Solution structure of human insulin-like growth factor 1: A nuclear magnetic resoiiancc aiid 30, 5484-5491. reqtrained molecular dynamics study. Biochemisfrj~ 21 Blundell, T. L. and Humbel, R. E. (1980). Hormone families: pancreatic hormones and homologous growth factors ilralurc 287, 781-787. 22 Rinderknecht, E. and Humbel. R. E. (1978). The amino-acid sequence of human insulin-likc growth factor I aiid its structural homology with proinsulin. J . Biol. Cheni. 253, 2769-2776. 23 Kinderknecht, E. and Humbel, R. ti. (1978). Primary structurc of human insulin-likc growth factor 11. FEBS Lettr. 89. 283-286.
24 Schwahe, C., McDonald, J. K. and Steinetz, R. G . (1977). Primary structure of the B-chain of porcine relaxin. Biochenz. Biophys. Res. Comm. 75,503-510. 25 Nagasawa, H., Suzuki, A. and Ishizaki, H. (1986). Aminoacid sequence of a prothoracicotrophic hormone of the bilkworm Bombyk mori. Proc. Natl Acud. S u . U S 2 83. 5830-5843. 26 S d t , A. B., Vreugdenhil, E., Ebberink, R. H. M., Geraerts, W. P. M., Klootwijk, J. and Joose, J. (1988). Growth controlling molluscan neurons produce the precursor of an insulin-related peptidc. Na/ure 331, 535-538. 27 Felsenstein, J. (1985). KITSCH computer program. Evolution 39, 739-791. 28 Ehina, Y., Ellis, L., Jarnigan, K., Edery, M., Graf, C., Clauser, E., Ou, J., Masiarz, F., Kan, Y. W., Goldfine, I. D., Roth, R.A. and Rutter, W. J. (198.5). ’The human insulin reccptor cDNA: the structural basis for hormone-activated transmembrane signalling. Cell 40, 747-758. 29 Ullrich, A., Gray, A., Tam, A. W., Yang-Feng, T., Tsuhokawa, M., Collins, C., Henzel, W., Lebon, T., Kathuna, S., Chen, E. S., Jacobs, S., Franke, U., Ramachandran. J. and Fujita-Yamaguchi, Y. (1986). Insulin-like growth factor 1 receptor primary structure: comparisoii with insulin receptor suggests structural determinants that define functional specificity. EMBO J. 5, 25032512. 30 Bajaj, M., Waterfield, M. D., Schlessinger, J., Taylor, W. R. and Bluudell, T. I,. (1987). O n the tertiary structures of the extracelliilar domains of the E G F and insulin receptors. Biochirn. BiuphyA. Arro 916, 220-226. 31 Schumacher, R., Mosthaf, L., Schlessinger, J., Brandenburg, D. and Ullrich, A. (1991). Insulin and Indin-like growth factor-1 binding specificity is determined by distinct regions of their cognate receptors. J. Bid. Chem. 266, 19288-19295. 32 Schaefer, E. K., Siddle, K. and Ellis, L. (1990). Deletion analysis of the human insulin receptor cctodomain rcvcals indepciidcutly folded soluhle subdomains and insulin binding by a inonomeric n-subunit. J . B i d . Chem. 265, 13248-13753. 33 Gustahon, T. A. and Rutter, W. J. (1990). The cysteine-rich domains pf the insulin and inaulin-like growth factor 1 receptors are primary determinants of hormone binding specificity. I . B i d . Clzwn. 265, 18663-18667. 34 Grmntield, C., Hiles, I., Waterfield, kl. D., Federwisch, M., Wollmer, A., Rlundell, T. L. and McDonald, N. (1989). Epidcrmal growth factor binding iuduces a conformatioual change in the external domain of its receptor. EMBO J , 8. 4115-4123. 35 Maddux, B. A. and Goldfine, I. D. (1991). Evidence that inaulin plus ATP may induce a conformational change in the subunit of the insulin receptor without inducing receptor autopliosphnrylation. J. Biol. Chem. 266.6731-6736. 36 Dodson, E. J., Dodson, G. G., Huhbard, R. E. and Reynolds, C. D. (1983). Insulin’s structural behaviour and its relation to activity. Biopolymers 22, 281291. 37 Brems, D. N., Brown, P. L., Nakagawa, S. H. and Tager, H. S. (1991). The conformational stability and flexibility of insulin with an additional intramolecular cross-link. J . B i d . Chem. 266, 1611-1615. 38 Markusscn, J., JWgenscn, K,, Surensen, A. and Arsthim, I. (1985). Single chain des-(U30) insulin. Irzf. J . Peptlde Prot~irrReseawh 26. 