Features of P-selectin with different conformations of the <i>R</i>3 loop.

<p>(<i>A</i>) Conformational differences in the <i>R</i>3 loop between <i>S</i>1 (<i>silver</i>), <i>S</i>1’ (<i>cyan</i>) and <i>S</i>2 (<i>blue</i>). The Lec domain is presented in <i>newcartoon</i> format in <i>topview</i>; the other components, except for the <i>R</i>3 loop and calcium ion, are shown in <i>transparent</i> format for clarity. The <i>S</i>1’ conformation is the final snapshot of the 20-<i>ns</i> equilibration of the 1G1Q/P-LEC system of the 1CR set. (<i>B</i>) Differences in receptor-ligand interactions for P-selectin with distinct <i>R</i>3 conformations of <i>S</i>1, <i>S</i>1’ and <i>S</i>2 during the free equilibration process of SGP-3-ligated systems in the 0CR, 1CR and 2CR sets. Non-covalent energy, including van der Waals and electrostatic interactions between P-selectin and the SGP-3 ligand, were quantified, and only the snapshots showing an <i>R</i>3 loop RMSD of less than or equal to 4.0 Å in relation to the reference state <i>S</i>1, <i>S</i>1’ or <i>S</i>2 were counted. The trajectory was counted in statistical analyses of state <i>S</i>1’ if its snapshots fit the rules of both <i>S</i>1 and <i>S</i>1’. The data are presented as the mean ± SD of the different equilibration systems. (<i>C</i>) Differences in dissociation time between the complexes with distinct <i>R</i>3 loop conformations of <i>S</i>1, <i>S</i>1’ and <i>S</i>2 under a constant force of 300 <i>pN</i> pulling on the SGP-3 ligand with EGF end fixation. The data were presented as the mean ± SD of six dissociation runs for each system. The dissociation simulations were based on the final state after a 20 <i>ns</i> equilibration of the 1G1Q/P-LECC-SGP3, 1G1Q/P-LEC-SGP3, and 1G1S/P-LECC-SGP-3 systems for <i>S</i>1, <i>S</i>1’ and <i>S</i>2, respectively.</p

Typical features of the constant velocity forced dissociation process in the SGP-3-ligated 1G1Q 2CR complex system.

<p>The typical dissociation processes for EGF end fixation (<i>A-C</i>), CR1 end fixation (<i>D-F</i>) and CR2 end fixation (<i>G-I</i>) are shown. (<i>A</i>, <i>D</i>, <i>G</i>) The profiles of force-extension (<i>black</i>), receptor-ligand separation-extension (<i>red</i>), and Fuc-calcium ion separation-extension (<i>blue</i>) in the dissociation processes at 0.01 Å/<i>ps</i>. (<i>B</i>, <i>E</i>, <i>H</i>) Corresponding evolution of the <i>R</i>3 loop conformation (<i>black</i>) and EGF orientation (<i>red</i>). The former was quantified according to the RMSD of the <i>R</i>3 loop for the <i>S</i>1’ state based on alignment of the rigid parts of the Lec domain, and the latter was calculated based on the reference of crystallized 1G1Q. (<i>C</i>, <i>F</i>, <i>I</i>) Conformational features immediately after the critical moment of the <i>R</i>3 loop RMSD. Only P-LE (<i>silver</i>, <i>Newcartoon</i>), calcium ion (<i>silver</i>, <i>VDW</i>) and the SGP-3 ligand are presented for clarity. The peptide, Core-2 sugar, and three sulfonated tyrosines of the SGP-3 ligand are shown using the <i>orange newcartoon</i>, <i>blue CPK</i> and <i>name licorice</i> formats, respectively.</p

Impact of the CR domain on the constant force dissociation process.

<p>(<i>A</i>) Dissociation time distribution for the EGF end-fixed (<i>black</i>), CR1 end-fixed (<i>white</i>) and CR2 end-fixed (<i>gray</i>) dissociation processes at 300 <i>pN</i> based on 20-<i>ns</i>-equilibrated snapshots of the SGP-3-ligated 1G1Q 2CR complex with the <i>R</i>3 loop in the <i>S</i>1 state (<i>left</i>), the 1G1Q 1CR complex with the <i>R</i>3 loop in the <i>S</i>1’ state (<i>middle</i>) and the 1G1S 2CR complex with the <i>R</i>3 loop in the <i>S</i>2 state (<i>right</i>), respectively. The data are presented as the mean ± SD of six independent runs for each fixation setting. (<i>B</i>, <i>C</i>, <i>D</i>) Corresponding separation-extension profiles of the dissociation processes based on the initial <i>S</i>1 (<i>B</i>), <i>S</i>1’ (<i>C</i>) or <i>S</i>2 (<i>D</i>) system. Six repeated runs for EGF end fixation, CR1 end fixation and CR2 end fixation are shown in the same color (<i>black</i>, <i>red</i> or <i>blue</i>, respectively).</p

Impact of the CR domain on the constant velocity forced dissociation process.

