Protease-based signaling and logic
- New antiparallel and destabilized coiled coil pairs were designed and functionally characterized in mammalian cells.
- Coiled coils were combined with split luciferase fragments to design functions with logical negation.
- Light and chemically inducible proteases were used as mediators in a functional proof of concept for fast regulated logic gates.
As the main challenge of our project was to create fast responsive synthetic circuits in cells, we sought to implement logic operations based on protein post-translational modification, rather than slower transcriptional activation. Introduction of a protease targeting site has already been used to engineer linear signaling pathways that functioned in vitro using linked autoinhibitory peptides
We realized that the same design could be adapted for our orthogonal proteases by replacing the cleavage sites with appropriate protease target motif, such as for the orthogonal proteases PPVp and TEVp.
Initial testing based on published coiled coil modules
The constructs B:nLuc, cLuc:A, A’:TEVs:B:nLuc and cLuc:A:PPVs:B’2A, which do not possess autoinhibitory segments, were tested for CC binding by measuring luciferase reconstitution. Constructs without protease cleavage sites (B:nLuc, cLuc:A ) were used as a control (4.12.1.). A’:TEVs:B:nLuc and cLuc:A:PPVs:B’2A were tested in the presence of TEVp and PPVp, which cleave off the autoinhibitory CC, resulting in split luciferase reconstitution. Additionally, different ratios of constructs were tested in order to obtain the best luciferase activity (4.12.1.).
Results showed that very high amounts of the constructs based on same coiled coil sequences used by Shekhawat et al.
Alpha-helical segment interaction is a common feature in protein tertiary and quaternary structures, where helices form complexes of two or more
coils (220.127.116.11). The most frequent interaction is between two alpha-helices, which form a dimeric coiled coil. Interactions can occur both in the two parallel
or antiparallel orientation of the coil pairs
Sequences of coiled coils that form interactions have a characteristic seven amino acid repeat, called heptad repeat. The position of each amino acid
within a heptad is presented in a unified nomenclature (a,b,c,d,e,f,g). Interaction between two coils occurs on a continuous patch along the side of each
alpha-helix with each patch facing the core of the dimer’s interface (18.104.22.168B). The amino acid residues which occupy this strip correspond to the
a and d positions of the heptad; they are generally hydrophobic and represent the driving force behind dimerization
Two alpha-helices that form a coiled coil can interact either in a parallel or in an antiparallel orientation
Antiparallel CC orientation allows for fusion of C-termini of N-part of split protein to N-termini of CC via a shorter linker, thereby likely resulting in more efficient reconstitution upon binding with appropriate CC partner. As represented in the wheel helical projection in 22.214.171.124 parallel CC are stabilized by electrostatic interactions g:e’ and e:g’, while interactions between g:g’ and e:e’ positions stabilize antiparallel CC. While CC orientation is mainly influenced by electrostatic interactions specific amino acid residues such as Asn inside CC core can contribute to the orientation as well. Due to polarity of the Asn residue two asparagines prefer interaction with each other rather than with other hydrophobic residues in vicinity such as Leu and Ile. These interactions stabilize the core of intended CC orientation and destabilize the core of CC in the opposite orientation.
Antiparallel coiled coils
In order to compare the reconstitution efficiency of split protein dictated by parallel or antiparallel coiled coil interaction, we prepared fusion proteins with split firefly luciferase where we designed a new antiparallel peptide (AP4) and tested their activity in cells. Antiparallel coiled coils (AP4:P3) worked significantly better than parallel coiled coils (P4:P3) (4.12.4), thus demonstrating that a shorter linker between reporters and dimerizing units helps in the reconstitution of the split protein.
To investigate whether the newly designed antiparallel CC is suited for implementation as logic unit into our system, the constructs nLuc:AP4 and P3:cLuc were compared
to the coiled coil cLuc:A and B:nLuc from Shekhawat et al.
The system presented by Shekhawat et al is able to process AND or OR logic functions but not those including negation (such as NOR, NAND etc.) We realized that this type of logic functions could be accomplished by introducing an additional cleavage site between the split reporter and coiled coil segment (126.96.36.199).
Constructs were therefore modified by the addition of TEVp cleavage site (TEVs) between nLuc and AP4 and PPVp cleavage site (PPVs) between P3 and cLuc. This represents a logic NOR gate based on the input signals, represented by TEVp and PPVp.
Destabilized coiled coils
For implementation of the system with additional logic operations further modifications on our own CCs collection were needed. Analysis of the equilibrium model reveals that the affinity of the autoinhibitory segment should not be too strong, otherwise the inhibition will remain; but should also not be too weak, otherwise the system would be leaky and active already without cleavage. Stability of the coiled coil interaction can be tuned by introduction of non-favorable interactions e.g. by introducing Ala residues at a and d positions
Those variably destabilized peptides were used as autoinhibitory coiled coil forming segments to test the difference in activity between the uncleaved and TEVp cleaved forms.
To test which one of our four destabilized CCs worked best, all constructs were tested in vivo with and without the presence of TEVp (4.12.9.). We concluded that P3mS and P3mS-2A demonstrated the highest fold increase in the luciferase activity upon the addition of TEVp. The other two constructs showed little to no increase in luciferase activity upon the addition of TEVp, suggesting that the peptides were destabilized too much leading to the leakage in the uninduced form.
The final test was to investigate if the system could indeed be controlled by two signals at the same time. In order to test this we constructed NOR gate with logic processing (nLuc:TEVs:AP4 and P3:PPVs:cLuc) and inducible components (split PPVp and TEVp inducible by the rapamycin and light, respectively) (4.12.13.).
The types building blocks that we developed made possible to construct all of the possible 16 two-input logic function based on the proteolysis. To demonstrate this we tested additional logic operations (A and A nimply B), where the response was measured directly after 15 minutes of induction to demonstrate the increased speed of protein-based processing, as shown on 4.12.11..
In both cases the system performed very well, producing clear difference between the active and inactive output states within 15 min after the stimulation by combinations of two signals. Logic function A nimply B is relatively difficult to implement but on the other hand it can be quite useful as for example the signal B may identify the cell type and trigger activation by an external signal only in a selected cell types or cells in a selected state.
The availability of a set of orthogonal proteases as well as an orthogonal coiled coil dimers toolset enabled the construction of a fast complex logic processing circuits. The previous CC toolbox has been further expanded with a strategy of generating antiparallel and destabilized CC. Furthermore, designed system based on split proteases can also be linked to many other input signals such as intracellular calcium increase. An important advance is the adaptation of the system to function in vivo in mammalian cells. Further, reporter as the output signal could be substituted by a split protease, enabling multi-layered processing, or used as an trigger for other cellular processes, such as the release of therapeutics. Therefore, we believe that those results represent a valuable foundational advance in synthetic biology.