title: Mutagenesis of the bovSERPINA3-3 demonstrates the requirement of aspartate-371 for intermolecular interaction and formation of dimers.
abstract: 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.
authors.affiliation: Faculté des Sciences et Techniques, INRA, UMR1061 Unité de génétique Moléculaire Animale, Université de Limoges, FR 3503 GEIST, 87060 Limoges, France.
authors.forename: X
authors.initials: X
authors.lastname: Blanchet
authors.forename: A
authors.initials: A
authors.lastname: Péré-Brissaud
authors.forename: N
authors.initials: N
authors.lastname: Duprat
authors.forename: E
authors.initials: E
authors.lastname: Pinault
authors.forename: D
authors.initials: D
authors.lastname: Delourme
authors.forename: A
authors.initials: A
authors.lastname: Ouali
authors.forename: C
authors.initials: C
authors.lastname: Combet
authors.forename: A
authors.initials: A
authors.lastname: Maftah
authors.forename: P
authors.initials: P
authors.lastname: Pélissier
authors.forename: L
authors.initials: L
authors.lastname: Brémaud
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015843:Serpins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; C402207:SERPINA3 protein, Bos taurus; D015843:Serpins; D001224:Aspartic Acid