Deciphering the role of the electrostatic interactions involving Gly70 in eglin C by total chemical protein synthesis

Biochemistry. 2000 Apr 4;39(13):3575-84. doi: 10.1021/bi992292q.

Abstract

Eglin c from the leech Hirudo medicinalis is a potent protein inhibitor of many serine proteinases including chymotrypsin and subtilisins. Unlike most small protein inhibitors whose solvent-exposed enzyme-binding loop is stabilized primarily by disulfide bridges flanking the reactive-site peptide bond, eglin c possesses an enzyme-binding loop supported predominantly by extensive electrostatic/H-bonding interactions involving three Arg residues (Arg48, Arg51, and Arg53) projecting from the scaffold of the inhibitor. As an adjacent residue, the C-terminal Gly70 participates in these interactions via its alpha-carboxyl group interacting with the side chain of Arg51 and the main chain of Arg48. In addition, the amide NH group of Gly70 donates an H-bond to the carbonyl C=O groups of Arg48 and Arg51. To understand the structural and functional relevance of the electrostatic/H-bonding network, we chemically synthesized wild-type eglin c and three analogues in which Gly70 was either deleted or replaced by glycine amide (NH(2)CH(2)CONH(2)) or by alpha-hydroxylacetamide (HOCH(2)CONH(2)). NMR analysis indicated that the core structure of eglin c was maintained in the analogues, but that the binding loop was significantly perturbed. It was found that deletion or replacement of Gly70 destabilized eglin c by an average of 2.7 kcal/mol or 20 degrees C in melting temperature. As a result, these inhibitors become substrates for their target enzymes. Binding assays on these analogues with a catalytically incompetent subtilisin BPN' mutant indicated that loss or weakening of the interactions involving the carboxylate of Gly70 caused a decrease in binding by approximately 2 orders of magnitude. Notably, for all four synthetic inhibitors, the relative free energy changes (DeltaDeltaG) associated with protein destabilization are strongly correlated (slope = 0.94, r(2) = 0. 9996) with the DeltaDeltaG values derived from a decreased binding to the enzyme.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Chymotrypsin / antagonists & inhibitors
  • Glycine / chemistry*
  • Glycine / metabolism*
  • Kinetics
  • Leeches
  • Molecular Sequence Data
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Denaturation
  • Proteins
  • Serpins / chemical synthesis*
  • Serpins / chemistry
  • Serpins / metabolism*
  • Static Electricity
  • Structure-Activity Relationship
  • Subtilisins / antagonists & inhibitors

Substances

  • Proteins
  • Serpins
  • eglin proteinase inhibitors
  • Subtilisins
  • Chymotrypsin
  • alpha-chymotrypsin
  • Glycine