Insight into the Structural and Biological Relevance of the T/R Transition of the N-Terminus of the B-Chain in Human Insulin

Abstract

The N-terminus of the B-chain of insulin may adopt two alternative conformations designated as the T- and R-states. Despite the recent structural insight into insulin–insulin receptor (IR) complexes, the physiological relevance of the T/R transition is still unclear. Hence, this study focused on the rational design, synthesis, and characterization of human insulin analogues structurally locked in expected R- or T-states. Sites B3, B5, and B8, capable of affecting the conformation of the N-terminus of the B-chain, were subjects of rational substitutions with amino acids with specific allowed and disallowed dihedral φ and ψ main-chain angles. α-Aminoisobutyric acid was systematically incorporated into positions B3, B5, and B8 for stabilization of the R-state, and N-methylalanine and d-proline amino acids were introduced at position B8 for stabilization of the T-state. IR affinities of the analogues were compared and correlated with their T/R transition ability and analyzed against their crystal and nuclear magnetic resonance structures. Our data revealed that (i) the T-like state is indeed important for the folding efficiency of (pro)insulin, (ii) the R-state is most probably incompatible with an active form of insulin, (iii) the R-state cannot be induced or stabilized by a single substitution at a specific site, and (iv) the B1–B8 segment is capable of folding into a variety of low-affinity T-like states. Therefore, we conclude that the active conformation of the N-terminus of the B-chain must be different from the “classical” T-state and that a substantial flexibility of the B1–B8 segment, where GlyB8 plays a key role, is a crucial prerequisite for an efficient insulin–IR interaction.

Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin

“…monomer is considered biologically active, 56,57 these results correlate well with the observed dimeric nature of HMWP. The fact that a sigmoidal full agonistic response was observed was probably due to a 0.5%-1% content of insulin monomer in the sample identified by analytical SEC (data not shown).…”

Human insulin analogues modified at the B26 site reveal a hormone conformation that is undetected in the receptor complex

“…The nature of the B-chain N-terminus (B1-B8), which in storage forms is generally found in the so-called T-state (extended) or R-state (helical, expanding the B9-19 helix), also remains undetermined in these complexes. However, our recent findings indicate that neither classical R-or T-states fulfil the criteria for an active hormone conformation and that high flexibility of the B-chain N-terminus is crucial for full hormone activity (Kosinová et al, 2013).…”

Amino acid modifications for conformationally constraining naturally occurring and engineered peptide backbones: Insights from the Protein Data Bank

“… However, not all examples of peptides incorporating Aib constrained the peptide backbone, as predicted. For example, introducing a single Aib (at the eighth position) in insulin chain A did not stabilize a helical conformation of the N‐terminus as seen in the R‐state of insulin (PDB ID 2mpg), suggesting that other parameters may have a role in stabilizing that conformation.…”