Selection of specific protein binders for pre-defined targets from an optimized library of artificial helicoidal repeat proteins (alphaRep).

We previously designed a new family of artificial proteins named αRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized αRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a "filtration" procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.7×10(9) independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific αRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with Kd values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between αRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that αRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties

Calorimetric data for competition binding experiments.

<p>ITC titration of a mixture of A3 (25 μM) pre-bound to bA3–17 (43 μM) with bA3–2 (350 μM). In these conditions, the apparent binding constant for bA3–2 decreases within the range required for ITC. Determination of the K_app is given by: K_app  = K_a^bA3–2/(1+ K_a^bA3–17 [bA3–17]). ΔH^bA3–17 and K_a^bA3–17 used in the data analysis had been determined in Fig. 2–B.</p

Biophysical characterization of the bEbs1-6 and Ebs1/bEbs1–6 complex.

<p>(A) ITC calorimetric titrations. (▪): Titration of Ebs1 (35 μM) with bEbs1–6 (387 μM). (□): The bEbs1–6 (30 μM) binding specificity was tested by titration with NCS-wt (350 μM). (B) Size Exclusion Chromatography (Superdex 200 prep grade Hiload 16/60) of the selected bEbs1–6 and Ebs1 (?):SEC elution profile of bEbs1–6 alone; (∇): elution profile of Ebs1 alone; (?): elution profile of an equimolar mixture of Ebs1 (55 nmol) and the binder bEbs1–6 (55 nmol).</p

Selection of binders from the αRep libraries.

a<p>The libraries indicated were used for the selections against the corresponding targets.</p>b<p>The phage-ELISA results are indicated as the number of clones giving a positive signal <i>versus</i> the number of clones tested. A Clone was scored as positive if its measured signal/noise <i>ratio</i> was greater than five.</p>c<p>The sequences were determined among the phage-ELISA positive clones. For each target, the number of distinct sequences is indicated over the total number of sequences determined.</p>d<p>The soluble expression of phage-ELISA positive clones was probed using CoFi blot or Western blot experiments after liquid expression cultures. Reported <i>ratios</i> indicate the number soluble proteins over the number of clones tested.</p>e<p>The properties of the clones used for further characterization were determined by ITC, DSC and/or SEC as described below. The number of internal repeats for each binder is indicated in parentheses.</p

Biophysical characterization of the bGFP-A and GFP/bGFP-A complex.

<p>(A) ITC calorimetric titrations. Concentrations values are expressed in monomer concentrations. (▪): Tiration of GFP (35 μM) with bGFP-A (350 μM). (□): The bGFP-A binding specificity was tested by titration with NCS-wt (bGFP-A 30 μM, NCS-wt 350 μM). (B) The GFP-binding specificity was evaluated by ITC analysis of injection of bA3–1 (360 μM) in a solution of GFP (30 μM). (C) Size Exclusion Chromatography (Superdex 75 10/300) of the selected bGFP-A and GFP. (▾): SEC Elution profile of a mixture of GFP (2.25 nmol) and the binder bGFP-A (6.75 nmol). (∇): elution profile of the bGFP-A alone (2.25 nmol). (<b></b>): elution profile of the GFP alone (6.75 nmol). (D) Affinity determination of selected bGFP-A using SPR. Different concentrations of bGFP-A (71,3; 118; 142,6; 237,6; 713; 1426 nM) were applied to flow cell with immobilized biotinylated EGFP for 120 s followed by washing buffer flow. The sensorgrams were corrected for non-specific binding by subtraction of a channel without EGFP bound (grey curve). The fits of k_on and k_off rates are indicated by black dashed line. K_d values were computed using k_off  = 1.7×10⁻⁴ s⁻¹ for all concentrations and k_on  = 4.3, 4, 2.2, 2.6, 2, 2×10⁴M⁻¹ s⁻¹ for the increasing concentrations respectively.</p

Characteristics of the initial and optimized libraries.

a<p>Lib1.0 was constructed using single stranded circles for the RCA amplification.</p>b<p>Lib2.0 was constructed using double stranded circles for the RCA amplification.</p>c<p>Lib2.1 was constructed from Lib2.0 in two steps. First, in-frame sequences were recovered by filtration of the phage library on an anti-Flag antibody; second, the filtrated library was shuffled using only modules preselected to be in frame. The total number of clones for each library is indicated in the library size column.</p>d, e<p>The ratio of coding sequences was determined from the sequence of randomly picked clones as the number of correct sequences versus the total number of sequences. “In-frame clones” (e) include all coding sequences while “in-frame clones (n ≥ 1)” (f) include coding sequences with at least one motif between the N-cap and the C-cap.</p>f, g<p>The average number of motifs was calculated as the mean of inserted motifs in all coding sequences (g) or in all coding sequences with at least one motif between the N-cap and the C-cap (h).</p

X-Ray data collection and refinement statistic.

<p>Values in parentheses are for highest resolution shell.</p>a<p>R_sym = y_h∑_i|I_hi – h>|/-_h∑_iI_hi, were I_hi is the <i>i</i>th observation of the reflection h, while h> is the mean intensity of reflection h.</p>b<p>R_factor  = ctorF_o| – |F_c||/|F_o|. R_free was calculated with a small fraction (5%) of randomly selected reflexions.</p

Representation of the bNCS-16/NCS-3.24 complex.

<p>(A) Ribbon representation of the two bNCS-16/NCS-3.24 complexes present in the asymmetric unit. (B) Comparison of the NCS-3.24/testosterone complex (NCS-3.24 is in grey and testosterone hemisuccinate are in blue) with the bNCS-16/NCS-3.24 complex (same colour code as panel A). For clarity, only loops from NCS-3.24 that undergo conformational changes are shown.(C) Representation of the interface between bNCS-16 and NCS-3.24 (same colour code as panel A). Residues involved in the interaction are shown as sticks. Residues from the interface, which belong to the invariant scaffold of αRep proteins are shown as ball and sticks. Hydrogen bonds are depicted by dashed lines.</p

Sequence diversity of the optimized library (Lib2.1).

<p>The experimental amino acids frequency at each of the 6 randomized positions as compared to the natural and encoded frequencies. Black bars: Amino acid frequencies calculated from the natural collection of αRep like repeats; White bars Frequencies: expected from the coding scheme; Grey bars: experimental diversity observed in library Lib2.1.</p

Properties of the selected binders.

a<p>The library form which the binder was selected is indicated.</p>b<p>The number of internal repeats inserted between the N-cap and the C-cap is reported for each binder.</p>c<p>The K_d values were determined by ITC and result from the fitting of individual experiments. For the titration of bA3–2/A3 the value obtained in the competition experiment (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071512#pone-0071512-g003" target="_blank">Fig. 3</a>) is reported here.</p>d<p>The RX structures of two αRep/Target complexes were determined.</p>e<p>For each binder, the residues found in the variable positions in the N-cap and the internal repeats are indicated in one-letter code.</p