title: Probing intermolecular backbone H-bonding in serine proteinase-protein inhibitor complexes.
abstract: 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.
authors.affiliation: Gryphon Sciences, 250 East Grand Avenue, Suite 90, South San Francisco, CA 94080, USA. wuyuanlu@midway.uchicago.edu
authors.forename: W
authors.initials: W
authors.lastname: Lu
authors.forename: M
authors.initials: M
authors.lastname: Randal
authors.forename: A
authors.initials: A
authors.lastname: Kossiakoff
authors.forename: S B
authors.initials: SB
authors.lastname: Kent
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000378:metabolism*
chemical_list: D046911:Macromolecular Substances; D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases