Fig 1.
Amino acid sequence and structure of PGK1.
N-terminal domain (violet), C-terminal domain (green), hinge region (blue) (PDB: 2XE7, open conformation). (A) Secondary structural elements are shown at the top of the amino acid sequence. Mutated residues are depicted in bold red. The dots under the sequence represent the residues involved in 3-PG or 1,3-BPG binding (pink dot) and in ATP or ADP binding (green dot). (B) Location of the mutations on PGK1 structure (open conformation). Mutated residues are depicted in scaled ball and stick and 3-PG and ADP in stick.
Table 1.
Kinetic parameters of PGK1 wild type and variants.
Fig 2.
Effect of temperature on kinase activity of PGK1 wild type and variants.
(A) Temperature dependence of kinase activity of PGK1 wild type and variants. (B) Non-linear fit of the temperature dependence of PGK1 activity to the Arrhenius equation Eq (1). Assays were performed under the conditions described in Materials and Methods, using 0.18–12.0 nM enzyme.
Table 2.
Effect of temperature on kinase activity of PGK1 wild type and variants.
Table 3.
Melting temperatures and thermodynamic parameters for urea-induced unfolding equilibrium of PGK1 wild type and variants measured by far-UV CD and fluorescence spectroscopy.
Table 4.
Superimposition between the PGK1 wild type and the crystallized variants.
Fig 3.
Detail of the 3-PG binding site in the variant R38M in comparison with PGK1 wild type.
Overlap of R38M variant (green) and PGK1 wild type crystallized (A) in the absence of 3-PG (PDB 2ZGV) (light grey) or (B) in the presence of 3-PG (PDB: 2XE7) (grey). The view in B is rotated about 90° anticlockwise with respect to A. ADP, 3-PG and the residues involved in 3-PG binding are shown as sticks.
Fig 4.
Detail of the 3-PG binding site in the variant G166D in comparison with PGK1 wild type.
Overlap of G166D variant (cyan) and PGK1 wild type crystallized (A) in the absence of 3-PG (PDB 2ZGV) (light grey) or (B) in the presence of 3-PG (PDB: 2XE7) (grey). The view in B is rotated about 90° anticlockwise with respect to A.
Fig 5.
ADP binding site in the variant V216F in comparison with PGK1 wild type.
(A) Detail of the binding of ADP to V216F variant (orange). ADP and the residues involved in the interaction are depicted as sticks, Mg2+ ion as a green sphere. (B) Superimpostition with wild type PGK1 crystallized with ADP and 3-PG (PDB: 2XE7) (grey), highlighting how the mutation V216F impairs the movement of the loop 212–218. The residue V/F216 and ADP are shown as sticks.
Fig 6.
M189I variant in comparison with PGK1 wild type.
(A) Detail of the binding of 3-PG to M189I variant in the partially-closed conformation (magenta) superimposed with partially-closed wild type PGK1 (PDB: 2XE7) (grey). 3-PG and the residues involved in the interaction are depicted as sticks. (B) Superimpostition of M189I variant in the closed conformation (pink) with wild type PGK1 crystallized with ADP, 3-PG and MgF3- (PDB: 2WBZ) (deep teal). MgF3- is shown as green and cyan spheres. Three water molecules (red spheres) bound to M189I mimic the position of F- in MgF3-.