70-77. 39 Derewenda, U., Derewenda, Z., Dodson, E. J., Dodson, G. G., Bing, X. and Markusson, J. (1991). X-ray analysis of the single chain B29-A1 peptide-linked insulin molecule. J . Mol. B i d . 220, 425-433. 40 Nakagawa, S. H. and Tager, H. S. (1991). Implications of invariant residue LeuB6 in insulin-receptor interactions. J . B i d . Chew. 266, 11502-11509. 41 McLeod, A. N., Auf der Mauer, A. and Wood. S. P. (1990). Higirperformance liquid chromatography of insulin: accessibility and flexibility. J . Chromatography 502, 325-336. 42 Hua, Q . X., Shoclson, S. E., Knchuyan, M. and Weiss, M. A. (1991). Receptor binding redefined by a structural switch in a mutant human insulin. Nalure 354, 238-241. 43 Tager, H. S. (1990). Mutant human insulins and insulin structurc-function relationships. In: Hundbook of Enperimmtal Pharmacology Vol 92 1990, (eds Cuatrecasas. P. aiid Jacobs, S.) pp. 41-63. Springer-Verlag. 44 Fischer, W. H., Saunders, D., Brandenburg, D., Wollmer, A. and Zahn, H. (1985). A rhnrtcncd insulin \%ithfull in vitro potency. Biol. Chem. Hoppe-Seyler 366. 521-525. 45 Hartmann, H., Oherhaus, K., Spahr, R., Brandenburg, D., Creutzfeldt, W. and Prohst, I. (1989). Biological activity of des-(B26-B3O)-iiisuliiianLide and related analogues in rat hepatocyle cultures. Diahetologia 32. 416-420. 46 Nakagawa, S. H. and Tager, H. S. (1986). Role of the phenylalanine 825 side chain in dirccting insulin interactiou with its receptor. J. Biol. Chem. 261, 7332-7341. 47 hfirmima, R. G., Nakagawa, S. H. and Tager, H. S. (1991). Importance of the character and configuration of residues BW. B25 and B26 in insulinreceptor interactions. J . Bid. Chem 266, 1428-1436.
T. L. Blundell, J. Murray-Rust, A. N. McLeod and S. P. Wood are at Laboratory of Molecular Biology and ICRF Unit of Structural Molecular Biology, Department of Crystallography, Birkbeck College, Malet Street, London WClE 7HX, UK.
Introd uction Tn the years since 1955when the amino acid sequence of insulin was determined by Sanger and co-workers(l?’) our knowledge of insulin and related proteins has increased dramatically. We now have sequences of more than 80 insulins and insulin-related proteins as well as site-directed mutants and chemically modified insulins. There are structures in several oligomeric states defined in the crystals by X-ray analysic, and in solution by 2-dimensional nmr. The relationship between sequence variation and the role of parts of the structure in processing, stora e, transport and receptor binding have been explored(3 . The insulin receptor has been identified, sequenced, cloned and expressed. However, although we have extensive knowledge of insulins and their receptors, the nature of the interaction between insulin and its receptor and the events that transduce the signal are less well understood. Early proposals for a receptor-binding region on the insulin molecule centred around the first 3-dimensional structure(4) as defined in crystals of 2-zinc insulin hexamers. Receptor interaction was postulated to involve a surface region of the insulin molecule, which partly overlaps with the hydrophobic surface involved in dimer formation. However, evidence from assays of binding and activity of modified and mutated insulins, and from structural variations in the monomer in different environments, has suggested the possibility that the monomer may adopt a slightly different conformation at the receptor than it does in the dimer. Here we review the crystal and NMR structures, studies of insulin sequences, mutagenesis and chemical modification, and studies of the receptor itself. These contribute to our present understanding of the molecular mechanism of receptor binding.
