A Straight Path to Circular Proteins

Folding and stability are parameters that control protein behavior. The possibility of conferring additional stability on proteins has implications for their use in vivo and for their structural analysis in the laboratory. Cyclic polypeptides ranging in size from 14 to 78 amino acids occur naturally and often show enhanced resistance toward denaturation and proteolysis when compared with their linear counterparts. Native chemical ligation and intein-based methods allow production of circular derivatives of larger proteins, resulting in improved stability and refolding properties. Here we show that circular proteins can be made reversibly with excellent efficiency by means of a sortase-catalyzed cyclization reaction, requiring only minimal modification of the protein to be circularized.Sortases are bacterial enzymes that predominantly catalyze the attachment of surface proteins to the bacterial cell wall (1, 2). Other sortases polymerize pilin subunits for the construction of the covalently stabilized and covalently anchored pilus of the Gram-positive bacterium (3–5). The reaction catalyzed by sortase involves the recognition of short 5-residue sequence motifs, which are cleaved by the enzyme with the concomitant formation of an acyl enzyme intermediate between the active site cysteine of sortase and the carboxylate at the newly generated C terminus of the substrate (1, 6–8). In many bacteria, this covalent intermediate can be resolved by nucleophilic attack from the pentaglycine side chain in a peptidoglycan precursor, resulting in the formation of an amide bond between the pentaglycine side chain and the carboxylate at the cleavage site in the substrate (9, 10). In pilus construction, alternative nucleophiles such as lysine residues or diaminopimelic acid participate in the transpeptidation reaction (3, 4).When appended near the C terminus of proteins that are not natural sortase substrates, the recognition sequence of Staphylococcus aureus sortase A (LPXTG) can be used to effectuate a sortase-catalyzed transpeptidation reaction using a diverse array of artificial glycine-based nucleophiles (Fig. 1). The result is efficient installation of a diverse set of moieties, including lipids (11), carbohydrates (12), peptide nucleic acids (13), biotin (14), fluorophores (14, 15), polymers (16), solid supports (16–18), or peptides (15, 19) at the C terminus of the protein substrate. During the course of our studies to further expand sortase-based protein engineering, we were struck by the frequency and relative ease with which intramolecular transpeptidation reactions were occurring. Specifically, proteins equipped with not only the LPXTG motif but also N-terminal glycine residues yielded covalently closed circular polypeptides (Fig. 1). Similar reactivity using sortase has been described in two previous cases; however, rigorous characterization of the circular polypeptides was absent (16, 20). The circular proteins in these reports were observed as minor components of more complex reaction mixtures, and the cyclization reaction itself was not optimized.Open in a separate windowFIGURE 1.Protein substrates equipped with a sortase A recognition sequence (LPXTG) can participate in intermolecular transpeptidation with synthetic oligoglycine nucleophiles (left) or intramolecular transpeptidation if an N-terminal glycine residue is present (right).Here we describe our efforts toward applying sortase-catalyzed transpeptidation to the synthesis of circular and oligomeric proteins. This method has general applicability, as illustrated by successful intramolecular reactions with three structurally unrelated proteins. In addition to circularization of individual protein units, the multiprotein complex AAA-ATPase p97/VCP/CDC48, with six identical subunits containing the LPXTG motif and an N-terminal glycine, was found to preferentially react in daisy chain fashion to yield linear protein fusions. The reaction exploited here shows remarkable similarities to the mechanisms proposed for circularization of cyclotides, small circular proteins that have been isolated from plants (21–23).