<i>In-silico</i> analysis of non-binding of C35.

<p>(a) LD2 crystal structure from PDB id: 1K05 (left) being compared with the LD2 structure in the side view and top view of C35 structure showing the masking of the hydrophobic binding region predicted through HMM based SAM-T08 software. The LD2 binding region and the masking regions are depicted by the bracketed region. (b) Docking control showing FAT (co-ordinates from PDB id: 1K05) and LD2 (co-ordinates from PDB id: 2L6F, NMR model # 1) interaction using Hex 6.3 software. (c) Docking of C35 with FAT showing non-interaction due to masking effect. The sidechains of the active residues are shown as red sticks. The hydrophobic patch—HP2 in FAT molecule, which preferentially binds to LD2 is shown as a space filling model in orange (part of helix 1 of FAT) and grey (part of helix 4 of FAT) colors.</p

Dissection of paxillin constructs (residues 1–313) followed by expression and interaction studies.

<p><b>(a)</b> Timeline for overall-strategy. <b>(b)</b> Illustration of solubility and activity level of linear dissected human paxillin (residues 1–313). <b>(c)</b> Phosphor screen image of filter assay for optimization of temperature for paxillin constructs (left). Tabular representation of paxillin constructs, negative and positive controls corresponding to each well in filter assay [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150153#pone.0150153.ref001" target="_blank">1</a>]. <b>(d)</b> Phosphor screen image of 10% SDS PAGE of ³⁵S labeled cell-free expressed samples after GST pull-down assay of the paxillin constructs A1–E1; The right panel shows fraction of interaction of each construct with respect to B2 (since B2 showed maximum level of interaction) <b>(e)</b> Illustration of solubility and activity of dissected C3 constructs. All experiments were performed in triplicates and averaged. To rule out non-specific interactions that might occur with GST tagged FAT, GFP that was expressed in cell-free system and a reaction without DNA template were used as negative controls.</p

K_D values and curve fitting details of B2, C35, C35_1, C35_2 and C35_3 using Bio layer interferometry.

<p>K_D values and curve fitting details of B2, C35, C35_1, C35_2 and C35_3 using Bio layer interferometry.</p

Binding studies of paxillin constructs using Bio-layer Interferometry on OctetRed96.

<p>(a) Switch off in C3 and D2 on LD2 and LD4 respectively; Hypothesis of partial switch on when regulatory region of LD2 is absent, as evidenced in C2. (b) Concentration calibration curves depicting binding of constructs B2, C35, ‘54–189’, ‘79–189’, ‘105–189’ with GST-FAT. The data is representative of a single experiment. Each experiment was performed at-least thrice. (c) Illustrations of C35, C35_1, C35_2 and C35_3.</p

Regulatory and masking regions around paxillin’s LD2 and LD4 and their circular dichroism spectra.

<p>(a) CD spectra of paxillin LD peptides (LD1-LD5) and constructs: B2, C3, C35 and D2. CD spectra of LD2, LD4, C35 and D2 constructs showed negative bands at 222nm and 206nm and a positive band at 192nm that confirms the presence of alpha helical content thus may behave as folded effector binding sites. However, LD1, LD3, LD5, B2 and C3 do not show the characteristic peaks of secondary structures, thus may behave as unfolded effector binding sites. (b) LD2 regulatory region (54–130) and masking region (167–224) evidenced by constructs B3, B4 and B5. (c) LD4 regulatory region (216–257) and masking region (280–313) evidenced by constructs D1, D2 and E1.</p