Chapter 3. Synthetic protein-protein interaction domains created by shuffling C2H2 ZFs
3.8 Discussion
3.8.1 DZF-derived C2H2 ZFs can be “mixed and matched”
The principal goal of DNA-binding ZF design was to engineer ZFs that can bind any DNA site of interest. To achieve this, different groups took advantage of the simple modular structure of DNA-binding ZFs and applied various methods including modeling, sequence
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Ik-Z23-GP IkD18Q-Z23-GP fold-activation of β- galactosidase expression
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Ik-Z23-GP IkD18Q-Z23-GP fold-activation of β- galactosidase expression
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Figure 3.24 Analysis of DZF mediated dimerization of DNA-binding ZFs.
Plasmids encoding Z23-GP fused to either the wild-type Ik DZF domain (blue bars) or the Ik DZF domain harboring the D18Q mutation (purple bars) together with the RNAP α-Gal4 expression plasmid were introduced into the 0-site B2H reporter strain harboring a composite DBS (see Table 3.4) and activity of these constructs was assessed over a range of IPTG concentrations.
comparison and selections to alter the specificity of individual fingers within a multifinger protein. These fingers together with naturally occurring ZFs can then be combined by
“mixing and matching” to create designer ZFs capable of recognizing novel DNA sequences.
In addition, various ZFs can be linked together into tandem arrays that are capable of recognizing extended DNA sequences. Before this study, it was not known whether protein-binding ZFs could be mixed and matched as well in order to create synthetic proteins with novel interaction specificities. It has previously been shown that the two ZFs in the Pegasus and Eos DZF domain are separable and capable of folding independently of each other (Westman et al., 2004). These data suggested that ZFs in the DZF domain may behave in a modular manner. In addition, a functional hybrid DZF domain consisting of two individual ZFs derived from different wild-type proteins has been described (McCarty et al., 2003).
Thus, we decided to systematically investigate whether C2H2 ZFs and DZFs could be “mixed and matched” to create domains with novel interaction specificities. Our results showed that shuffling of DZF-derived C2H2 ZFs can yield synthetic DZFs with new specificities that can then be linked together into more extended interaction interfaces. Interestingly, only a small number (less then 0.5%, 26 selected DZF pairs/5344 total potential pairs) of the potential combinations of DZFs were identified as positive for interactions. Since the theoretical number of combinations in all libraries was completely over sampled, and because our sequencing results revealed that we identified many of the interacting pairs multiple times, we are confident that we identified nearly all interacting DZFs. In addition, attempts to create synthetic DZFs without using the B2H selection strategy by simply constructing all possible combinations of C2H2 ZFs and linkers from the Ikaros and Drosophila Hunchback DZFs were unsuccessful and none of these synthetic domains could mediate interaction with each other. Thus, we conclude that individual fingers in the DZF domain do not always operate as completely modular units and, as is the case for DNA-binding C2H2 ZFs (Isalan et al., 1997;
Isalan et al., 1998; Wolfe et al., 1999; Hurt et al., 2003; see also section 1.2.5), context-dependent interactions are important for the binding affinity. This also emphasizes the importance and necessity of using selection methods to identify synthetic DZF pairs that are fully optimized for protein-binding.
3.8.2 Anti-parallel interaction mode
Analysis of the interaction specificities of the synthetic DZFs revealed a novel antiparallel interaction geometry for DZF domains. Previous studies suggested that DZF domains interact in a parallel fashion based on the interaction specificity of an engineered chimeric Ikaros/Hunchback DZF (McCarty et al., 2003). However, the identities of our selected synthetic DZFs together with their homo- and heterotypic interaction specificities indicated that these domains interact in an anti-parallel manner. Furthermore, the relative interaction affinities of the double-DZFs are most consistent with a model in which the component synthetic DZFs interact in an anti-parallel fashion. It is noteworthy that our selections also identified a synthetic DZF pair that suggests a parallel interaction mode (Pe-Hd-Hd interacting with Hd-Hd-Hl). Thus, both parallel and anti-parallel interaction geometries seem to exist for synthetic DZFs which further highlight the functional versatility of C2H2 ZFs. It is worth noting that these interaction modes were defined for synthetic isolated DZFs and may not reflect the geometry of naturally occurring DZFs in the context of a full-length protein.
3.8.3 Applications of synthetic DZFs
Our synthetic DZF domains have proven to be efficient in mediating assembly of a bi-partite transcriptional activator capable of stimulating expression of the endogenous VEGF-A gene in human cells. This represents an example of synthetic control as we managed to by-pass the normal regulatory signals such as hypoxia to activate VEGF-A. Furthermore, because this activation depends upon the presence of two DZF-linked proteins, it provides a mechanism for making VEGF-A expression dependent on two inputs (as in an ‘AND’ gate circuit, Kramer et al., 2004). These synthetic DZF domains can also be used to activate expression of a specific gene in a bacterial cell suggesting that they may also have implications for constructing synthetic circuits in bacteria.
The fact that our synthetic DZF domains mediate preferentially heterotypic interactions provides important advantages compared to naturally occurring wild-type DZFs. Heterotypic interactions may be particularly useful for applications requiring asymmetric complex assembly. In fact, these synthetic DZFs are more efficient in mediating assembly of a bi-partite transcription factor in our “activator reconstitution” assay than for example naturally
occurring homotypic DZFs. On explanation for this observation could be that the formation of unwanted homodimers of DNA-binding domain fusions (or activation domain fusions) is less likely to occur since our domains prefer heterotypic interactions. These undesired homodimers could compete with the formation of the wanted DNA-binding domain/activation domain heterodimer and would thereby impair the ability of the DZF domains to mediate activation of VEGF-A.
Furthermore, our synthetic DZF domains demonstrated a high level of specificity which may have helped avoiding any unwanted cross-interactions with endogenous competitors. In fact, our synthetic DZFs were functional in three cellular compartments (i.e. the cytoplasm of bacterial and mammalian cells and the nucleus of mammalian cells) indicating that none of the potential competitors present in these cellular contexts was able to interfere with the interactions. Moreover, the specificity did not depend on over-expression of the synthetic DZF domains since both lowering the expression levels of these DZFs and over-expression of potential interfering proteins did not impair the ability to interact with defined partners in human cells.
Another notable feature of our synthetic DZF domains is that they can be linked together into more extended arrays. This suggests that DZF domains can be used as modules to create synthetic multi-DZF ‘scaffold’ or ‘adaptors’ upon which various DZF-linked proteins might be assembled. Our initial attempts to design a transcriptional scaffold consisting of two transcriptional activators has proven to be challenging and the obtained activation of the VEGF-A expression was very weak. It will be interesting to explore whether it is possible to engineer a scaffold in the cytoplasm of a human cell by applying our synthetic DZF domains.
Such a scaffold could be used to create novel synthetic signaling pathways by simple tethering defined signaling molecules of interest (Park et al., 2003; Harris et al., 2001).
3.8.4 Future directions
Finally, these findings have important implications for future prospects to design C2H2 ZFs capable of interacting with any target protein of interest. The versatility and modularity of the DZF domain suggest potential strategies for re-engineering zinc finger protein-protein interfaces. Thus, like for their DNA-binding counterparts, it may be possible to randomize residues within the DZF domain that are known to be important for mediating protein-protein interactions (see Chapter 4) to create C2H2 ZFs with completely novel specificities. Such an
approach depends on a precise structural and biochemical information about the interaction surface. Mutagenesis as well as structural analysis of DZF domains is necessary and will help narrow down the choice of residues feasible for randomization (see Chapters 4 and 5).
Resulting re-engineered ZFs in combination with naturally occurring ZFs could then be shuffled and linked together to create finger arrays with novel interaction specificities.
In the long-term, the capability to engineer synthetic C2H2 ZFs with desired protein-protein interaction specificities, together with existing strategies for engineering designer C2H2 ZF DNA-binding proteins, should yield a powerful toolbox useful to construct artificial cellular networks. Hence, the development of these technologies should have significant applications in synthetic biology as well as biomedical research.