Inhibition of Caspase-2 by a Designed Ankyrin Repeat Protein: Specificity, Structure, and Inhibition Mechanism

Abstract

Specific and potent caspase inhibitors are indispensable for the dissection of the intricate pathways leading to apoptosis. We selected a designed ankyrin repeat protein (DARPin) from a combinatorial library that inhibits caspase-2 in vitro with a subnanomolar inhibition constant and, in contrast to the peptidic caspase inhibitors, with very high specificity for this particular caspase. The crystal structure of this inhibitor (AR_F8) in complex with caspase-2 reveals the molecular basis for the specificity and, together with kinetic analyses, the allosteric mechanism of inhibition. The structure also shows a conformation of the active site that can be exploited for the design of inhibitory compounds. AR_F8 is a specific inhibitor of an initiator caspase and has the potential to help identify the function of caspase-2 in the complex biological apoptotic signaling network.

Structural and functional analysis of phosphorylation-specific binders of the kinase ERK from designed ankyrin repeat protein libraries

“…Apart from being useful as capturing molecules in protein arrays or affinity precipitations, DARPins are especially suited for functional studies as intracellular protein-specific reagents (intrabodies), because they do not require stabilizing disulfide bonds (12,18,39) and because they can thus fold in the cytoplasm, where they neither aggregate nor are degraded. Intrabodies targeting PTM sites are of particular importance for detection of target protein level and the present status of modification.…”

Repeat-protein folding: New insights into origins of cooperativity, stability, and topology

“…As described below, designed consensus repeats have been shown to adopt their target structures, and in many cases appear to possess very high thermodynamic stability. Moreover, consensus repeat proteins have been used as structural platforms to generate high-affinity protein interaction domains [37][38][39][40].…”

“…As described below, designed consensus repeats have been shown to adopt their target structures, and in many cases appear to possess very high thermodynamic stability. Moreover, consensus repeat proteins have been used as structural platforms to generate high-affinity protein interaction domains [37][38][39][40].2 For a few repeat proteins such as individual clathrin heavy-chain repeats and the β-helix domains of pertactin and pelC, primary sequence similarity between adjacent repeats are not easily detected. 3 Some repeat proteins curve so much that they form a closed, circular structure.…”

Amyloid-β Peptide-specific DARPins as a Novel Class of Potential Therapeutics for Alzheimer Disease

“…Furthermore, we could confirm the N-terminal A␤ domain to be involved in DARPin recognition, and of the several monoclonal antibodies with comparable affinities tested (33), 6E10 was most impaired in binding the complex of D23 and A␤ and thus competes for the epitope. A peptide scanning approach enlarged this binding region to the central A␤ domain, suggesting the binding of a discontinuous rather than linear epitope, as generally described for DARPins binding to different targets (10,12,34). A␤ is thought to contain a largely unstructured N terminus (residues 1-11) and two domains spanning residues 12-21 and 24 -42, respectively, with varying contacts among each other, whereas the connecting residues (22 and 23) face the solvent (35).…”