Modelling of the disulphide-swapped isomer of human insulin-like growth factor-1: implications for receptor binding.
Insulin-like growth factor-1 (IGF-1) is a serum protein which unexpectedly folds to yield two stable tertiary structures with different disulphide connectivities; native IGF-1 [18-61,6-48,47-52] and IGF-1 swap [18-61,6-47, 48-52]. Here we demonstrate in detail the biological properties of recombinant human native IGF-1 and IGF-1 swap secreted from Saccharomyces cerevisiae. IGF-1 swap had a approximately 30 fold loss in affinity for the IGF-1 receptor overexpressed on BHK cells compared with native IGF-1. The parallel increase in dose required to induce negative cooperativity together with the parallel loss in mitogenicity in NIH 3T3 cells implies that disruption of the IGF-1 receptor binding interaction rather than restriction of a post-binding conformational change is responsible for the reduction in biological activity of IGF-1 swap. Interestingly, the affinity of IGF-1 swap for the insulin receptor was approximately 200 fold lower than that of native IGF-1 indicating that the binding surface complementary to the insulin receptor (or the ability to attain it) is disturbed to a greater extent than that to the IGF-1 receptor. A 1.0 ns high-temperature molecular dynamics study of the local energy landscape of IGF-1 swap resulted in uncoiling of the first A-region alpha-helix and a rearrangement in the relative orientation of the A- and B-regions. The model of IGF-1 swap is structurally homologous to the NMR structure of insulin swap and CD spectra consistent with the model are presented. However, in the model of IGF-1 swap the C-region has filled the space where the first A-region alpha-helix has uncoiled and this may be hindering interaction of Val44 with the second insulin receptor binding pocket.
D016475:3T3 Cells; D017667:Adipocytes; D000818:Animals; D002845:Chromatography; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007328:Insulin / Q000737:chemistry; D007334:Insulin-Like Growth Factor I / Q000737:chemistry*; D007536:Isomerism; D051379:Mice; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D013329:Structure-Activity Relationship; D013936:Thymidine / Q000378:metabolism; D013997:Time Factors