title: 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.
authors.affiliation: Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic , v.v.i., Flemingovo nám 2, 166 10 Prague 6, Czech Republic.
authors.forename: Lucie
authors.initials: L
authors.lastname: Kosinová
authors.forename: Václav
authors.initials: V
authors.lastname: Veverka
authors.forename: Pavlína
authors.initials: P
authors.lastname: Novotná
authors.forename: Michaela
authors.initials: M
authors.lastname: Collinsová
authors.forename: Marie
authors.initials: M
authors.lastname: Urbanová
authors.forename: Nicholas R
authors.initials: NR
authors.lastname: Moody
authors.forename: Johan P
authors.initials: JP
authors.lastname: Turkenburg
authors.forename: Jiří
authors.initials: J
authors.lastname: Jiráček
authors.forename: Andrzej M
authors.initials: AM
authors.lastname: Brzozowski
authors.forename: Lenka
authors.initials: L
authors.lastname: Žáková
mesh_terms: D000621:Aminoisobutyric Acids / Q000737:chemistry; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry*; D008958:Models, Molecular; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular
chemical_list: D000621:Aminoisobutyric Acids; D007328:Insulin; C100049:2-aminoisobutyric acid