The First Crystal Structure More than 20 years ago, determination of the X-ray crystal structure of 2-zinc porcine insulin(’) provided the first direct experimental evidence for the insulin fold. In this structure, insulin protomers form a hexamer composed of three identical dimers assembled around a three-fold axis (Fig. 1). The original structure has undergone subsequent refinement. but the essential components are unchanged(‘). In the dimer the two monomers are not completely identical, but both have their A and B chains folded to form compact globular structures with a number of common features (Fig. 2). These include: 1. an A chain with two helices (A2-A7 and A13-A19), joined by an extended loop(A8-Al2); 2. a B chain with an extended N-terminus, followed by a helix (B9-B19), followed by a sharp turn (B20-B23) and an extended C-terminal section; 3, three disulphide bridges (A6-All, A7-B7, A20-Bl9); and 4. a hydrophobic core. In the dimcr, an antiparallel {j-sheet is formed between the C-termini of the B chains of adjacent monomers and the B chain helices pack together. This leads to the burying of hydrophobic sidechains. Additional, but less strong, hydrophobic contacts are made between dimers in hexamer formation. Early Models for Receptor Binding An early suggestion that some of the elements involved in dimer formation wcre involved in receptor interaction was supported by the relatively high conservation of putative binding residues across the inyulin family. The biological activity of analogues with chemical modifications in the dimer-forming surface and the
?
d
Fig. 1. Assembly of an insulin hexamer. Pairs of insulin molecules come together about 2-fold axes in the plane of the diagram to form three dimers, which then assemble around a three-fold axis perpendicular to the plane of the diagram, along with two zinc ions, to form the ‘2-zinc’ insulin hexamer.
Fig. 2. The insulin monomer. A ribbon diagram through the main-chains of the insulin molecule. The A-chain is shaded, and the sulphur atoms of the disulphidebonds are represented as dotted spheres.
crystal structures of some modified in~ulins(~) were consistent with this view. Studies on sequence variant and modified insulins have also indicated that, some residues, which are not part of the diiner foi-ming surface may be iiivolved in receptor binding. For example, a low-activity mutant human insulin has been identified where A3 valine is replaced with leucine; substitution of B10 histidine by aspartic acid (as found in some patients with hyperproinsulinaemia) has been shown to make insulin more active; and the presence of histidine at A8, as found in some bird insulins, can also be associated with high activity. The spatial location of the residues on the insulin monomer, both 'dimerforming' and further removed (e.g. A3, BlO) (Fig. 3 ) gives rise to the idea of an 'extended binding region' of residues which have all been shown to be important in receptor interaction, but not all of which are solvent accessible in the protomer as found in the crystal structure. More X-ray Studies Since the initial structure determination, the threedimensional structure of insulin has been studied intensively. Crystal structures have been solved for insulins in different aggregation states (monomer, dimer, hexamer), for insulin crystallised in differing solvent conditions which alter the monomer conformation, for sequence variant insulins from different species, for chemically modified insulins and for des(B26-B30)insulin (DPI). It has become clear that, although most of the common features listed above are
Fig. 3. A receptor's view of the insulin molecule'? Residues which are labelled have been shown to be important in receptor binding.
