Potent and Specific Inhibition of Glycosidases by Small Artificial Binding Proteins (Affitins)

Abstract

Glycosidases are associated with various human diseases. The development of efficient and specific inhibitors may provide powerful tools to modulate their activity. However, achieving high selectivity is a major challenge given that glycosidases with different functions can have similar enzymatic mechanisms and active-site architectures. As an alternative approach to small-chemical compounds, proteinaceous inhibitors might provide a better specificity by involving a larger surface area of interaction. We report here the design and characterization of proteinaceous inhibitors that specifically target endoglycosidases representative of the two major mechanistic classes; retaining and inverting glycosidases. These inhibitors consist of artificial affinity proteins, Affitins, selected against the thermophilic CelD from Clostridium thermocellum and lysozyme from hen egg. They were obtained from libraries of Sac7d variants, which involve either the randomization of a surface or the randomization of a surface and an artificially-extended loop. Glycosidase binders exhibited affinities in the nanomolar range with no cross-recognition, with efficient inhibition of lysozyme (Ki = 45 nM) and CelD (Ki = 95 and 111 nM), high expression yields in Escherichia coli, solubility, and thermal stabilities up to 81.1°C. The crystal structures of glycosidase-Affitin complexes validate our library designs. We observed that Affitins prevented substrate access by two modes of binding; covering or penetrating the catalytic site via the extended loop. In addition, Affitins formed salt-bridges with residues essential for enzymatic activity. These results lead us to propose the use of Affitins as versatile selective glycosidase inhibitors and, potentially, as enzymatic inhibitors in general.

Switching an anti-IgG binding site between archaeal extremophilic proteins results in Affitins with enhanced pH stability

“…The deletion of three residues from the ␣-helix end of D1Sac7d could also explain the decrease in activity observed for D1Sso7d. In fact, we observed in the three-dimensional structure of a lysozyme/anti-lysozyme Affitin complex that this part of the Affitin was in contact with lysozyme (Correa et al, 2014). This work illustrates the difficulty to anticipate possible effects on binding activity of a mutational/grafting approach, even when applied to a rigid region (Kahsai et al, 2005) of the protein.…”

“…The method of improving a scaffold for binding is often driven by structure/function relationships with other proteins. For instance, the binding mode of antibodies involving CDR loops has inspired the engineering of new generations of Monobodies, DARPins, and Affitins with artificially extended loop(s) (Correa et al, 2014;Koide et al, 2012;Schilling et al, 2014).…”

“…Binders with high pH and thermal stabilities have been obtained by coupling the generation of combinatorial libraries of Sac7d variants corresponding to the randomization of 10-14 residues of the ␤-sheet surface originally involved in the binding of DNA and selections against different targets. We recently reported that natural (Béhar et al, 2013) or artificially extended loops (Correa et al, 2014) of Sac7d can be advantageously recruited to generate Affitins, some of them showing potent inhibition properties by penetrating deep into a glycosidase active site.…”

The archaeal “7 kDa DNA-binding” proteins: extended characterization of an old gifted family

“…DSC experiments were carried out in PBS as described previously49 using a VP-DSC instrument (Microcal, Northampton, MA) and data was analyzed with the software supplied with the equipment.…”

Artificial proteins as allosteric modulators of PDZ3 and SH3 in two‐domain constructs: A computational characterization of novel chimeric proteins

“…These two mini‐proteins are ideal candidates to study in atomic detail the interplay between native interactions and interactions at the protein surface. Affitin H4 is an artificial protein able to selectively bind antigens that can be easily engineered to recognize various protein or DNA targets.…”