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Review
. 2017 May;26(5):910-924.
doi: 10.1002/pro.3148. Epub 2017 Mar 24.

Monobodies and Other Synthetic Binding Proteins for Expanding Protein Science

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Free PMC article
Review

Monobodies and Other Synthetic Binding Proteins for Expanding Protein Science

Fern Sha et al. Protein Sci. .
Free PMC article

Abstract

Synthetic binding proteins are constructed using nonantibody molecular scaffolds. Over the last two decades, in-depth structural and functional analyses of synthetic binding proteins have improved combinatorial library designs and selection strategies, which have resulted in potent platforms that consistently generate binding proteins to diverse targets with affinity and specificity that rival those of antibodies. Favorable attributes of synthetic binding proteins, such as small size, freedom from disulfide bond formation and ease of making fusion proteins, have enabled their unique applications in protein science, cell biology and beyond. Here, we review recent studies that illustrate how synthetic binding proteins are powerful probes that can directly link structure and function, often leading to new mechanistic insights. We propose that synthetic proteins will become powerful standard tools in diverse areas of protein science, biotechnology and medicine.

Keywords: biologic therapeutics; directed evolution; protein engineering; protein-protein interaction; structure-function relationship.

Figures

Figure 1
Figure 1
Generation of synthetic binding proteins using molecular scaffolds. (A) A schematic representation of the processes for generating synthetic binding proteins. The rectangle at the left indicates an inert scaffold. The yellow circles denote sequence diversity at chosen positions. The right is a binding protein‐target complex with the optimized interface shown in yellow. (B) The three‐dimensional structures of representative scaffold architectures, including the natural antibody for which only the Fab portion is shown. Only those scaffolds for which structure‐guided design of libraries have led to improved performance are shown. References are given in the main text.
Figure 2
Figure 2
Examples of Monobodies and Adnectins binding to a functional site within the target protein. The target proteins are shown in gray with the epitope in orange. Natural ligands are in red, and Monobodies and Adnectins in blue. The identities of the target molecules and PDB entry codes are indicated. For the Fluc channel structure, the natural ligand, F ion, is not shown because of its small size.
Figure 3
Figure 3
Concavity analysis of binding protein‐target interfaces. (A–C) Three representative structures of Monobody‐target complexes with different levels of concavity. For each crystal structure, a spherical shell (tan) was fit to all the atoms that compose the target‐contacting residues on the Monobody (i.e., the paratope; yellow spheres). A spherical shell with a large radius approximates a flat interaction. Spherical shells with smaller radii, centered within the Monobody or target represent convex or concave paratopes, respectively. To distinguish between the two orientations, the radii of shells corresponding to concave paratopes were assigned negative values. Monobody and target structures are shown as blue and gray cartoons, respectively. Atoms composing the Monobody‐contacting residues on the target (i.e., the epitope) are shown as gray spheres. (D) Concavity analysis on 34 synthetic binding protein‐target complex structures from the PDB. Nanobody complexes are also included for comparison. Curvature is defined as the inverse of the radius of the spherical shell as described above. An arbitrary threshold of |rshell| ≥ 100Å (|curvature| ≤ 0.01 Å−1) was defined as an effectively flat interface.
Figure 4
Figure 4
Synthetic binding proteins identify novel modes of protein‐protein interactions. Comparisons of the binding mode of a natural ligand (red) and that of a synthetic binding protein (blue) for Caspase‐3 (A), SHP2 SH2 domain (B), Abl SH2 (C) and H‐RAS (D) are shown. For C and D, the side chains of the ligand and synthetic binding protein located within 4.5 Å of the target protein are shown in the lower panels.
Figure 5
Figure 5
Schematics showing allosteric regulation using synthetic binding proteins. (A) Monobody binding to the SH2 domain of ABL kinase disrupts the intramolecular, domain‐domain interaction, which leads to kinase inhibition.45, 70 (B) Monobody binding to a dimerization interface of RAS disrupts the RAS‐RAF hetero‐trimer and inhibits RAS‐mediated activation of RAF.59 (C) Tandem DARPin disrupts the dimer formation of HER2, thereby inhibiting the kinase activity of HER2. Figure based on Jost et al.71.

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