Novel miniproteins engineered by the transfer of active sites to small natural scaffolds

Abstract

Small multidisulfide‐containing proteins are attractive structural templates to produce a biologically active conformation that mimics the binding surface of natural large proteins. In particular, the structural motif that is evolutionary conserved in all scorpion toxins has a small size (30–40 amino acid residues), a great structural stability, and high permissiveness for sequence mutation. This motif is composed of a β‐sheet and an α‐helix bridged in the interior core by three disulfides. We have used this motif successfully to transfer within its β‐sheet new functional sites, including the curaremimetic loop of a snake neurotoxin and the CDR2‐like site of human CD4. Accumulated evidence indicated that the two miniproteins produced, the curaremimetic miniprotein and the CD4 mimetic, contain the α/β fold that is characteristic of the scaffold used and bind respectively to the acetylcholine receptor and to the envelope gp120 of HIV‐1. Furthermore, the latter was shown to prevent viral infection of lymphocytes. These examples illustrate that, by the transfer of active sites to small and stable natural scaffolds, it is possible to engineer miniproteins reproducing, in part, the function of much larger proteins. Such miniproteins may be of great utility as tools in structure‐function studies and as leads in drug design. © 1998 John Wiley & Sons, Inc. Biopoly 47: 93–100, 1998

Direct Expression and Validation of Phage-selected Peptide Variants in Mammalian Cells

“…First, it binds gp120 with higher affinity and neutralizes HIV-1 more efficiently than CD4mim1. Second, some steric interference between the N terminus of CD4mim1 and gp120 has been observed (22), precluding N-terminal fusions with this mimetic. By eliminating the first disulfide bond of CD4mim1, the N terminus of CD4mim6 can now be readily fused to a CCR5-mimetic peptide, positioning the latter mimetic much closer to its binding pocket.…”

Endoradiotherapy with Peptides - Status and Future Development

“…Binding surfaces for proteins have been engineered on the faces of helical bundles or similar molecules ( Table 3, [137][138][139][140][141][142][143][144][145][146][147][148][149][150][151][152]). Some scaffolds, such as those based on lipocalins, the tenth type III fibronectin domain or helical bundles such as affibodies which are derivatives from the Zdomain of S. aureus protein A, have been successfully used for the selection of high-affinity binders [153].…”

Engineered proteins as specific binding reagents

“…In this respect, the scorpion toxins charybdotoxin [69][70][71], scyllatoxin [72 ,73] and a-conotoxin [74] (Figure 1; Table 1), the cellulose-binding domain of cellulases [75,76], the insect defensin A [77] (secreted by certain larvae to attack bacterial membranes), and the Ecballium elaterium trypsin inhibitor II [78] have been used as scaffolds for generating new binding molecules. While the cellulose-binding domain and charybdotoxin were used to generate novel binding specificities via surface residue randomization and selection, the charybdotoxin and other scaffolds were also used in loop grafting studies with loops of defined sequence.…”