Efforts to obtain a high resolution crystal structure of the 2HDLL:M100 complex were unsuccessful. However, the SAXS data has provided new insight into the flexibility of the complex. Further investigations into the conformation of the complex with further SAXS experiments may extend our knowledge more. These investigations are currently underway, as more SAXS data was gathered in November 2018. This data focussed on 2HDLL and M100c20b, but also on other protein constructs in combination with M100: 2HDN, LLHD3, and NHD3. Although initial analysis of this additional SAXS data looks promising, thorough analysis and modelling could not be conducted in the timeframe of this thesis. These other protein constructs may be useful in gathering further structural insights. The flexibility seen in the conformation of 2HDLL, both alone and in complex with M100, may potentially be due to movement between the LIM:LID regions and the homeodomains respectively. If this is the case, SAXS data for the 2HDN:M100 complex may not be influenced by the flexibility
that prevents rigorous structural modelling of the 2HDLL:M100 complex. Since 2HDN appears to bind DNA in a very similar manner to 2HDLL (Section 5.7), it may then also be of interest to attempt to solve the structure of 2HDN:M100 through X-ray crystallography.
The binding behaviour of 2HDLL to M100 could also be further characterised to provide more information about how binding is facilitated. This includes the use of surface plasmon resonance (SPR) to probe the kinetic properties of binding, as well as uncovering which specific amino acids are involved in binding through HSQC (heteronuclear single quantum coherence) NMR titrations.
A larger focus on the DNA side of the interaction may also provide new information. Varying the spacing of the CATTAG and AAATTA sites within the M100 oligonucleotide and checking the binding with 2HDLL could help to indicate whether a direct Isl1:Lhx3 interaction influences the DNA binding of the overall complex. It would also be intriguing to check the binding behaviour of GST-NHD3 dimers to such DNA mutants, to see if they still follow the same binding trends as 2HDLL.
6.4.3 Conclusion
Without a clearer structural picture of how the motor neuron complex is binding DNA, it is impossible to precisely define the DNA-binding behaviour of Isl1. At this stage, it appears likely that Isl1 does contribute to DNA-binding when in complex with a binding partner, such as Lhx3. It is possible that this behaviour is functionally relevant, and acts as a mechanism by which Isl1 can act in combination with many different DNA-binding proteins, allowing precise targeting of specific DNA sequences in an efficient manner. This could provide a partial explanation for how Isl1 plays a role in regulating gene expression in such a diverse range of tissues, even though it does not appear to bind DNA when in isolation.
7 Conclusions
This thesis has investigated the protein-DNA and protein-protein interactions that allow Isl1 to function as a transcriptional regulator with diverse roles across many tissues, in the hopes of gaining new insights into how Isl1 acts in a wide variety of cellular contexts.
Chapters 3 and 4 investigated the protein interaction domains of Isl1, searching for novel binding partners through yeast two-hybrid library screening. These screens were successful in identifying putative novel binding partners for Isl1, with Mkln1 being a potential binding partner of interest based on its ability to bind both Isl1LIM and Ldb1LID/Isl1LIM in two
orthologous systems. Other potential binding partners identified herein may also be of interest, although close assessment of the literature suggests that some may not represent biologically relevant interactions (Chapter 4). This identification of binding events that may be real but are not biologically relevant highlights the need for good moderation of large screening datasets of this type, and for thorough validation of putative interactions.
Chapters 5 and 6 shifted the focus onto the mechanisms by which Isl1 and Lhx3 interact with DNA as isolated LIM-HD proteins or when in combination. Chapter 5 probed the DNA- binding behaviour of the two homeodomains and reveals that Lhx3 and Isl1 have very different DNA binding behaviours, despite being very similar in terms of sequence, fold and stability. It appears that Isl1 is not able to bind to DNA, including its reported recognition sequences, with high affinity in the absence of a protein binding partner that promotes DNA- binding. In Chapter 6, the behaviour of the Isl1 and Lhx3 homeodomains in complex was further investigated, with the key goal being determination of a structure of the 2HDLL:M100 complex. Although an atomic resolution structure was not produced, SAXS has provided new insights into this apparently dynamic complex (Section 6.3). The combination of EMSA data presented in Chapter 5 and SAXS data presented in Chapter 6 provide biophysical evidence that the Isl1 homeodomain can bind to DNA directly to modulate the stronger binding of Lhx3. Additional structural and biophysical studies, informed by the presented data herein, should provide confirmation of how these two homeodomain proteins can bind different DNA sites as single entities or through combined efforts.