Molecular Recognition of Protein Surfaces:  High Affinity Ligands for the CBP KIX Domain

Abstract

Potent and specific inhibitors of protein•protein interactions have significant potential both as therapeutic compounds and biological tools, yet discovery of such molecules remains a significant challenge. Whereas small molecules typically bind proteins in small, well-defined, deep clefts, proteins generally recognize each other through formation of large, heterogeneous complementary surfaces. Our laboratory has recently described a general solution, called protein grafting, for the identification of highly functional miniature proteins by stabilization of α-helical binding epitopes on a protein scaffold. In protein grafting, those residues that comprise a functional epitope are grafted onto the solvent-exposed α-helical face of the small yet stable protein avian pancreatic polypeptide (aPP). In this study, we use protein grafting in combination with molecular evolution by phage display to identify phosphorylated peptide ligands that recognize the shallow surface of CBP KIX with high nanomolar to low micromolar affinity. Furthermore, we show that grafting of the CBP KIX-binding epitope of CREB KID onto the aPP scaffold yields molecules capable of high affinity recognition of CBP KIX even in the absence of phosphorylation. Importantly, both classes of designed ligands exhibit high specificity for the target CBP KIX domain over carbonic anhydrase and calmodulin, two unrelated proteins that bind hydrophobic or α-helical molecules that might be encountered in vivo.

Protein domain mimetics as in vivo modulators of hypoxia-inducible factor signaling

“…Given that both CBP and p300 regulate multiple signaling pathways, they provide an intriguing opportunity for an effective modulation of the expression of genes involved in cancer progression and metastasis (31). Our design strategy involves judicious mimicry of transcription factor fragments that contact p300/CBP to rationally develop artificial regulators of transcription (4,5).…”

“…High-resolution structures of transcription factors in complex with protein partners offer a foundation for rational drug design strategies (3)(4)(5). Although many transcription factors exhibit significant intrinsic disorder (6), their complexes with coactivator proteins often feature discrete protein secondary structures, such as α-helices, that contribute significantly to binding and may be used as templates for rational drug design (7,8).…”

Targeting protein–protein interactions by rational design: mimicry of protein surfaces

“…Schepartz et al have recently described an elegant strategy, termed protein grafting, for the identification of miniature proteins (MPs) that bind protein (and nucleic acid) targets with high affinity and selectivity, thereby inhibiting protein-protein interactions (Chin & Schepartz 2001a,b;Rutledge et al 2003;Montclare & Schepartz 2003;Golemi-Kotra et al 2004;Gemperli et al 2005). In this technique, which is often used in combination with molecular evolution, the residues that comprise a functional a-helical binding epitope are substituted ('grafted') onto the solvent-exposed, a-helical face of the small, yet well-folded avian pancreatic polypeptide (aPP).…”

Affinity Polymers Tailored for the Protein A Binding Site of Immunoglobulin G Proteins

“…[7][8][9] Various methods of protein grafting have been introduced that rely on epitope docking onto the surface of a miniature protein. [10,11] Dendrimeric multiplication of anionic binding sites has been used quite efficiently in lectin binding; thus multiple polyglycerol sulfates were shown to interact strongly, albeit in an unspecific fashion, with l-selectin. [12,13] Polymer nanoparticles decorated with charged functional groups possess interfaces that might act as capture agents for oppositely charged toxic peptides and proteins.…”