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 α-helix 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-^αthioesters 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.

Figure 1.

Structural alignment of pSer-containing fragments from structures in PDB (accessed in February 2020) with pSer located at Ncap, N1, and N2 positions of the corresponding α-helical fragments. The occurrences of pSer in a nonredundant version of the PDB were determined using the database and methods previously described. pSers at Ncap positions form two clusters, due to a bimodal distribution of its ψ-dihedrals. In both clusters, all of the pSers form H-bonds with downstream amides. However, only 7 out of 23 pSers in the N1 position hydrogen bond to the helix backbone. The geometry of N2 pSers appears as stabilizing as Ncap pSers; 13 out of 16 fragments participate in H-bonds with their preceding and/or succeeding amides. pSers at the Ncap and N2 positions are observed contributing bivalent H-bonds, whereas only 1 H-bond is observed for pSer at the N1 position. Example H-bonds are represented by dashed lines, and phosphate groups that do not H-bond with the helix backbone are transparent. The PDB entries of the structures used in the alignment are listed in Table S1.

Figure 2.

Design of heterodimers. (A) Left: charged residue patterning by grafting negative charges (Glu in red) on the strand containing the Cys catalytic residue and positive charges (Arg in blue) onto the other helix. Right: resulting coulombic surface is shown. (B) Helical wheel diagram of the proteins that corresponds to the halfstructure depicted above the dashed line in A. Leucine residues constitute the hydrophobic core (a and d positions). Glu and Arg residues at e and g positions promote heterodimerization. (C) Sequences of the two monomers of Het-N2. (D) Sequences of the three control constructs with Cys at the Ncap position in Het-Ncap, a 12-residue N-terminal fragment trunc-Cys-N2, and negative control Neg-N2 with Ala at the N2 position. The underlined sequence fragments correspond to that depicted on the wheel diagram in B.

Figure 3.

X-ray structures of the synthesized DSD analogues Het-N2 (left) and Het-Ncap (right). Catalytic sites are shown revealing solvent-exposed cysteine residues depicted in magenta with sulfur atom in yellow in ball-and-sticks representation. The simulated annealing 2F_o–F_c electron density map is shown at the 1.0σ level. The X-ray structures of Het-N2 and Het-Ncap (depicted in cyan) are superimposed onto the original DSD structure (in yellow). PDB IDs: 6Z0L (Het-N2) and 6Z0M (Het-Ncap). Table S3 provides the refinement statistics. Figures S6 and S7 show crystal packing and conformational variability of N-termini.

Figure 4.

Active site in Het-N2 is located at the interface of two N-termini of α-helices with nucleophilic Cys^N2 positioned next to a small cavity. (A) Horizontal view illustrating the helix–helix interface with the unpaired N–H groups pointing toward the cavity; (B) surface representation; (C) clipped section along the helical axis. Sulfur atom of Cys^N2 is in yellow.

Figure 5.

Cys-S-sulfonate mimicking tetrahedral intermediates. (A) Similarity of geometry and H-bonding interactions with catalytic protein for Cys-SO3⁻ and tetrahedral intermediates for transthioesterification and amidation steps (X = S or NH2⁺). (B) Two distinct conformations of Cys-SO3⁻ and helical N-termini observed in the crystal structure of Het-N2-SO3⁻ (PDB ID: 7BEY). Conformation 1 contains Cys-SO3⁻ H-bonded to adjacent amides. An H₂O molecule mediating additional H-bonding interactions is in cyan. The H-bond donor–acceptor distances are shown in Å. More information is shown in Table S3 and Figures S8 and S9.

Figure 6.

Characterization of catalytic activity. (A) Ac-VALENF-^αthioester consumption in the presence of Het-N2, Het-Ncap, trunc-N2-Cys, glutathione, and MPAA (4-mercaptophenylacetic acid) in comparison to background reaction and negative control Neg-N2. (B) Changes in concentration of peptide–protein branched thioester resulting from the transthioesterification between Ac-VALENF-^αthioester and Het-N2, Het-Ncap, trunc-Cys-N2, or glutathione. (C) Michaelis–Menten kinetics for the catalyzed reaction of Ac-VALENF-^αthioester with Het-N2. (D) Observed rate constants and outcome of the reaction between Ac-GRLEEIDR-^αthioester and Tris in the presence of Het-N2 as a function of the concentration of Gn·HCl and the degree of folding of Het-N2 based on CD measurements.

Scheme 1.

Mechanism for the Catalysis of the Acyl Transfer Reaction (e.g., Peptide Ligation) by De Novo Catalytic Protein (Depicted Schematically in Cyan)^{a a}TI-1 and TI-2 refer to the key tetrahedral intermediates of transthioesterification and amidation steps, respectively, where negatively charged oxyanions are stabilized by the H-bonding network (shown in blue).