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Mutagenesis of the bovSERPINA3‐3 demonstrates the requirement of aspartate‐371 for intermolecular interaction and formation of dimers
Author(s) -
Blanchet X.,
PéréBrissaud A.,
Duprat N.,
Pinault E.,
Delourme D.,
Ouali A.,
Combet C.,
Maftah A.,
Pélissier P.,
Brémaud L.
Publication year - 2012
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2078
Subject(s) - intermolecular force , mutagenesis , chemistry , site directed mutagenesis , intermolecular interaction , stereochemistry , biophysics , computational chemistry , biochemistry , biology , mutation , mutant , molecule , gene , organic chemistry
The family of serpins is known to fold into a metastable state that is required for the proteinase inhibition mechanism. One of the consequences of this conformational flexibility is the tendency of some mutated serpins to form polymers, which occur through the insertion of the reactive center loop of one serpin molecule into the A‐sheet of another. This “A‐sheet polymerization” has remained an attractive explanation for the molecular mechanism of serpinopathies. Polymerization of serpins can also take place in vitro under certain conditions (e.g., pH or temperature). Surprisingly, on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, bovSERPINA3‐3 extracted from skeletal muscle or expressed in Escherichia coli was mainly observed as a homodimer. Here, in this report, by site‐directed mutagenesis of recombinant bovSERPINA3‐3, with substitution D371A, we demonstrate the importance of D371 for the intermolecular linkage observed in denaturing and reducing conditions. This residue influences the electrophoretic and conformational properties of bovSERPINA3‐3. By structural modeling of mature bovSERPINA3‐3, we propose a new “non‐A‐sheet swap” model of serpin homodimer in which D371 is involved at the molecular interface.