retained, there is considerable conformational variation in the insulin monomer (Fig. 4). [For a detailed review, see reference 81. A recently determined X-ray crystal structure of human relaxin(') shows an insulin fold, differing only in that the truncated C-terminal region of the B chain is an extension of the B chain helix rather than an insulin-like turn. In the insulin molecule the most extreme variation is in the N-terminus of the B chain, which may exist either in an extended conformation or as an extension of the B chain helix. However, this is thought to be a property of the hexarner('") and therefore not directly relevant to receptor binding. Superimposing insulin structures defined in different crystal forms, it is seen that the major secondary structural elements are capable of individual rigid body movements without major changes to the structure. These movements are accommodated by alterations in side chain conformations and are generally determined by packing interactions in the crystal. Relatively small changes in one part of the molecule may be transmitted by this mechanism to distant regions of the molecule. The insulin monomer is, therefore, rather flexible('''. Crystallographic studies on proinsulin(12)show that the A and B chains of the insulin moiety retain a similar structure to that in other crystal structures, while the C-
\
A
B
Fig. 4. Superimpositionof some insulin crystal structures. Achains are shown in dotted, and B-chains in continuous 1ines.The structures used were: the two molecules from 2-zinc insulin, two molecules from 4-zinc insulin. one molecule from the monoclinic insulin hexamer and one molecule from des-
pentapeptide insulin. peptide is not well-defined, because of either static disorder or mobility. Molecular dynamic simulations(13) starting from several different crystallographically defined protomer structures reinforce these concepts, highlighting relative movements of the helices and flexibility of the chain termini. Computer modelling showed that the other members of the family can adopt an insulin-like fold for the regions corresponding to the A and B chains of insulin("-17), although some are single chain molecules with B and A chains linked by a C-peptide, and A or B chain extensions may be present (Fig. 5).
Nuclear Magnetic Resonance Insulin would appear to be a natural candidate for the recently developed 2-dimensional NMR methods for large molecules, but problems in obtaining a spectrum of the molecule in a single known aggregation state have impeded a complete sequential assignment. The presence of multiple aggregation states in solution is one cause of line broadening in the spectra; but dilution to a point where the solution contains only monomers leads to weak signals and consequent long data collection times. However, com lete assignments have now been made for insulin(ls2') and IGFlC2"),and spectra measured for various related molecules. The solution structures of insulin are broadly similar to the crystal structure, and the IGFl structure is similar to the model based on the insulin fold, although greater flexibility is reported in the helix extremities and chain
r:
Fig. 5. Computer models of members of the insulin family. The models werc built on computer graphics systems using the coordinatcs of the insulin crystal structures as a starting point. The regions corresponding to the insulin A chains are shown as heavy lines. (a) IGFl, (b) relaxin, ( c ) bombyxin-2, (d) molluscan insulin-like peptide-1.
termini. Again, relative movements of the structural elements are a salient feature. In both IGFl and proinsulin the C-peptides are apparently mobile.
Sequences of Members of the Insulin Family Insulins and related peptides("] have been identified in several phyla of the animal kingdom. Most have been found in the vertebrates, but examples have also been identified in proto-chordates, insects and molluscs. This distribution may reflect the relative extent to which these groups have been studied rather than the actual distribution of the molecules in nature. The family comprises the insulins, the insulin-like growth factors (IGFs)(zz~z3j, the relaxins(24)),the bombyxins(") and the molluscan insulin-like peptides (MIPS)('@. Apart from the IGFs, all contain separate A and B chains. The C peptides of the IGFs are not cleaved. They are single-chain peptides organised in a manner analogous to proinsulin, with the C peptide linking the B chain C-terminus to the A chain Nterminus. A C-terminal extension to the A chain forms the D region and, in some forms of IGF, there is an additional C-terminal E region which may be removed in post-translational processing. Within the family the
PTTHs (bombyxins) dogfish relaxin shark relaxin skate relaxin human relaxin porcine and whale relaxins rat relaxin
lengths of the A and B chains are rather variable. In particular. the MIPs havc a long N-terminal extension to the H chain containing a cysteine which may form an additional disdphide bridge with the cysteine found at A4 in these molecules. The sequences of the insulin family have been clustered and a tree (Fig. 6), similar to a phylogenetic tree, constructed by cluster analysis techniques(27). Since bombyxins and MIPs each represent multiple sequences from the same species, this is not a true phylogenetic tree in the sense that it parallels the evolutionary relationship between species, but is rather a ‘molecular phylogeny’. The tree represents evolutionary divergence up to the point where the insulins have diverged from the IGFs. After the IGFs, the bombyxins are next most closely related to the insulins. The next are the relaxins which fall into two groups, mammalian and fish, which diverge before the bombyxins diverge from the lGFs and insulins. Surprisingly the insuliiis and IGFs are less divergent than the relaxins of different species and most remarkably, the MIPS, which are all derived from a single species, show even more divergence. In considering the common features of the sequences, it is useful to define a ‘common framework’ for
Fig. 6. Relationships in the insulin fdmily. Comparison of the sequences of the members of the famly allows them to be arranged in drder of similarity to form a ‘molecular phylogeny’.
