Structural plasticity in the loop region of engineered lipocalins with novel ligand specificities, so-called Anticalins

Abstract

Anticalins are generated via combinatorial protein design on the basis of the lipocalin protein scaffold and constitute a novel class of small and robust engineered binding proteins that offer prospects for applications in medical therapy as well as in vivo diagnostics as an alternative to antibodies. The lipocalins are natural binding proteins with diverse ligand specificities which share a simple architecture with a central eight-stranded antiparallel β-barrel and an α-helix attached to its side. At the open end of the β-barrel, four structurally variable loops connect the β-strands in a pair-wise manner and, together, shape the ligand pocket. Using targeted random mutagenesis in combination with molecular selection techniques, this loop region can be reshaped to generate pockets for the tight binding of various ligands ranging from small molecules over peptides to proteins. While such Anticalin proteins can be derived from different natural lipocalins, the human lipocalin 2 (Lcn2) scaffold proved particularly successful for the design of binding proteins with novel specificities and, over the years, more than 20 crystal structures of Lcn2-based Anticalins have been elucidated. In this graphical structural biology review we illustrate the conformational variability that emerged in the loop region of these functionally diverse artificial binding proteins in comparison with the natural scaffold. Our present analysis provides picturesque evidence of the high structural plasticity around the binding site of the lipocalins which explains the proven tolerance toward excessive mutagenesis, thus demonstrating remarkable resemblance to the complementarity-determining region of antibodies (immunoglobulins).