<p>(<i>A</i>-<i>C</i>, <i>E</i>-<i>G</i>) Force-extension profiles at 0.01 Å/<i>ps</i> based on the initial conformations of the 20-<i>ns</i>-equilibrated SGP-3-ligated 2CR set of the 1G1Q system (<i>A</i>-<i>C</i>) with an <i>R</i>3 loop state of <i>S</i>1 and the 1G1S system (<i>E</i>-<i>G</i>) with an <i>R</i>3 loop state of <i>S</i>2. Three runs (<i>black</i>, <i>red</i>, <i>blue</i>) were performed for each fixation setting including EGF end fixation (<i>A</i>, <i>E</i>), CR1 end fixation (<i>B</i>, <i>F</i>) and CR2 end fixation (<i>C</i>, <i>G</i>). (<i>D</i>, <i>H</i>) The corresponding separation-extension profiles were summarized for the 1G1Q (<i>D</i>) and 1G1S (<i>H</i>) systems. Each three runs for EGF end fixation, CR1 end fixation or CR2 end fixation are shown in the same color (<i>black</i>, <i>red</i> or <i>blue</i>, respectively). (<i>I</i>, <i>J</i>) The dissociation times (<i>I</i>) and the separation time (<i>J</i>) were compared. The data are presented as the mean ± SD of six independent dissociation runs.</p

P-selectin components and simulation system.

<p>(<i>A</i>) Schematic representation of P-selectin domain components. (<i>B</i>) Maximal components of the P-selectin-PSGL-1 complex for the simulations, which included the Lec, EGF, CR1 and CR2 domains of P-selectin and the SGP-3 ligand of PSGL-1.</p

Typical features of the equilibration processes for the 1G1Q/P-LEC and 1G1S/P-LEC systems.

<p>(<i>A</i>, <i>D</i>) The conformation of the Lec domain quantified according to the RMSDs of the <i>R</i>3 loop with respect to the references <i>S</i>1 (<i>black</i>), <i>S</i>1’ (<i>red</i>) and <i>S</i>2 (<i>blue</i>) based on the alignment of the rigid regions of the Lec domain. (<i>B</i>, <i>E</i>) The EGF domain orientation with respect to the references crystallized 1G1Q (<i>black</i>) and 1G1S (<i>red</i>). (<i>C</i>, <i>F</i>) The orientation of the CR1 domain relative to the initial conformation. The 20-<i>ns</i> equilibration process for the unligated 1G1Q/P-LEC (<i>A</i>-<i>C</i>) and 1G1S/P-LEC (<i>D</i>-<i>F</i>) systems of the 1CR set is featured here, and the corresponding final snapshots (<i>cyan</i>, <i>purple</i>) developed from 1G1Q (<i>G</i>, <i>H</i>) and 1G1S (<i>J</i>, <i>I</i>) are illustrated to highlight the EGF orientations (<i>G</i>, <i>J</i>) and <i>R</i>3 loop (<i>H</i>, <i>I</i>). The crystallized references 1G1Q (<i>silver</i>) and 1G1S (<i>blue</i>) are superposed for comparison.</p

Impact of the CR domain on the equilibration process.

<p>Differences of the conformational features of Lec domain <i>R</i>3 loop (<i>A</i>, <i>C</i>) and the EGF orientation (<i>B</i>, <i>D</i>) between equilibration processes of non-ligated 0CR (<i>black</i>) and 1CR (<i>white</i>) sets of 1G1Q (<i>A</i>, <i>B</i>) and 1G1S (<i>C</i>, <i>D</i>) systems. The conformational features of Lec domain <i>R</i>3 loop was quantified by the RMSD for the <i>S</i>1’ state based on alignment of the rigid parts of the Lec domain, and the EGF orientation was calculated based on the references of crystallized 1G1S and 1G1Q for the 1G1Q and 1G1S systems, respectively. The data were from six equilibration processes for each system.</p