Destabilizing an interacting motif strengthens the association of a designed ankyrin repeat protein with tubulin.

Shoeb Ahmad,Marcel Knossow,Birgit Dreier,Benoît Gigant,Andreas Plückthun,Magali Aumont-Nicaise,Ludovic Pecqueur,Djemel Hamdane

doi:10.1038/srep28922

Abstract

Affinity maturation by random mutagenesis and selection is an established technique to make binding molecules more suitable for applications in biomedical research, diagnostics and therapy. Here we identified an unexpected novel mechanism of affinity increase upon in vitro evolution of a tubulin-specific designed ankyrin repeat protein (DARPin). Structural analysis indicated that in the progenitor DARPin the C-terminal capping repeat (C-cap) undergoes a 25° rotation to avoid a clash with tubulin upon binding. Additionally, the C-cap appears to be involved in electrostatic repulsion with tubulin. Biochemical and structural characterizations demonstrated that the evolved mutants achieved a gain in affinity through destabilization of the C-cap, which relieves the need of a DARPin conformational change upon tubulin binding and removes unfavorable interactions in the complex. Therefore, this specific case of an order-to-disorder transition led to a 100-fold tighter complex with a subnanomolar equilibrium dissociation constant, remarkably associated with a 30% decrease of the binding surface.

Highlights

Bind tubulin two orders of magnitude stronger than the parental one, resulting in complexes with subnanomolar dissociation constant
In the course of the study of microtubules, we have selected DARPins that bind tubulin as multipurpose tools and in particular as crystallization chaperones[9]. Whereas these DARPins have proven instrumental to crystallize tubulin and its complexes with interacting proteins, the tubulin−DARPin affinity is within the range of values usually observed for interactions that are transient on a time scale of seconds to minutes[4], the measured equilibrium dissociation constant (KD) being in the 100 nM range[9]
We identified DARPins with an improved affinity for tubulin associated with the destabilization of the C-terminal capping motif (C-cap)

Summary

Introduction

Bind tubulin two orders of magnitude stronger than the parental one, resulting in complexes with subnanomolar dissociation constant. Biochemical and structural characterizations demonstrated that the affinity increase is coupled with the destabilization of the ankyrin C-terminal capping motif (C-cap) of the DARPin, which in the parent DARPin interacts with tubulin but needs to undergo a rotation to avoid a clash. This remarkable order-to-disorder transition illustrates a new mechanism for affinity maturation

Methods

Results

Conclusion