title: The folding unit of phosphofructokinase-2 as defined by the biophysical properties of a monomeric mutant.
abstract: Escherichia coli phosphofructokinase-2 (Pfk-2) is an obligate homodimer that follows a highly cooperative three-state folding mechanism N2 ↔ 2I ↔ 2U. The strong coupling between dissociation and unfolding is a consequence of the structural features of its interface: a bimolecular domain formed by intertwining of the small domain of each subunit into a flattened β-barrel. Although isolated monomers of E. coli Pfk-2 have been observed by modification of the environment (changes in temperature, addition of chaotropic agents), no isolated subunits in native conditions have been obtained. Based on in silico estimations of the change in free energy and the local energetic frustration upon binding, we engineered a single-point mutant to destabilize the interface of Pfk-2. This mutant, L93A, is an inactive monomer at protein concentrations below 30 μM, as determined by analytical ultracentrifugation, dynamic light scattering, size exclusion chromatography, small-angle x-ray scattering, and enzyme kinetics. Active dimer formation can be induced by increasing the protein concentration and by addition of its substrate fructose-6-phosphate. Chemical and thermal unfolding of the L93A monomer followed by circular dichroism and dynamic light scattering suggest that it unfolds noncooperatively and that the isolated subunit is partially unstructured and marginally stable. The detailed structural features of the L93A monomer and the F6P-induced dimer were ascertained by high-resolution hydrogen/deuterium exchange mass spectrometry. Our results show that the isolated subunit has overall higher solvent accessibility than the native dimer, with the exception of residues 240-309. These residues correspond to most of the β-meander module and show the same extent of deuterium uptake as the native dimer. Our results support the idea that the hydrophobic core of the isolated monomer of Pfk-2 is solvent-penetrated in native conditions and that the β-meander module is not affected by monomerizing mutations.
authors.affiliation: Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile.
authors.forename: César A
authors.initials: CA
authors.lastname: Ramírez-Sarmiento
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Casilla 233, Santiago, Chile.
authors.forename: Mauricio
authors.initials: M
authors.lastname: Baez
authors.affiliation: Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile.
authors.forename: Ricardo A
authors.initials: RA
authors.lastname: Zamora
authors.affiliation: Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California.
authors.forename: Deepa
authors.initials: D
authors.lastname: Balasubramaniam
authors.affiliation: Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile.
authors.forename: Jorge
authors.initials: J
authors.lastname: Babul
authors.affiliation: Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California. Electronic address: ekomives@ucsd.edu.
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Komives
authors.affiliation: Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile. Electronic address: vguixe@uchile.cl.
authors.forename: Victoria
authors.initials: V
authors.lastname: Guixé
mesh_terms: D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D025481:Phosphofructokinase-2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D021122:Protein Subunits; D025481:Phosphofructokinase-2