the molecule. Sequence data suggest that structural elements conserved throughout the family include the disulphide bridges, the three helices and their spatial relationship, A and B chain turns and the buried hydrophobic residues. We exclude the extended regions of the B chain, and chain extensions or links found in some non-insulin members of the family, An analysis of sequence variability is given in Figure 7; within the common framework all sequences can be aligned without insertions or deletions. Of the structurally important residues in insulin, only thc six halfcystines and the glycine at B8 are rigidly conserved in all known sequences. The glycine at B20 is conserved in most insulins, being substituted only in hystricomorph insulins which do not dimerise. It is conserved in all relaxins and all but but one IGF (rat IGF2). B23 glycine is conserved in all known insulins and IGFs, but is substituted in the relaxins, MTPs and bombyxin-IV. Lack of glycines at B20 and B23 in MIPs makes the presence of an insulin-like B chain turn in these molecules rather unlikely. Across the family, other residues generally conserved are those buried in the insulin monomer (A2, A16, B11, B15) and residues at the N- and C-termini of the A chain. Of the residues involved in forming the insulin dimer, B12 is conserved
25
2
1 5
s
x c ._ Q
m ._
1
2
’
05
0
-0 5
-1
‘I I I I I I I I I I 1 I I I I I 1 I I I I I I I I 1 I I
I I
I I I I I I I I I I
1 I I I I I I I
I I
II
G IVEQCCHSTCSLYQLENYCNFVNRHLCGSHLVEA LYLVCGERGFFYTPKA
Residue number
Fig. 7. Sequence variability at each residue in the insulin molecule. The histogram represents the reciprocal of the percentage conservation for each residue in the consensus sequence for the insulin family. The data are scaled with A4 arbitrarily taken as zero, so that negative values represent more conserved residues and positive values more variable residues. The consensus sequence is shown on the X axis, and the numbers associated with each residue are the sequence numbers in the A and B chains. A database of 80 sequences of members of the insulin family was used.
