Structural Insights into the Regulation of <i>Staphylococcus aureus</i> Phosphofructokinase by Tetramer–Dimer Conversion

Most reported bacterial phosphofructokinases (Pfks) are tetramers that exhibit activity allosterically regulated via conformational changes between the R and T states. We report that the Pfk from <i>Staphylococcus aureus</i> NCTC 8325 (<i>Sa</i>Pfk) exists as both an active tetramer and an inactive dimer in solution. Multiple effectors, including pH, ADP, ATP, and adenylyl-imidodiphosphate (AMP-PNP), cause equilibrium shifts from the tetramer to dimer, whereas the substrate F6P stabilizes <i>Sa</i>Pfk tetrameric assembly. Crystal structures of <i>Sa</i>Pfk in complex with different ligands and biochemical analysis reveal that the flexibility of the Gly150-Leu151 motif in helix α7 plays a role in tetramer–dimer conversion. Thus, we propose a molecular mechanism for allosteric regulation of bacterial Pfk via conversion between the tetramer and dimer in addition to the well-characterized R-state/T-state mechanism

Human Cbx3 chromodomain binds to methylated histone H1K26 and G9aK185.

<p>ITC data for Cbx3 chromodomain binding to (A) H1K26 peptides (residues 18–29) and (B) G9aK185 peptides (residues 179–190). Lower panel show fit to a one-site binding model to the binding isotherms.</p

Comparison of three structures of Cbx3 chromodomain binding to methylated histone H3, H1 and G9a peptides.

<p>(A) Superposition of human Cbx3 chromodomain in complex with methylated histone H1 peptide (yellow), histone H3 peptide (orange), G9a peptide (cyan), Cbx3 chromodomains are colored as magenta, gray and green, respectively. (B) Superposition of histone H1 peptide (yellow), histone H3 peptide (orange). (C) Structure of Cbx3-H3K9me3 complex (magenta) was superposed to one protomer of the tetramer of Cbx3-G9aK185me3 complex (green) formed in one asymmetric unit. (D) The α helix (residues 70 to 79) of the chromodomain in the structure of Cbx3-G9aK185me3 complex (green) shifts 4.9 Å away from its counterpart in the structures of Cbx3-H3K9me3 (magenta).</p

X-ray Data collection and refinement statistics.

a<p>The values in parentheses refer to statistics in the highest shell.</p>b<p>Rmerge = |Ii−<i>|/|Ii| where Ii is the intensity of the ith measurement, and <i>is the mean intensity for that reflection.</i></i></p><i><i>c<p>Rwork = Σh|Fo(h)−Fc(h)|/ΣhFo(h), where Fo and Fc are the observed and calculated structure factor amplitudes, respectively.</p>d<p>Rfree was calculated with 10% of the reflections in the test set.</p>e<p>Categories were defined by MolProbity.</p></i></i

Structure basis for Cbx3 binding to methylated histone H1K26 and G9aK185 peptide.

<p>(A and C) Electrostatic surface depiction of human Cbx3-histone H1K26me2, and Cbx3-G9aK185me3 complex. Peptide substrates are shown in a stick mode. Surfaces with positive electrostatic potential are blue, and negative potential are red. The side chain of residue H1A24 (G9aA183) inserts into the small hydrophobic pocket formed by Phe48 and Leu49 of human Cbx3. The size of the pocket is only sufficient to accommodate a methyl group but not other residue side chains. (B and D) Binding of histone H1 peptide and G9a peptide in the binding groove of Cbx3 chromodomain, respectively. Hydrogen-bonds are shown as dashed lines. Yellow: histone H1 peptide; Gray: Cbx3 chromodomain in Cbx3-histone H1K26me2 complex. Cyan: G9a peptide; Green: Cbx3 chromodomain in Cbx3-G9aK185me3 complex.</p

Peptide binding specificity of human Cbx3 chromodomain.

*<p>N/B: No binding was detected.</p