Stereospecific Phosphorylation by the Central Mitotic Kinase Cdk1-Cyclin B

The <i>cis</i> vs <i>trans</i> conformation, or shape, of phosphoserine-proline (pSer-Pro), a prevalent motif in cell cycle proteins, may play a significant role in regulating mitosis. We demonstrate that Cdk1-cyclin B, the central mitotic kinase, is specific for the <i>trans</i> conformation, not <i>cis</i>, of synthetic, locked Ser-Pro 11-residue peptide substrates, using LC-MSMS detection and sequencing of phosphorylated products. This substrate stereospecificity may contribute an additional level of mitotic regulation

Pin1 is proposed to stretch the prolyl ring by binding phosphate and C-terminal residues tightly, creating a <i>trans</i>-pyrrolidine conformation of the substrate and forcing pyramidalization of the prolyl nitrogen in the twisted-amide mechanism.

<p>Distance measurements are from calculated structures of Ac–Pro–OH in the ground state and the <i>trans</i>-pyrrolidine transition state.</p

Ketone inhibitors were designed to mimic the tetrahedral intermediate of proposed mechanism B.

<p>(A) Proposed Pin1 hydrogen-bond assisted twisted amide mechanism <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044226#pone.0044226-Schroeder1" target="_blank">[25]</a>, (B) Pin1 Cys113 nucleophilic-addition mechanism tetrahedral intermediate proposed by Ranganathan et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044226#pone.0044226-Ranganathan1" target="_blank">[26]</a>. (C) Electrophilic ketone inhibitor designed to mimic the proposed tetrahedral intermediate upon Cys113-S nucleophilic addition.</p

X-ray crystal structures of intermediates (1<i>S</i>,3<i>R</i>,4<i>R</i>)-11 and <i>rac</i>-11 are shown above as displacement ellipsoid drawings (50%).

<p>The positional disorder of the benzyl group in <b><i>rac</i></b><b>-11</b> is shown as lighter lines. Hydrogen atoms are omitted for clarity. Structural depiction of the stereochemistries of <b>(1</b><b><i>S</i></b><b>,3</b><b><i>R</i></b><b>,4</b><b><i>R</i></b><b>)-11</b> and <b><i>rac</i></b><b>-11</b> are shown below each crystal structure.</p

Pin1 inhibitors discussed are cyclohexyl ketones 1 and <i>rac</i>-2 (this work); reduced amides 3 and 4 [<b>27</b>]; (<i>Z</i>)-alkene 5 [<b>13</b>]; and α-ketoamides 6a and 6b [<b>14</b>].

<p>Pin1 inhibitors discussed are cyclohexyl ketones 1 and <i>rac</i>-2 (this work); reduced amides 3 and 4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044226#pone.0044226-Xu3" target="_blank">[<b>27</b>]</a>; (<i>Z</i>)-alkene 5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044226#pone.0044226-Zhao1" target="_blank">[<b>13</b>]</a>; and α-ketoamides 6a and 6b <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044226#pone.0044226-Xu2" target="_blank">[<b>14</b>]</a>.</p

Models of cyclohexyl ketone inhibitors were docked with dynamic minimization.

<p>(A) <b>(1</b><b><i>S,</i></b><b>3</b><b><i>R,</i></b><b>4</b><b><i>R</i></b><b>)-1</b> in orange, (B) <b>(1</b><b><i>R,</i></b><b>3</b><b><i>R,</i></b><b>4</b><b><i>R</i></b><b>)-2</b> in blue, (C) <b>(1</b><b><i>S,</i></b><b>3</b><b><i>S,</i></b><b>4</b><b><i>S</i></b><b>)-2</b> in green, and (D) superposition of all atoms of <b>1</b> and <b><i>rac</i></b><b>-2</b>. Models were based on PDB 2Q5A <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044226#pone.0044226-Zhang2" target="_blank">[32]</a>, and minimized using Sybyl 8.1.1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044226#pone.0044226-1" target="_blank">[42]</a>. Images were prepared using MacPyMol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044226#pone.0044226-DeLano1" target="_blank">[44]</a>.</p

Synthesis of the Ac–L-pSer–Ψ[(<i>Z</i>)CH = C]-D-Pip–NEA inhibitor (2<i>R</i>,5<i>S</i>)-1.

<p>Synthesis of the Ac–L-pSer–Ψ[(<i>Z</i>)CH = C]-D-Pip–NEA inhibitor (2<i>R</i>,5<i>S</i>)-1.</p

Determination of the (<i>Z</i>)-alkene stereochemistry of intermediate (2<i>R</i>,5<i>S</i>)-6.

<p>The 1D nOe ¹H NMR. Irradiation of ¹H_f shows an nOe at ¹H_m and not at ¹H_h.</p

Synthesis and determination of the stereochemistry of derivative (2<i>S</i>,3<i>R</i>)-13.

<p>Cyclic compounds <b>13</b> were synthesized. ¹H NMR coupling constants (<i>J</i>) were used to determine the relative stereochemistry at the carbons to which H_a and H_b are attached.</p

Designed enantiomeric inhibitors synthesized.

<p>Designed enantiomeric inhibitors synthesized.</p