Structure of the apo form of Bacillus stearothermophilus phosphofructokinase

Abstract

The crystal structure of the unliganded form of Bacillus stearothermophilus phosphofructokinase (BsPFK) was determined using molecular replacement to 2.8 Å resolution (Protein Data Bank entry 3U39 ). The apo BsPFK structure serves as the basis for the interpretation of any structural changes seen in the binary or ternary complexes. When the apo BsPFK structure is compared with the previously published liganded structures of BsPFK, the structural impact that the binding of the ligands produces is revealed. This comparison shows that the apo form of BsPFK resembles the substrate-bound form of BsPFK, a finding that differs from previous predictions.

Figure 1

Apo BsPFK tetramer, subunit A colored blue, subunit B colored green, subunit C colored yellow, subunit D colored red, and calcium ions colored black. (A) View of apo BsPFK showing the substrate binding interface between red and blue monomers and green and yellow monomers, allosteric interface between red and yellow monomers and blue and green monomers. (B) Alternate view, rotated 90° along the horizontal axis.

Figure 2

Comparison of wild-type BsPFK crystal structures. (A) Apo BsPFK (B) Substrate-bound BsPFK (accession code 4PFK) (12) with active-site and effector-site ADP in space filled purple. (C) PGA-bound BsPFK (accession code 6PFK) (13) with PEP in space filled cyan. The blue monomer was aligned to the blue monomer of the apo structure for each ligand bound structure. Each monomer is colored blue, green, yellow, and red. The substrate binding interfaces are between the red and blue monomers and the green and yellow monomers. The effector binding interfaces are between the green and blue monomers and the red and yellow monomers. Areas where there are differences in the tertiary and quaternary structures are pointed out with yellow arrows.

Figure 3

Electron density of the apo PFK Fru-6-P binding site (A) and effector site (B). Figures were rendered in CCPRMG molecular graphics software (38). Shown in coral colored mesh is a 2F₀-F_c “kick” map (39) contoured at 1 sigma. Protein residues are shown as sticks colored by element, with carbon as blue. Side chains lacking density, or with ambiguous density, are not shown.

Figure 4

Overlay of residues in the Fru-6-P binding site of BsPFK. (A) Apo residues in blue, substrate-bound BsPFK residues in green, and Fru-6-P in black ball and stick. (B) Apo residues in blue and PGA-bound BsPFK residues in red. All residues are labeled.

Figure 5

Overlay of residues in the effector binding site of BsPFK. (A) Apo residues in blue, substrate-bound BsPFK residues in green, ADP in gray ball and stick, and Mg ion in yellow. (B) Apo residues in blue, PGA-bound BsPFK residues in red, and PGA in purple ball and stick.