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

Background: Intermolecular backbone H-bonding (N-H ... O=C) is a common occ urrence at the interface of protein-protein complexes. For instance, the am ide NH groups of most residues in the binding loop of eglin c, a potent ser ine proteinase inhibitor from the leech Hirudo medicinalis, are H-bonded to the carbonyl groups of residues in the target enzyme molecules such as chy motrypsin, elastase and subtilisins, We sought to understand the energetic significance of these highly conserved backbone-backbone H-bonds in the enz yme-inhibitor complexes, Results: We synthesized an array of backbone-engineered eater analogs of eg lin c using native chemical ligation to yield five inhibitor proteins each containing a single backbone eater 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 (Delta Delta G(NH-->O)) associated with the bindin g of eater analogs at P3, P1 and P2' with bovine cc-chymotrypsin, subtilisi n 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 enz yme-eglin c complexes. The interplay of rigidity and adaptability of the bi nding loop of eglin c seems to play a prominent role in defining the bindin g action.