Fluorescence polarization binding studies of modified peptides.

<p>Representative FP isotherms are shown for the displacement of CALP:reporter binding by (A) Ac-iCAL36, (B) scaffold peptides containing acetylated lysine substitutions at each non-motif position, or (C) peptides containing halogenated substituents at the P⁻¹ position (C). In each panel, iCAL36 displacement is also shown for reference. A fluoresceinated iCAL36 peptide (<i>F*</i>-iCAL36) was used as the reporter for all measurements. For illustrative purposes, a logistic curve-fit is shown, but <i>K</i>_I values and fractional reporter occupancy values were determined by least-squares fitting of the complete binding equation. Summary values of independent experiments (n = 3) are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103650#pone-0103650-t001" target="_blank">Table 1</a>.</p

Data collection and refinement statistics for chemically-modified iCAL36 peptide derivatives, bound to CALP.

a<p>Values in parentheses are for data in the highest-resolution shell.</p>b<p><i>R</i>_sym = Σ<i>_h</i>Σ<i>_i</i> |I(<i>h</i>)−I<i>_i</i>(<i>h</i>)|/Σ<i>_h</i>Σ<i>_i</i> I<i>_i</i>(<i>h</i>), where I<i>_i</i>(<i>h</i>) and I(<i>h</i>) values are the <i>i-</i>th and mean measurements of the intensity of reflection <i>h</i>.</p>c<p><i>R</i>_work = Σ∥F_obs|_h−|F_calc∥_h/Σ |F_obs|_h, h ∈ {working set}.</p>d<p><i>R</i>_free is calculated as <i>R</i>_work for the reflections h ∈ {test set}.</p>e<p>Core/allowed/generously allowed/disallowed.</p

Chemically modifiable scaffolds at each non-motif position in the CALP binding cleft.

<p>The C_α trace (gray) shows all CALP structures, aligned by main chain atoms (RMSD ≤0.3 Å compared to CALP:iCAL36, PDB ID: 4E34). CALP is also shown as a transparent surface (gray). The peptides are shown as black C_α traces, with the acetylated lysine residue of each respective peptide highlighted (carbon atoms colored by position, as indicated). Peptide positions are labeled, and non-carbon atoms are colored separately: O = blue, N = red.</p

Differential stereochemical and electrostatic environments at each modifiable position.

<p>The electrostatic potential surface map of CALP is shown, rendered at ±10 k_BT/e (negative surface in red, positive surface in blue). Boxes and labels (S⁻¹, S⁻², etc.) indicate an approximate area that chemical modifications could bind when attached to the lysine N_ζ moiety at that position. Overlap in accessible area suggests a possible region for side-chain cyclization. Peptide backbones are shown as black C_α traces, with acetyl-lysine residues shown as sticks and colored by position. Non-carbon atoms are colored separately: O = blue, N = red.</p

Electron density of chemically modified peptides.

<p>A) Prior to ligand modeling, positive electron density was observed in <i>F</i>_O – <i>F</i>_C difference maps, as illustrated here (contour level: 2σ) for the halogenated substituents attached to the Lys side chain at P⁻¹. B) After iterative rounds of refinement and model building, final 2<i>F</i>_O – <i>F</i>_C electron density maps (contour level: 1σ) showed excellent agreement with the model, as shown here for the corresponding P⁻¹ side chains. The final refined peptide structures are shown as stick figures and colored by atom/position (C = light blue for P⁻¹ or black for other positions, O = blue, F = sky blue, N = red, Br = dark red). The van der Waals surface of CALP is shown in gray. The substituents are indicated as follows: Tfa, trifluoroacetic acid; FB, 4-fluorobenzoic acid; BB, 4-bromobenzoic acid.</p

Chemical modifications at the P⁻¹ position reveal similar modes of binding to CALP.

<p>A) Schematic representations of the three additional chemically modified peptides are shown for reference and labeled by modification: , , . B) The structures of CALP (gray C_α trace and van der Waals surface) in complex with these peptides (stick figures, black carbons), or in complex with peptides bearing acetylated lysines at P⁻¹ (blue carbons), P⁻³ (green carbons), and P⁻⁴ (yellow carbons) are shown following alignment. Superposition of these structures reveals that all halogenated substituents interact with a similar region (arrow) at the edge of the CALP peptide-binding cleft, in close proximity to the P⁻³ binding site. Non-carbon atoms are colored by element: O = blue, F = sky blue, Br = dark red, N = red.</p

Comparison of binding affinities measured by various techniques.

a<p><i>K</i>_D values as reported in ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103650#pone.0103650-Saro1" target="_blank">[20]</a>.</p>b<p>Value previously reported in ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103650#pone.0103650-Amacher2" target="_blank">[17]</a>.</p>c<p>Value in parentheses indicates fold-deviation from the value obtained by FP.</p>d<p>Ac- indicates N-terminal acetylation.</p>e<p>[Ac-K] indicates N_ζ-acetyl-lysine.</p>f<p>n = 1.</p>g<p>[BB-K] indicates N_ζ-4-bromobenzoyl-lysine.</p>h<p>[FB-K] indicates N_ζ-4-fluorobenzoyl-lysine.</p>i<p>[Tfa-K] indicates N_ζ-trifluoroacetyl-lysine.</p

Data collection and refinement statistics for iCAL36 acetylated peptide derivatives in complex with CALP.

a<p>Values in parentheses are for data in the highest-resolution shell.</p>b<p><i>R</i>_sym = Σ<i>_h</i>Σ<i>_i</i> |I(<i>h</i>)−I<i>_i</i>(<i>h</i>)|/Σ<i>_h</i>Σ<i>_i</i> I<i>_i</i>(<i>h</i>), where I<i>_i</i>(<i>h</i>) and I(<i>h</i>) values are the <i>i-</i>th and mean measurements of the intensity of reflection <i>h</i>.</p>c<p><i>R</i>_work = Σ∥F_obs|_h−|F_calc∥_h/Σ |F_obs|_h, h ∈ {working set}.</p>d<p><i>R</i>_free is calculated as <i>R</i>_work for the reflections h ∈ {test set}.</p>e<p>Core/allowed/generously allowed/disallowed.</p

Experimental validation of poorly-ranked predictions.

<p>Experimental validation of poorly-ranked predictions.</p

(A) Structural model of the CAL PDZ domain (green and blue) bound to a CFTR C-terminus mimic (gray) used as input for computational designs (PDB id: 2LOB).

<p>Residues shown in blue were modeled as flexible during the design search. (B) Model of the CFTR trafficking pathway with PDZ domain containing proteins NHERF1 and CAL. CAL is associated with lysosomal degradation of CFTR, while NHERF1 is associated with insertion of CFTR into the cell membrane.</p