Abstract
[i]
ABSTRACT
Transcriptions factors are proteins that play a central role in regulating key life processes from development to metabolism. It has been observed that most transcription factors have small DNA binding domains and long intrinsically disordered regions. Owing to the importance of their action, the production and functioning of these proteins are under tight regulation in the cell. Mutation or aberrant expression of transcription factors lead to several diseases like cancer, developmental disorders, diabetes and cardio-vascular diseases. For example, mutation of the transcription factor p53 hampers its ability to bind DNA leading to consequences like cancer. Mechanisms regulating the functioning of transcription factors include post-translational modification, DNA-binding auto- inhibition, and protein partnerships. Typically, these functional regulations are carried out by transactivation regions, which are distinct from the DNA-binding domains of these proteins. Often the transactivation regions are intrinsically disordered.
This work focuses on the structural and functional analysis of two partner transcription factors (TFs): Deformed (DFD), a member of the HOX family and Extradenticle (EXD), a member of the TALE family. The HOX transcription factors play critical roles in the development of bilateral vertebrates. In vitro, isolated HOX TFs bind a consensus AT rich DNA sequence.
However, in vivo these proteins bind highly specific DNA sequences to carry out specific functions. This functional specificity is achieved when HOX proteins bind with their partner proteins from the TALE family. The HOX and TALE proteins bind adjacent sites in the DNA and also interact via a disordered region in the HOX protein. In humans misregulated function of HOX and TALE proteins are implicated in various forms of leukaemia.
This work presents a thorough structural and dynamic characterization of the two partner transcription factors DFD and EXD. We show that both have a well-folded DNA-binding homeodomain that are appended to functionally important intrinsically disordered regions. Fast picosecond-nanosecond timescale backbone dynamics experiments reveal that the disordered region in the N-terminus of DFD homeodomain and C-terminus of EXD homeodomain exhibit
Abstract
[ii]
varying degree of flexibility. These dynamics experiments of DFD reveal that its disordered region has small segments of amino acids (3 to 9 residues) that are relatively rigid. We show that one such rigid segment specifically interacts with EXD and is key to the HOX-TALE partnership. We provide an NMR dynamics based method to identify functionally important rigid segments in intrinsically disordered regions of proteins. The EXD homeodomain binds the DFD disordered region via a hydrophobic pocket formed by an extended loop region between helices H1 and H2.
The C-terminal disordered region of EXD, on the other hand, is poised to form an additional helix upon DNA binding. Using gel-based mobility assay, we found that the C-terminal disordered region enhances the DNA-binding affinity of EXD homeodomain.
These results show that the EXD homeodomain has three distinct interaction interfaces: i) the DNA-binding helix H3, ii) the hydrophobic pocket that interacts with DFD, and iii) the new helix H4 packing interface. We postulate that each binding event affects the others and thus, there must be allosteric communication between these sites. To gain structural insights, the solution structure of EXD homeodomain was solved using NMR spectroscopy and several interesting differences were observed between the free EXD homeodomain and the DNA-bound EXD homeodomain structures. The packing of the three helices are slightly different in both structures.
The DNA-recognition helix H3 is shorter by one turn in the free protein. Analysis of contact maps of the two structures shows major differences in loop between the helices H1 and H2, the turn between the helices H2 and H3, and the helix H3 itself. Thus, the subtle structural changes in the EXD homeodomain provides a communication pathway between its DNA recognition and partner protein functions, which is further modulated by the appended C-terminal sequence. Overall, this work provides a structural and dynamic basis of functional partnership between the transcription factors Deformed and Extradenticle.
Keywords: Transcription factors, Intrinsically Disordered Regions (IDRs), fast time-scale dynamics, Electrophoretic mobility shift assay (EMSA), protein structure, solution NMR spectroscopy, protein-protein interaction, protein-DNA interaction.