Introduction
The SpyCatcher-SpyTag system is a widely used protein ligation method. SpyCatcher is derived from a modified domain of the surface protein of Streptococcus pyogenes and specifically recognizes the homologous 13-amino acid peptide called SpyTag. Upon recognition, a peptide bond forms between the lysine in SpyCatcher and the aspartic acid side chain in SpyTag. Additionally, related systems called SnoopCatcher and DogCatcher were discovered in Streptococcus pneumoniae. Prior to conducting wet experiments, we aimed to comprehend the conformation of SpyCatcher, SnoopCatcher, and DogCatcher after ligation to the protein cage. To achieve this, we utilized the ColabFold online version for structural modeling to explore the fusion protein's structure involving Catcher and the protein cage subunit.
Results & Interpretation
Fig. 1. Structures predictedbyColabfold.(A)SpyCatcher-Mi3_subunit.(B)SnoopCatcher-Mi3_subunit.(C)DogCatcher-Mi3_subunit.
Furthermore, we employed molecular dynamics simulations to conduct an initial characterization of the three proteins. To evaluate the impact of linker reduction between the two proteins on the simulation outcomes, we utilized only GGS as the linker during the simulation. The Charmm36 force field was chosen for simulating the proteins in an aqueous solution at 25°C for a duration of 50ns.
Their partial trajectories are animated as follows
Fig. 2. The trajectory of SpyCatcher-Mi3_subunit
Fig. 3. The trajectory of SnoopCatcher-Mi3_subunit
Fig. 4. The trajectory of DogCatcher-Mi3_subunit
Fig. 5. RMSD for structures of SpyCatcher-Mi3_subunit, SnoopCatcher-Mi3_subunit, and DogCatcher-Mi3_subunit.
The RMSD analysis reveals significant conformational changes in DogCatcher-mi3_subunit during the 50ns simulation compared to its initial conformation. In contrast, the structural changes observed in SpyCatcher-mi3_subunit and SnoopCatcher-mi3_subunit remain within a reasonable and comparatively stable range over the course of the simulation.
Fig. 6. Rg for structures of SpyCatcher-Mi3_subunit, SnoopCatcher-Mi3_subunit, and DogCatcher-Mi3_subunit.
The gyrate radius (Rg) results demonstrate a similar trend, where the structure of SpyCatcher-mi3_subunit and SnoopCatcher-mi3_subunit gradually converges and stabilizes as the simulation progresses. Conversely, for DogCatcher-mi3_subunit, the Rg continuously fluctuates within a broad range throughout the entire simulation, indicating ongoing structural changes and lack of stabilization.
Conclusion
In summary, our analysis of RMSD and Rg has shed light on the stability of the three fusion proteins, focusing on dry experiments. Based on these findings, it appears that SpyCatcher and SnoopCatcher are promising protein tags that can be effectively incorporated into our protein cage system, given their relatively stable behavior and structural characteristics.
Future Directions
In our analysis, we have exclusively focused on a single subunit of Mi3. To glean more intricate insights, conducting a stepwise simulation of Catcher-Mi3 could prove valuable. This approach would involve comparing and analyzing the subunits of SpyCatcher-Mi3, SnoopCatcher-Mi3, and DogCatcher-Mi3 at various hierarchical levels, allowing for a more comprehensive understanding of their behavior and interactions within the protein cage system.
