Abstract
Apart from its role in insulin receptor (IR) activation, the C terminus of the B-chain of insulin is also responsible for the formation of insulin dimers. The dimerization of insulin plays an important role in the endogenous delivery of the hormone and in the administration of insulin to patients. Here, we investigated insulin analogues with selective N-methylations of peptide bond amides at positions B24, B25, or B26 to delineate their structural and functional contribution to the dimer interface. All N-methylated analogues showed impaired binding affinities to IR, which suggests a direct IR-interacting role for the respective amide hydrogens. The dimerization capabilities of analogues were investigated by isothermal microcalorimetry. Selective N-methylations of B24, B25, or B26 amides resulted in reduced dimerization abilities compared with native insulin (K(d) = 8.8 μM). Interestingly, although the N-methylation in [NMeTyrB26]-insulin or [NMePheB24]-insulin resulted in K(d) values of 142 and 587 μM, respectively, the [NMePheB25]-insulin did not form dimers even at high concentrations. This effect may be attributed to the loss of intramolecular hydrogen bonding between NHB25 and COA19, which connects the B-chain β-strand to the core of the molecule. The release of the B-chain β-strand from this hydrogen bond lock may result in its higher mobility, thereby shifting solution equilibrium toward the monomeric state of the hormone. The study was complemented by analyses of two novel analogue crystal structures. All examined analogues crystallized only in the most stable R(6) form of insulin oligomers (even if the dimer interface was totally disrupted), confirming the role of R(6)-specific intra/intermolecular interactions for hexamer stability.
Highlights
Insulin is an important polypeptide hormone that controls a wide range of cellular processes such as the regulation of blood glucose uptake and has a large impact on protein and lipid metabolism
The solid state-based structural insight into the insulin molecule is limited to inactive dimeric or hexameric storage forms [1,2,3], whereas the insulin monomer represents the active form of the hormone when binding to the insulin receptor (IR)
Binding Affinities—Insulin analogues were prepared by trypsin-catalyzed semisynthesis, and their binding affinities to the insulin receptor in membranes from rat adipose tissue were determined (Table 1 and Fig. 1)
Summary
Insulin is an important polypeptide hormone that controls a wide range of cellular processes such as the regulation of blood glucose uptake and has a large impact on protein and lipid metabolism. The regions of the insulin molecule responsible for the interaction with the IR [3, 14] or for its dimerization and hexamerization [15, 16] have been functionally and structurally identified in a number of insulin analogues. Our previous studies on the structural and functional relevance of the B26 [17, 25, 26] and B25 [27] positions of insulin focused on modulation of hormone conformation by the incorporation of an N-methyl group on the amide nitrogen of the peptide bond in shortened and full-length insulin analogues. We investigated the selective application of this N-methylation within the B24 –B26 region of insulin by the systematic elimination of the inter/intramolecular hydrogen bonds, which are important for the stability and folding of the insulin monomer, dimer formation, and interaction with IR. The approach reported here represents an application of a very specific, chemically controlled molecular tool for the functional assessment of the hydrogen bond network related to the dimer interface
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