Mapping the functional surface of insulin by design: structure and function of a novel A-chain analogue.

Functional surfaces of a protein are often mapped by combination of X-ray crystallography and mutagenesis. Such studies of insulin have yielded paradoxical results, suggesting that the native state is inactive and reorganizes on receptor binding. Of particular interest is the N-terminal alpha-helix of the A-chain. Does this segment function as an alpha-helix or reorganize as recently proposed in a prohormone-convertase complex? To correlate structure and function, we describe a mapping strategy based on protein design. The solution structure of an engineered monomer ([AspB10, LysB28, ProB29]-human insulin) is determined at neutral pH as a template for synthesis of a novel A-chain analogue. Designed by analogy to a protein-folding intermediate, the analogue lacks the A6-A11 disulphide bridge; the cysteine residues are replaced by serine. Its solution structure is remarkable for segmental unfolding of the N-terminal A-chain alpha-helix (A1 to A8) in an otherwise native subdomain. The structure demonstrates that the overall orientation of the A and B chains is consistent with reorganization of the A-chain's N-terminal segment. Nevertheless, the analogue's low biological activity suggests that this segment, a site of clinical mutation causing diabetes mellitus, functions as a preformed recognition alpha-helix.

Insulin-like compounds related to the amphioxus insulin-like peptide.

Three insulin-like compounds consisting of two disulfide-linked polypeptide chains have been synthesized. The A-chains of these compounds correspond either to the A- or to the A + D-domain of the putative amphioxus insulin-like peptide (amphioxus ILP), and their B-chains correspond either to the B-chain of insulin or to a slightly modified (i.e., [1-Thr]) B-domain of amphioxus ILP. The biological potency of these compounds was evaluated in mammalian cells or cell fractions containing either human or rat insulin receptors or human or mouse insulin-like growth factor I (IGF-I) receptors, with respect to binding affinity, insulin-like metabolic activity (lipogenesis), and growth factor activity (mitogenesis). Amphioxus ILP A/bovine insulin B and amphioxus ILP A + D/bovine insulin B exhibited potencies ranging from 2.0 to 9.8% relative to natural insulin, and both compounds were full agonists in lipogenesis assays, stimulating lipogenesis to the same maximal extent as seen with natural insulin. Amphioxus ILP A/amphioxus ILP [1-Thr]B stimulated lipogenesis with a potency of 0.01% relative to natural insulin. We consider this compound also likely to be a full agonist. In assays measuring binding to IGF-I receptors and stimulation of mitogenesis, these compounds displayed some activity although the activity was too low for exact quantification. These results suggest that amphioxus ILP has retained an overall structural similarity to mammalian insulin and IGF-I but has also accumulated substantial mutations which markedly reduce its ability to bind and activate their cognate receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Possible involvement of the A20-A21 peptide bond in the expression of the biological activity of insulin. 1. [21-Desasparagine,20-cysteinamide-A]insulin and [21-desasparagine,20-cysteine isopropylamide-A]insulin.

The C-terminal region of the A chain of insulin has been shown to play a significant role in the expression of the biological activity of the hormone. To further delineate the contribution of this segment, we have synthesized [21-desasparagine,20-cysteinamide-A]insulin and [21-desasparagine,20-cysteine isopropylamide-A]insulin, in which the C-terminal amino acid residue of the A chain of insulin, asparagine, has been removed and the resulting free carboxyl group of the A20 cysteine residue has been converted to an amide and an isopropylamide, respectively. Both insulin analogues display biological activity, 14-15% for the unsubstituted amide analogue and 20-22% for the isopropylamide analogue, both relative to bovine insulin. In contrast, a [21-desasparagine-A]insulin analogue has been reported to display less than 4% of the activity of the natural hormone [Carpenter, F. (1966) Am. J. Med. 40, 750-758]. The implications of these findings are discussed, and we conclude that the A20-A21 amide bond plays a significant role in the expression of the biological activity of insulin.

Possible involvement of the A20-A21 peptide bond in the expression of the biological activity of insulin. 3. [21-Desasparagine,20-cysteine ethylamide-A]insulin and [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin.

We have synthesized [21-desasparagine,20-cysteine ethylamide-A]insulin and [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin, which differ from natural insulin in that the C-terminal amino residue of the A chain, asparagine, has been removed and the resulting free carboxyl group of the A20 cysteine residue has been converted to an ethylamide and a trifluoroethylamide group, respectively. [21-Desasparagine,20-cysteine ethylamide-A]insulin displayed equivalent potency in receptor binding and biological activity, ca. 12% and ca. 14%, respectively, relative to bovine insulin. In contrast, [21-desasparagine,20-cysteine 2,2,2-trifluoroethylamide-A]insulin displayed a divergence in these properties, ca. 13% in receptor binding and ca. 6% in biological activity. This disparity is ascribed to a difference in the electronic state of the A20-A21 amide bond in these two analogues. A model is proposed to account for the observation of divergence between receptor binding and biological activity in a number of synthetic insulin analogues and naturally occurring insulins. In this model, changes in the electronic state and/or the orientation of the A20-A21 amide bond can modulate biological activity independently of receptor binding affinity. The A20-A21 amide bond is thus considered as an important element in the "message region" of insulin.

Hybrid molecules containing the B-domain of insulin-like growth factor I are recognized by carrier proteins of the growth factor.

The insulin-like growth factors (IGFs) are polypeptides in plasma that are chemically related to insulin and have mitogenic and insulin-like activity. Unlike insulin, the IGFs circulate in plasma bound to specific high molecular weight carrier proteins that regulate their delivery to target tissues. To define the sites on the IGFs that allow them to be recognized by carrier proteins, we constructed hybrid molecules containing different portions of the insulin, IGF-I, and IGF-II molecules. The presence of the B domain of IGF-I, but not the D domain of IGF-II, enables these insulin-IGF hybrid molecules to be recognized by acid-stripped IGF carrier proteins from rat serum and other sources. By contrast, neither the BIGF-I nor DIGF-II domain is sufficient to enable binding to type II IGF receptors, despite the fact that type II receptors, like the carrier protein, specifically bind IGF-I and IGF-II but do not interact with insulin. By differentiating those sites on the IGF molecule required for binding to IGF carrier protein and receptors, the insulin-IGF hybrid molecules should help delineate the role of the carrier protein in presenting biologically active IGF to target tissues.