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.

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Backbone-ester analogs of eglin c, a leech serine proteinase inhibitor, probed intermolecular H-bonds with chymotrypsin, subtilisin and elastase; contributions were context dependent, and an NH-to-O substitution at P2 strengthened binding up to 500-fold.

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