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

Background Intermolecular backbone H-bonding (Ndouble bond; length as m-dashH…Odouble bond; length as m-dashC) 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. double bond; length as m-dashCONHdouble bond; length as m-dash to double bond; length as m-dashCOOdouble bond; length as m-dash). The structure at the ligation site (P6-P5) is essentially unaltered as shown by a high-resolution analysis of the subtilisin-BPNʹ-eglin c complex. The free-energy changes (ΔΔGNH→O) associated with the binding of ester analogs at P3, P1 and P2′ with bovine α-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.