title: Deciphering the role of the electrostatic interactions involving Gly70 in eglin C by total chemical protein synthesis.
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.
authors.affiliation: Gryphon Sciences, South San Francisco, California 94080, USA.
authors.forename: W Y
authors.initials: WY
authors.lastname: Lu
authors.forename: M A
authors.initials: MA
authors.lastname: Starovasnik
authors.forename: J J
authors.initials: JJ
authors.lastname: Dwyer
authors.forename: A A
authors.initials: AA
authors.lastname: Kossiakoff
authors.forename: S B
authors.initials: SB
authors.lastname: Kent
authors.forename: W
authors.initials: W
authors.lastname: Lu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D005998:Glycine / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D007865:Leeches; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D011506:Proteins; D015843:Serpins / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism*; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000037:antagonists & inhibitors
chemical_list: D011506:Proteins; D015843:Serpins; C028656:eglin proteinase inhibitors; D013381:Subtilisins; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D005998:Glycine