Probing intermolecular backbone H-bonding in serine proteinase-protein inhibitor complexes

BACKGROUND: Intermolecular backbone H-bonding (N-H.O=C) is a common occurrence at the interface of protein-protein complexes. For instance, the amide NH groups of most residues in the binding loop of eglin c, a potent serine proteinase inhibitor from the leech Hirudo medicinalis, are H-bonded to the carbonyl groups of residues in the target enzyme molecules such as chymotrypsin, elastase and subtilisins. We sought to understand the energetic significance of these highly conserved backbone-backbone H-bonds in the enzyme-inhibitor complexes. RESULTS: We synthesized an array of backbone-engineered ester analogs of eglin c using native chemical ligation to yield five inhibitor proteins each containing a single backbone ester bond from P3 to P2' (i.e. -CONH-to -COO-). The structure at the ligation site (P6-P5) is essentially unaltered as shown by a high-resolution analysis of the subtilisin-BPN'-eglin c complex. The free-energy changes (DeltaDeltaGNH-->O) associated with the binding of ester analogs at P3, P1 and P2' with bovine alpha-chymotrypsin, subtilisin Carlsberg and porcine pancreatic elastase range from 0-4.5 kcal/mol. Most markedly, the NH-->O substitution at P2 not only stabilizes the inhibitor but also enhances binding to the enzymes by as much as 500-fold. CONCLUSIONS: Backbone H-bond contributions are context dependent in the enzyme-eglin c complexes. The interplay of rigidity and adaptability of the binding loop of eglin c seems to play a prominent role in defining the binding action.

Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.

Intermolecular backbone H-bonding (N-H.O=C) is a common occurrence at the interface of protein-protein complexes.

Leech-derived molecules this paper discusses, each with a full profile in the ASH compound registry:

Erfahren Sie, wie diese Forschung mit der klinischen Praxis verknüpft ist

More peer-reviewed studies in Arzneimittelentwicklung.

• Novel Leech Antimicrobial Peptides — Hirunipins — Kumar et al. (2025)
• Medicinal Leech Genome Assembly — Anticoagulant Gene Catalog — Kvist et al. (2020)
• Effects of hirudotherapy on liver functions, lipid profile, and insulin sensitivity in rats with metabolic syndrome — Bilden A et al. (2026)
• Hirudotherapy in Medicine and Dentistry — Jha K et al. (2015)
• Medicinal leech therapy (hirudotherapy): a brief overview — Singh A (2010)
• HIRUDOTHERAPY IN PRESBYACUZIS PRAECOX IN CLEAN-UP WORKERS AFTER THE CHORNOBYL ACCIDENT DURING THE POST-CATASTROPHE YEARS — Zabolotnyi D, Mishchanchuk N (2019)
• Investigation of oxidant-antioxidant status in patients treated with hirudotherapy: an experimental study — Sarikan I, Savas H (2024)
• Leech therapeutic applications — Abdualkader A et al. (2013)
• Non-Occlusive Mesenteric Ischemia Due to Hirudotherapy: A Case Report — Akalın Ç, Ekmen N (2020)
• Leech therapy: a history — Hyson J (2005)

Browse all ASH-analysed research →