Macrocyclisation is a strategy that has been widely adopted by nature to produce polypeptides with remarkable stability. The advantages of cyclising proteins and peptides are being realised in drug discovery and development where an increased number of cyclic natural products and peptide-based drug leads are being reported. Similarly, protein cyclisation has also found applications in biotechnology, where circularised nanodiscs (cNDs) have been reported to be highly stable and homogeneous and yield stable lipid bilayers suitable as drug carriers and model membranes for biophysical studies of membrane proteins.
Among the common biological methods for macrocyclisation, 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 optimisation of cyclisation over the competitive polymerisation reaction. The undesirable polymerisation reaction can be minimised by reducing the substrate concentration but that results in very slow cyclisation rates — leading to unfeasible reaction conditions.
We have developed a novel, general, self-cyclising class of 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 polymerisation can be minimised without affecting the reaction rate. We investigated the utility of this platform in producing a range of differently sized cNDs. Using autocyclases, we successfully incorporated ion channel voltage-sensing domain in cND, identified the lipid-binding interface of an ion channel-targeting peptide and produced the first data on the in vivo metabolism of cNDs as a function of cND size and cyclisation.
Autocyclases offer a simple and scalable platform to access a vast diversity of macrocyclised peptides and proteins and facilitate future research across fields of structural biology, drug design and delivery.