Acyl Transfer Catalytic Activity in De Novo Designed Protein with N-Terminus of α-Helix As Oxyanion-Binding Site

Abstract

The design of catalytic proteins with functional sites capable of specific chemistry is gaining momentum and a number of artificial enzymes have recently been reported, including hydrolases, oxidoreductases, retro-aldolases, and others. Our goal is to develop a peptide ligase for robust catalysis of amide bond formation that possesses no stringent restrictions to the amino acid composition at the ligation junction. We report here the successful completion of the first step in this long-term project by building a completely de novo protein with predefined acyl transfer catalytic activity. We applied a minimalist approach to rationally design an oxyanion hole within a small cavity that contains an adjacent thiol nucleophile. The N-terminus of the α-helix with unpaired hydrogen-bond donors was exploited as a structural motif to stabilize negatively charged tetrahedral intermediates in nucleophilic addition−elimination reactions at the acyl group. Cysteine acting as a principal catalytic residue was introduced at the second residue position of the αhelix N-terminus in a designed three-α-helix protein based on structural informatics prediction. We showed that this minimal set of functional elements is sufficient for the emergence of catalytic activity in a de novo protein. Using peptide-α thioesters as acyl-donors, we demonstrated their catalyzed amidation concomitant with hydrolysis and proved that the environment at the catalytic site critically influences the reaction outcome. These results represent a promising starting point for the development of efficient catalysts for protein labeling, conjugation, and peptide ligation.

Biocatalysts Based on Peptide and Peptide Conjugate Nanostructures

“…141 Degrado's group recently showed that three-helix bundles formed by domain-swapped 48-residue dimers (DSDs) can be designed as catalysts containing oxyanion-binding sites which were active in acyl transfer reactions such as peptide ligation through transthioesterification and aminolysis. 142 The heteromeric dimers contain pairs of peptides bearing complementary E or R residues, the anionic peptide being functionalized with an active cysteine residue near its N-terminus to facilitate the reaction with a peptide-α thioester substrate.…”

From peptides to proteins: coiled-coil tetramers to single-chain 4-helix bundles

“…Breaking the symmetry of de novo CC homo-oligomers to make heteromeric systems has many advantages and potential applications. 37, 52, 76 We sought to expand the utility of apCC-Tet* by redesigning it to make an A2B2 heterotetramer, apCC-Tet*-A 2 B 2 . For this, we maintained the g, a, d and e sites as Gln, Leu, Ile, and Ala, respectively, and made two potentially complementary peptides: an acidic peptide, apCC-Tet*-A, with Glu at all b and c positions; and a basic peptide, apCC-Tet*-B, with Lys at those sites.…”

“…Breaking the symmetry of de novo CC homo-oligomers to make heteromeric systems has many advantages and potential applications. 37,52,76 We sought to expand the utility of 1 and S2). The four peptides were synthesized by SPPS, purified, verified by mass spectrometry (Figures S23-S26), and characterized alone and as equimolar paired mixtures as follows.…”

pH-Controlled Protein Orthogonal Ligation Using Asparaginyl Peptide Ligases

“…The practice of enzymatic peptide ligation for protein synthesis and protein tagging began with the engineering of subtiligase in the 1990s, which also witnessed the original development of chemical ligation methods that operate in water. − Subtiligase and its variants are engineered from bacterial proteases (i.e., subtilisins) and catalyze the aminolysis of an ester or thioester substrate for peptide bond formation. − Most recently, de novo proteins were designed to catalyze the acyl transfer reaction of peptide thioesters for aminolysis, representing a promising initial step toward the development of artificial peptide ligases . In contrast, naturally occurring peptide ligases such as sortase A and PALs effect peptide ligation through catalyzing the transpeptidation reaction of straight peptide or protein substrates.…”

“…60−63 Most recently, de novo proteins were designed to catalyze the acyl transfer reaction of peptide thioesters for aminolysis, representing a promising initial step toward the development of artificial peptide ligases. 64 In contrast, naturally occurring peptide ligases such as sortase A 65 and PALs effect peptide ligation through catalyzing the transpeptidation reaction of straight peptide or protein substrates. With their extremely high catalytic activity toward P1-Asn substrates, PALs are the most powerful peptide ligases known to date.…”