Monobodies as enabling tools for structural and mechanistic biology

Abstract

Monobodies, built with the scaffold of the fibronectin type III domain, are among the most well-established synthetic binding proteins. They promote crystallization of challenging molecular systems. They have strong tendency to bind to functional sites and thus serve as drug-like molecules that perturb the biological functions of their targets. Monobodies lack disulfide bonds and thus they are particularly suited as genetically encoded reagents to be used intracellularly. This article reviews recent monobody-enabled studies that reveal new structures, molecular mechanisms and potential therapeutic opportunities. A systematic analysis of the crystal structures of monobody-target complexes suggests important attributes that make monobodies effective crystallization chaperones.

Figure 1:

De novo crystal structures enabled with monobody chaperones. (a) Designs of monobody combinatorial libraries. The diversified positions in the Loop and Side libraries are shown with spheres. (b–e) Crystal structures of isoprenylcysteine carboxyl methyltransferase (ICMT), CLC^F transporter, Bcr-Abl Dbl-homology (DH) and Pleckstrin-homology (PH) domains. monobodies are shown in blue. Crystal packing is also shown.

Figure 2:. Monobody-mediated packing interactions.

(a and b). Monobody-mediated reduction of disorder in target molecules. The Fluc F⁻ channel Ec2 dimer bound with one monobody chaperone (a) and that bound by two copies of a different monobody (b). The cartoons are colored based on the B factor (top) and the packing modes are shown with monobodies in blue, magenta, and green (bottom). (c) Crystal packing interactions between monobodies and their target molecule. Alignment of all monobody-target complexes, subdivided into structures with monobody-monobody contacts and those with no monobody-monobody contacts. The aggregate alignments are shown in the upper panel, with a magnified view rotated 90° from the upper panel shown below. (d) Alignment of monobody-monobody interactions observed in crystal structures of the monobody-target complexes. The aggregate alignment is shown in the upper panel, and a magnified view of these interactions subdivided into the loop and side libraries are shown below. (e) Representative modes of monobody-monobody interactions observed in crystal structures. The reference monobody is shown in blue, and the interacting monobody is shown in magenta. The diversified positions are indicated as spheres. The numbers in the boxes indicate the number of instances in which the indicated modes are observed. Symmetry-mates were generated and contacts were mapped by selecting any residue within 5 Å of the reference monobody in PyMOL. Protein molecules are considered interacting if three or more residues are contacting.

Fig. 3.. Monobodies that modulate biological functions of their respective targets and their crystal structures.

(a) Allosteric activator and inhibitor of Aurora A kinase. (b) Competitive inhibitors of the interaction between SH2 domains and pY peptides. (c) Competitive inhibitor of the interaction between WDR5 and a fragment of MLL1. (d) Allosteric inhibitor targeting the interface between the Bcr-Abl SH2 and kinase domains. (e) Allosteric inhibitor targeting the self-association surface of HRAS.