Ensemble-Based Segmental Helical Organization in an Aib-Rich Lipidated Peptide: Biomacromolecular Insights from High-Field NMR.

Abstract

Tirzepatide (TZP) is a dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonist that has demonstrated remarkable therapeutic efficacy for the treatment of type 2 diabetes and obesity. Despite its clinical importance, residue-specific information on its solution conformation remains limited due to its structural complexity, including noncanonical amino acids and a lipidated side chain. In this study, we performed a solution NMR investigation of tirzepatide to establish residue-specific resonance assignments and characterize its conformational properties in an aqueous solution. Backbone and side-chain resonances were assigned using multidimensional NMR spectroscopy, including 1H-15N HMQC, TOCSY, and NOESY experiments. Reliable sequential NOE connectivities were identified for several peptide segments, while ambiguous and unresolved resonances were conservatively excluded from the structural interpretation. The N-terminal Aib2 residue served as an important assignment anchor but did not define a stable terminal structure. In contrast, the Aib13 region exhibited a locally organized backbone environment with a short helical-like conformational tendency, suggesting Aib13-centered local ordering within the peptide chain. Structure calculations based on the 1.2 GHz NMR and CD experiments revealed that tirzepatide does not adopt a single rigid conformation in solution but instead exists as a conformational ensemble composed of locally ordered and flexible regions. These findings provide a residue-specific view of tirzepatide dynamics in solution and offer structural insights into the conformational landscape of this therapeutically important peptide.

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