as valine in all insulins, IGFs and mammalian relaxins; B24 is conserved as phenylalanine in all insulins and IGFs. Regions of high variability occur in the regions A8-Al0, the N-terminus of the B chain and at B27. Of the helices, residues in the second A chain helix are less well conserved than in the other two. Examination of the sequence variation enables us to assess the possibility of attaining the 2Zn insulin type of fold, and models have been constructed of IGFs, relaxins, bombyxins and MIPS (Fig. 5 ) . Most residues in the extended binding region of insulin are well conserved in other members of the family. Exceptions are at A8, where the consensus residue histidine is associated with high activity in avian and mammalian insulins but not in fish: and A17, A18 and B21. So far regions determining receptor specificity have not been identified for these molecules. The Insulin Receptor The first step in the action of both insulin and IGFs is binding to a cell surface receptor. Both insulin and IGFl receptors have been sequenced, and shown to be derived from distinct but related genes(28729)); insulin and IGFl can bind to both receptors, but each binds to its own with much greater affinity. The receptors are membrane proteins, which in their mature forms are disulphide bonded a& heterotetramers. with extracellular, mcmbrane-spanning and intracellular (tyrosine kinase) domains. A diagram of our proposed model of an ap subunit of the insulin and IGFl receptors, based on sequence alignment and secondary structure prediction(”) is shown in Figure 8. The large variable regions (L1 and L2) apparently consist of four structural motifs joined by variable loops. or five a-@ The cystine residues in the S domains appear to form both inter- and intra-chain disulphide bonds, although the connectivity is unknown. Ligand binding is confined to the extracellular domain of the receptor, which contains one high affinity binding site per receptor subunit. Detailed discussion of the IGF1-IGF1 receptor system is beyond the scope of this essay, but there is evidence that binding specificity is determined by distinct regions of their receptors(31).Each of the two ligand-receptor systems could thur have co-evolved from a common ancestor. When insulin binds to the extracellular domain, tyrosine kinase activity is stimulated, leading to phosphorylation in the intracellular domain of the /?subunit, the initial step in the cellular events. The ability to construct and express mutants of the extracellular domain of the insulin receptor now allows systematic investigation of areas of sequence involved in insulin binding. Monomeric a-subunit can bind insulin only slightly less well than secreted intact extracellular domain(32). Within the subunit, both L and S regions have been proposed as the insulin binding site. Chimaeric receptors formed by coupling the extracellular domain of one receptor to the intracellular
Alpha Subunit
Ll
1
Large subunits
\
L2
1
W Fig. 8. The insulin and lGFl receptors. The schematic diagram shows just an a$’ dimer of the a2J$receptor. Binding of the ligand to the extracellular domain causes a signal to be transmitted through the cell membrane which activates autophosphorylation between subunits in the cytoplasmic domain, the first of a cascade of events in the cell.
domain of another are functional, with ligand specificity determined by the extracellular domain(33). It has been shown(”) that binding of epidermal growth factor causes a conformational change in the ectodomain of its receptor (which is similar to insulin’s, but monomeric). In the insulin receptor two conformational changes have been postulated; the first, on binding insulin to the a-subunit increases ATP binding to the B-subunit; the @-subunitthen undergoes a further conformational chan e which is important for transmembrane signallingb”’. Ligand-receptor contacts can be assumed to be rather extensive, and not necessarily confined to one portion of primary, or even secondary structure.
Discussion If we assume that insulin binds to its receptor as a monomer, then we must consider the possible effects of stability, flexibility and other properties of the monomer in receptor binding. The two interchain disulphide bridges are essential to the stability of the insulin molecule, as shown by the ease with which the chains can be separated following oxidation of the disulphides
and the difficulties associated with chain recombination reactions. Whilst these disulphides restrict monomer flexibility, considerable potential remains, and this has long been recognised as important for receptor interaction(36). Flexibility can be reduced by crosslinking the C-terminus of the B chain and the Nterminus of the A-chain, which are quite close in space. Links between the sidechain of B29 and the LY amino group of A1 cause reduced activity(37);and a peptide bond between B29 and A1 in des-B30 single chain insulin (SCI)(38)causes a total loss of activity which cannot be completely explained by the loss of charge at A l . Al-B29 crosslinked insulin is known from crystallographic evidence to be similar to the native protomer in three-dimensional structure, except for the reduced flexibility(”). The N-terminus of the B chain shows considerable variation in that it cxists in helical and several extended conformations. A recent study has em hasized the importance of LeuB6 in receptor