Deciphering the role of the electrostatic interactions involving Gly70 in eglin C by total chemical protein synthesis
Structural biology study published in Biochemistry (2000)
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.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Resumen
Total chemical protein synthesis of eglin C variants probing the role of Gly70 in electrostatic interactions — reveals fold-stability determinants.
Por qué esto importa para la hirudoterapia
This research used total chemical protein synthesis to understand the structural role of Gly70 in eglin c, a potent serine proteinase inhibitor from the medicinal leech (Hirudo medicinalis). The study demonstrated that Gly70 is crucial for the inhibitor's structural stability and binding affinity through its participation in an electrostatic and hydrogen-bonding network. Understanding these structural dynamics is valuable for ASH members interested in the fundamental protein engineering and stability of leech-derived biotherapeutics. The caveat is that this is a highly specialized in vitro biophysical study focused purely on protein structure and kinetics, without any direct clinical or whole-organism hirudotherapy application.
Citación
Deciphering the role of the electrostatic interactions involving Gly70 in eglin C by total chemical protein synthesis.
Lu WY et al. · Biochemistry, 2000
Contexto clínico relacionado
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Añadido a la biblioteca ASH: May 27, 2026 · Última actualización del sitio: June 18, 2026