Macrocyclization is a strategy that is widely adopted by nature to produce polypeptides with remarkable stability. The advantages of cyclizing proteins and peptides are being realised in drug discovery and development where an increased number of cyclic peptide-based drug leads are being reported. Similarly, protein cyclization has also found applications in biotechnology, where circularized nanodiscs (cNDs) have been reported to be highly homogeneous and yield stable lipid bilayers that are suitable as drug carriers and model membranes for biophysical studies of membrane proteins.
Among the common biological methods for macrocyclization, enzymatic transpeptidation using sortase A (SrtA) is perhaps the most popular method. While very effective, the conventional bimolecular reaction involving the polypeptide substrate and SrtA require careful optimization of cyclization over the competitive polymerization reaction. The undesirable polymerization reaction can be minimised by reducing the substrate concentration but that results in very slow cyclization rates — leading to unfeasible reaction conditions.
We have developed a novel class of self-cyclizing proteins that we call “autocyclases”, in which the protein substrate is suitably linked to the ligase (SrtA). We demonstrate that this design enables the reaction to follow a unimolecular mechanism, where polymerization can be minimised without affecting the reaction rate. We investigated the utility of this platform in producing different cyclic peptides and a range of differently-sized cNDs. Using autocyclases, we successfully produced the first uniformly 15N/13C-labelled cyclized disulfide-rich peptide and acquired triple resonance NMR spectra of the peptide, incorporated an ion channel voltage-sensing domain in cND for the first time and produced the first data on the in vivo metabolism of cNDs as a function of cND size and cyclization.
Autocyclases offer a simple and scalable platform to access a vast diversity of macrocyclized peptides and proteins and facilitate future research across fields of structural biology, drug design and delivery.