Curvature of designed armadillo repeat proteins allows modular peptide binding

Abstract

Designed armadillo repeat proteins (dArmRPs) were developed to create a modular peptide binding technology where each of the structural repeats binds two residues of the target peptide. An essential prerequisite for such a technology is a dArmRP geometry that matches the peptide bond length. To this end, we determined a large set (n=27) of dArmRP X-ray structures, of which 12 were previously unpublished, to calculate curvature parameters that define their geometry. Our analysis shows that consensus dArmRPs exhibit curvatures close to the optimal range for modular peptide recognition. Binding of peptide ligands can induce a curvature within the desired range, as confirmed by single-molecule FRET experiments in solution. On the other hand, computationally designed ArmRPs, where side chains have been chosen with the intention to optimally fit into a geometrically optimized backbone, turned out to be more divergent in reality, and thus not suitable for continuous peptide binding. Furthermore, we show that the formation of a crystal lattice can induce small but significant deviations from the curvature adopted in solution, which can interfere with the evaluation of repeat protein scaffolds when high accuracy is required. This study corroborates the suitability of consensus dArmRPs as a scaffold for the development of modular peptide binders.

Structural analysis of biological targets by host:guest crystal lattice engineering

“…3A,B. We proceeded with DARPins, as they are more rigid than dArmRPs, which possess some internal flexibility 22 . Furthermore, the selection of tight binders from DARPin libraries against almost any target molecule is well established 23 .…”

Toxin Neutralization Using Alternative Binding Proteins

“…In this final library, and after significant engineering efforts [164,165,169,170], six randomization positions per repeat could be achieved, conferring a theoretical diversity of 9.9 × 10 6 per repeat [168], and by stacking of the repeats, target specificity is obtained. As target specificity was obtained by stacking of this modular system, the system has the potential to allow for generation of preselected repeats for certain short pieces of target peptide that can be custom designed and assembled on demand into a new protein that can bind a prescribed, extended peptide [171].…”

“…Conversely, mutations in nArmRPs (or ArmR-containing proteins) have been linked to a number of diseases, such as Parkinson’s disease [172], neuroblastoma progression [173], and Bilateral macronodular adrenal hyperplasia [174]. Also, designing preselected repeats may have the drawback that significant deviations occur in the ArmRP’s curvature upon changes in environment, such as specific solutions, making it more difficult to design for larger target peptides [171]. Another obstacle is the expected rapid clearance from the circulatory system, which may prevent the ArmRPs to efficiently exert their effects, although it is yet to be tested in vivo.…”

Symmetry at the Cellular Mesoscale

“…The inner surface of this helix interacts with an extended conformation of a target protein. The structure that is shown here is an engineered version that is designed to bind to a repeated peptide of lysine and arginine amino acids with a defined length [13].…”