bindinggo), probably through stabilisation of the tertiary structure, although dirwt receptor interactions are not excluded. The C-terminus of the B chain appears to play a pivotal role in the expression of insulin’s biological activity. Superimposition of crystal structures shows the C-terminus of the B chain to be relatively mobile and in DPI, which does not dimerise, it adopts a conformation (determined by crystal packing interactions) , different from that in any other insulin crystal structure. Investigation of insulin analogues by analytical HPLC has suggested that in the presence of organic solvent the B chain may unfold from B24(41).NMR studies of the insulin monomer in aqueous solution suggest that the residues up to B28 are in a similar position to that found in the crystal structure(”), although in solutions containing organic solvent it appears to be displaced(18). of a mutant insulin with B24 An NMR phenylalanine replaced by glycine, which is a full agonist, shows destabilisation of the B chain turn and a loss of contact between the C-terminal residues of the B chain and the rest of the molecule. This exposes residues such as A2 and A3 which form part of the binding region shown in Figure 3. The identification of mutant human insulins with reduced activity and substitutions at B24 and B25 (both Phe in native insulin) (for a review, see ref. 43) has reinforced interest in this part of the molecule. In DPI, the B chain from B25 to the C-terminus is missing, yet the molecule retains significant activity. Removal of B25 and B24 leads to progressively reduced activity and, where both residues are missing, the molecule is virtually inactive. However, amidation of the free carboxylate of B25 in DPI restores activity to levels similar to native insulin(44),suggesting that the C-terminal pentapeptide is not necessary for full activity, and indeed that DPI may be a good starting point from which to model the receptor-bound conformation of insulin. In studies with DPI amides, it has been shown that, where B25 is substituted with an aromatic amino acid,
particularly one with a polar sidechain. activity may be either normal or significantly enhanced(45). However, effects observed for specific substitutions in whole insulin may be significantly different in DPI amide, suggesting a modulating role for the terminal pentapeptide. B25 has been implicated as a potential primary contact between the hormone and its receptor. It has been suggested(46) that, following initial binding between the B25 sidechain and a specific pocket in the receptor, conformational changes take place in both the hormone and the receptor which lead to a secondary binding event involving other areas of the insulin monomer, leading to expression of full biological activity. Recent studies on the region B24 Phe, B25 Phe and B26 Tyr suggest(j7)that these residues all have a role in determining insulin-receptor interactions, influencing the conformation of the monomer and its ability to aggregate. But none of the native side chains is absolutely required for receptor binding, since analogues have been identified where these residues are individually either deleted or substituted by residues with non-aromatic sidechains but which retain the ability to bind the receptor, albeit with altered affinity. Conformational constraints imposed by a D amino acid at B24 can lead to enhanced affinity. The C-terminus of the insulin B chain has, therefore, an obvious role in receptor interaction. There is, however, considerable indirect evidence that this part of the molecule must undergo some conformational change on receptor binding. If it moves away from the surface of the monomer, a5 it does in non-aqueous solution, a surface similar to that in DPI will be exposed. The receptor binding region would then involve residues such as A3 which would otherwise be sterically hindered by the C-terminal residues of the B chain. It would also involve other residues such as A4. A8 A19, B9 and I310 that are not involved in dimer formation; this would explain the greater affinity of a monomer for the receptor than for another monomer. Aromatic residues in positions corresponding to B24B26 are found in all insulins (apart from some hystricomorphs where B 26 is non-aromatic) and IGFs, all of which show some affinity for the insulin receptor, but not in the relaxins, bombyxins or MIPS. There is no evidence that these latter molecules show any affinity for the insulin receptor, and their relative lack of conservation of the glycines at B20 and B23 suggest that the C-terminus of the B chain does not follow the insulin fold. It is likely, however, that the other elements of their common frameworks are similar to insulin. The receptor binding region described here involves regions occluded by the C-terminus of the B chain in the conformer stabilized in the porcine insulin dimer. This region (for example A3 Val) is relatively well conserved amongst the broader insulin family. It is interesting to speculate that the hormone and its receptor may have co-evolved. If this is the case, the receptor binding site
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T. L. Blundell, J. Murray-Rust, A. N. McLeod and S. P. Wood are at Laboratory of Molecular Biology and ICRF Unit of Structural Molecular Biology, Department of Crystallography, Birkbeck College, Malet Street, London WClE 7HX, UK.
