Mutations at hypothetical binding site 2 in insulin and insulin-like growth factors 1 and 2 result in receptor- and hormone-specific responses

Information on how insulin and insulin-like growth factors 1 and 2 (IGF-1 and -2) activate insulin receptors (IR-A and -B) and the IGF-1 receptor (IGF-1R) is crucial for understanding the difference in the biological activities of these peptide hormones. Cryo-EM studies have revealed that insulin uses its binding sites 1 and 2 to interact with IR-A and have identified several critical residues in binding site 2. However, mutagenesis studies suggest that Ile-A10, Ser-A12, Leu-A13, and Glu-A17 also belong to insulin's site 2. Here, to resolve this discrepancy, we mutated these insulin residues and the equivalent residues in IGFs. Our findings revealed that equivalent mutations in the hormones can result in differential biological effects and that these effects can be receptor-specific. We noted that the insulin positions A10 and A17 are important for its binding to IR-A and IR-B and IGF-1R and that A13 is important only for IR-A and IR-B binding. The IGF-1/IGF-2 positions 51/50 and 54/53 did not appear to play critical roles in receptor binding, but mutations at IGF-1 position 58 and IGF-2 position 57 affected the binding. We propose that IGF-1 Glu-58 interacts with IGF-1R Arg-704 and belongs to IGF-1 site 1, a finding supported by the NMR structure of the less active Asp-58–IGF-1 variant. Computational analyses indicated that the aforementioned mutations can affect internal insulin dynamics and inhibit adoption of a receptor-bound conformation, important for binding to receptor site 1. We provide a molecular model and alternative hypotheses for how the mutated insulin residues affect activity.


Mutations at hypothetical binding Site 2 in insulin and insulin-like growth factors 1 and 2 elicit receptor-and hormone-specific responses
Macháčková et al. Table of contents   Table S1 Binding affinities and receptor activation abilities of analogs for IR-A.

Table S2
Binding affinities and receptor activation abilities of analogs for IR-B.

Table S3
Binding affinities and receptor activation abilities of analogs for IGF-1R. Representative Western blots for relative abilities of insulin analogs to activate receptors'

Figure S7
Differential contact maps calculated from metadynamics of the B-chain C-terminus opening for insulin analogs HisA10, ThrA12, HisA13, HisA17.

Figure S8
Structural characterization of the metadynamics ensembles -hydrophobic collapse and hydrogen bond analysis.
Supplementary references Table S1. Receptor-binding affinities of human insulin, IGF-1, IGF-2 and analogs for human IR-A in membranes of IM-9 lymphocytes and relative abilities of the hormones/analogs to stimulate phosphorylation of human IR-A in membranes of transfected mouse fibroblasts (details are provided in Methods           1.09

Differential Contact Maps calculated from metadynamics of the insulin B-chain C-terminus opening
The inter-residue contacts with a negative difference in contact lifetime between the wild-type and mutants are those that are more frequently present in mutant compared to wild-type insulin (colored in orange in Figure S7). A range of hydrophobic contacts between the B-chain N-terminus (PheB1, ValB2) and the A chain (LeuA13, LeuA16) were established to maintain the collapsed state for HisA10 and HisA13 mutants, as well as the closed state for the HisA17 mutant. Contacts between the B-chain N-terminus and residues in the B-chain α-helix were more frequently present in the collapsed/closed states of the Hisinsulin mutants. For example, the contacts in HisA13 mutant between residues HisB5-GlyB8, GlnB4-HisB10, GlnB4-GluB13 contributed to the partial collapse of the B-chain α-helix. For the native-like affinity ThrA12 contacts that were formed more frequently than in the wild type (for example IleA2, ValA3 with LeuB15, LeuB11) maintained the hydrophobic interface between A/B chains in a more compact state.

Structural characterization of the metadynamics ensembles
The insulin hydrophobic core comprises residues IleA2, ValA3, GlyB8, LeuB11, ValB12, LeuB15, PheB24 and TyrB26 (8). We calculated its size in terms of the radius of gyration Rgyr comprising all atoms for the respective residues. The hydrophobic core is more compact for the mutants compared to the wild-type insulin, except for the HisA10-mutant which collapses to extended states ( Figure S8A). Connected to the extent of the hydrophobic collapse, we observed different protein-water hydrogen bonding local to the point of mutation. The number of water-protein hydrogen bonds was determined for a shell of the radius 1 nm around the Cα (CA) atom of a mutated residue. With OH and NH groups regarded as donors and O and N atoms as acceptors, the donor-acceptor cutoff distance was set to 0.35 nm and the angle hydrogendonor-acceptor to 30°. The number of water-protein H-bonds in a sphere of a 1 nm radius around the Cα atom of a mutated/wild-type residue is plotted in Figure S8B comparing the HisA17-mutant and ThrA12mutant in red to wild-type native insulin in black. The dynamics of H-bond formation is more stable in the closed compact state of HisA17-mutant. On the other hand, the bulkier ThrA12 is buried inside the core and forms less H-bonds with water than the wild-type SerA12.
A B Figure S8. A. Free energy profiles with respect to the radius of gyration of residues defining the hydrophobic core. B. The number of protein-solvent hydrogen bonds in the sphere of 1 nm around Cα atoms of mutated residues: Cα (ThrA12) in ThrA12-insulin mutant and Cα (SerA12) in wild-type insulin (up), Cα (HisA17) in HisA17-insulin mutant and Cα (GluA17) in native insulin (down). Insulin wt is a native wild-type insulin.