Effects of Protein-Ligand Associations on the Subunit Interactions of Phosphofructokinase from B. stearothermophilus

Abstract

Differences between the crystal structures of inhibitor-bound and uninhibited forms of phosphofructokinase (PFK) from B. stearothermophilus have led to a structural model for allosteric inhibition by phosphoenolpyruvate (PEP) wherein a dimer-dimer interface within the tetrameric enzyme undergoes a quaternary shift. We have developed a labeling and hybridization technique to generate a tetramer with subunits simultaneously containing two different extrinsic fluorophores in known subunit orientations. This construct has been utilized in the examination of the effects of allosteric ligand and substrate binding on the subunit affinities of tetrameric PFK using several biophysical and spectroscopic techniques including 2-photon, dual-channel fluorescence correlation spectroscopy (FCS). We demonstrate that PEP-binding at the allosteric site is sufficient to reduce the affinity of the active site interface from beyond the limits of experimental detection to nanomolar affinity, while conversely strengthening the interface at which it is bound. The reduced interface affinity is specific to inhibitor binding because binding the activator ADP at the same allosteric site causes no reduction in subunit affinity. With inhibitor bound, the weakened subunit affinity has allowed the kinetics of dimer association to be elucidated.

Phytochemical Drug Discovery for COVID-19 Using High-resolution Computational Docking and Machine Learning Assisted Binder Prediction

“…92 The NSP7-NSP8 heterodimer and NSP10 are both subunits of larger protein complexes, and ligands docked near the binding sites of the subunits could potentially affect the ability of the subunits to assemble into functional oligomers via allosteric modulation. 93 Clustering and Classification of Phytochemical Ligands. The Ward hierarchical clustering method and the Random Forest method were selected to cluster and classify phytochemicals, respectively.…”

Structure of the Apo Form of Bacillus stearothermophilus Phosphofructokinase

“…In the apo structure, His 249 forms a hydrogen bond with Tyr 164 across the binding interface. The breaking of the hydrogen bond between His 249 and Tyr 164 when PEP binds to the enzyme may contribute to the substrate-binding interface becoming weaker in response to PEP binding. , Perhaps the formation of a hydrogen bond across the substrate-binding interface between His 249 and either Tyr 164 or Glu 161 is important for tetramer stability. Therefore, the addition of PEP may cause a perturbation between these residues and in the quaternary structure of the enzyme to weaken the substrate-binding interface.…”

“…The breaking of the hydrogen bond between His 249 and Tyr 164 when PEP binds to the enzyme may contribute to the substrate-binding interface becoming weaker in response to PEP binding. 15,28 Perhaps the formation of a hydrogen bond across the substrate-binding interface between His 249 and either Tyr 164 or Glu 161 is important for tetramer stability. Therefore, the addition of PEP may cause a perturbation between these residues and in the quaternary structure of the enzyme to weaken the substrate-binding interface.…”

Structural Insights into the Regulation of Staphylococcus aureus Phosphofructokinase by Tetramer–Dimer Conversion

“…Interface II is responsible for binding of the substrates, while interface I contains the binding site for allosteric effectors (Figure A,B). Two allosteric effectors for bacterial Pfks have been reported, including the allosteric activator ADP-Mg and the inhibitor phosphoenolpyruvate (PEP), both of which are K-type allosteric effectors (change the affinity of the enzyme for the substrate so that the K m value is altered) of the enzyme for F6P without changing its maximal activity. , By comparison of the crystal structures of Pfk from Bacillus stearothermophilus ( Bs Pfk) in complex with phosphoglycolate [PGA; a PEP analogue; Protein Data Bank (PDB) entry 6PFK] and Bs Pfk bound to F6P and ADP-Mg (PDB entry 4PFK), an R-state/T-state model was proposed for the allosteric behavior of bacterial Pfks (Figure C). ,, In this model, the quaternary structure of the substrate- and activator-bound R state of Pfk undergoes a 7° relative rotation of dimer CD with respect to dimer AB as compared with the inhibitor-bound T state of Pfk, thereby repacking the dimers across interface II, which increases the binding affinity of the enzyme for the F6P substrate …”