Local structure and <i>N_eq</i> analyses.

<p><i>N_eq</i> values (average number of PBs weighted by their frequencies, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047304#s2" target="_blank">Materials and Methods</a>) of residues from the PSI, I-EGF-1, and I-EGF-2 domains were computed for L33 (blue lines) and P33 (red lines) β3 forms. For each domain, the absolute value of <i>N_eq</i> L33 and <i>N_eq</i> P33 differences (Δ<i>N_eq</i>) is also plotted (black lines). The sequence of the C26–C38 loop in the PSI domain is highlighted (pink-shaded area). The horizontal gray lines indicate an <i>N_eq</i> value of 1. A <i>N_eq</i> value of 1 indicates strong structure rigidity since only one PB is adopted by the residue; above 1, flexibility is proportional to the <i>N_eq</i> value. <i>N_eq</i> analyses demonstrated that the three domain structures are highly flexible, in particular, PSI has four small interspersed rigid areas. L33 and P33 (indicated by an arrow), belong to a rigid stretch (residues 33 to 35). Residues whose Δ<i>N_eq</i> was significantly modified (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047304#s2" target="_blank">Materials and Methods</a>) by the L33P substitution are identified by a vertical dotted line.</p

Snapshots of statistically representative structures of the PSI, I-EGF-1, and I-EGF-2 domains.

<p>Three structure snapshots of L33 (a, b, and c) and of P33 (e, f, and d) β3 were chosen among the statistically most frequent structures in terms of rASA and number of contacts. L33 and P33 atoms are shown as blue and red spheres, respectively. All remaining atoms belonging to the PSI, I-EGF-1, and I-EGF-2 domains are shown as orange, light green and dark green spheres, respectively. In these snapshots, L33 is largely exposed at the β3 surface while P33 can be remote. P33 is in relatively close contact with the I-EGF-2 domain, but L33 is not. These representations were generated using PyMOL software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047304#pone.0047304-DeLano1" target="_blank">[16]</a>.</p

Ectodomain 3D structure model of the L33 β3 form.

<p>A side view of β3 integrin (A) and an apical view of the β3 knee (B) are shown. Domains are differently colored and labeled and the L33 residue is shown in blue. These static views illustrate the HPA-1 polymorphic site that is located at the top of the β3 knee. These representations were generated using PyMOL software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047304#pone.0047304-DeLano1" target="_blank">[16]</a>.</p

Structural flexibility of domains PSI, I-EGF-1, and I-EGF-2.

<p>(A) RMSF values calculated for each residue of the L33 (blue line) and the P33 (red line) forms of β3. Both structures show high flexibility. (B) Atom movements analyzed by PCA are projected on the first two modes. The P33 structure movements (red cloud of dots) are higher than that measured for the L33 structure (blue cloud of dots) for the first mode. No significant differences were observed for the second mode. The proline increased the structural flexibility of the PSI, I-EGF-1 and I-EGF-2 domains. The first and second modes represent 45% and 13% of the informativeness of the data.</p

rASA and number of contacts for L33 and P33.

<p>Panels A and B respectively show rASA and number of contact distributions for L33 (blue) and P33 (red). These distributions show that L33 is more exposed and establishes fewer contacts than P33.</p

L33 and P33 distances to domains I-EGF-1 and I-EGF-2.

<p>Center-of-mass distances were measured between L33 (blue line) or P33 (red line) and the I-EGF-1 (A) or I-EGF-2 (B) domains. No significant differences in distribution were observed regarding the I-EGF-1 domain. However, P33 is close to the I-EGF-2 domain (≈10 Å) while L33 always remains farther than 15 Å.</p