Kinetic mechanism and structure.

<p>(A) The DHDPS-catalyzed reaction follows a classic bi-bi substrate model, requiring the first substrate (PYR; pyruvate) bind the enzyme for the second substrate (ASA) to be recruited to the active site and ultimately liberate the reaction product (HTPA). The initial pyruvate-binding portion of the reaction scheme is highlighted in cyan. (B) Quaternary structure of the DHDPS dimer. (C) Licorice representation of key active site residues. Protein chains A and B are shown in yellow and green, respectively.</p

Grouping of states from binding trajectories into a coarse-grained metastable state model.

<p>(A) Unbound states. One hundred randomly selected conformations of pyruvate from unbound states S2 (yellow) and S6 (green) are presented against a cartoon representation of DHDPS. Collectively, poses within these states lack any conserved interactions with the protein surface. (B) The DHDPS-pyruvate bound complex (S13). Conformations of pyruvate and active site residues from this state (green) are contrasted with a crystallographic reference structure of pyruvate bound to DHDPS (PDB ID 3DI1; silver). Key active site residues Thr46, Tyr109, Tyr135, and Lys163 are indicated. Conformations of pyruvate within this state deviate from the reference structure by as little as 1.85 Å. Note that Tyr109 is shown from the opposing DHDPS subunit. (C) Individual metastable states, labelled accordingly, are shown as nodes within rounded boxes. Edges depict bidirectional interstate transitions, where edge shade reflects the transition probability (darker arrows indicate higher probabilities). States classified as unbound (S2, S6) are shaded in red, whereas the DHDPS-pyruvate bound state (S13) is shaded in green. For clarity, only highly-populated states (equilibrium populations greater than 4%) are shown.</p

Umbrella sampling PMF.

<p>PMF curve calculated using 28 windows of umbrella sampling along an arbitrary <i>Z</i>-coordinate. Each curve, colored from red to blue, represents successive truncation of the data in 5% increments from the beginning of each simulation window until the final 50% of data (2.5 ns) remained. Several bound states identified are labelled according to their average <i>Z</i>-coordinate. State S13, which was bimodal with respect to the <i>Z</i>-coordinate, is highlighted as a gray box.</p

The major pyruvate-binding pathway is multi-step.

<p>(A) Pyruvate, indicated using green carbon atoms, must successively pass through several binding intermediates to reach the crystallographic bound pose. From bulk solvent, pyruvate forms a transient interaction with an arginine residue at the entryway to the active site (T1), moves into the active site cavity (T2), and in the penultimate step penetrates deeper into the active site to assume a ‘pre-bound’ pose (T3). Finally, from the ‘pre-bound’ pose pyruvate undergoes a twisting motion (T4) and achieves the crystallographic DHDPS-pyruvate complex. (B) Multiple sequence alignment of bacterial DHDPS enzymes. Several interacting residues from the binding pathway depicted in (A) are absolutely conserved across species. Sequence alignment was performed using CLUSTALO [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004811#pcbi.1004811.ref037" target="_blank">37</a>].</p

Binding data.

<p>(A) Ligand RMSD to crystal structure (PDB ID 3DI1) is shown as a function of time for 10 randomly selected trajectories, representing an eighth of the total simulation data set. Hydrogen atoms were excluded from RMSD calculations. (B) Ligand density plot. The <i>x</i> and <i>y</i> plane components of the geometric center of pyruvate were derived from each frame of the simulation data set and binned to form a 2-dimensional matrix. The color mapping reflects the number of counts within each of these bins (blue indicates low density, red indicates high density). For reference, the relative locations of several active site residues (Thr46, Tyr109, Tyr135, Arg140, and Lys163; <i>α</i>-carbons only) are indicated using black markers and labelled accordingly.</p