Abstract
The limited information available on the structure of complexes involving transcription factors and cognate DNA response elements represents a major obstacle in the quest to understand their mechanism of action at the molecular level. We implemented a concerted structural proteomics approach, which combined hydrogen-deuterium exchange (HDX), quantitative protein-protein and protein-nucleic acid cross-linking (XL), and homology analysis, to model the structure of the complex between the full-length DNA binding domain (DBD) of Forkhead box protein O4 (FOXO4) and its DNA binding element (DBE). The results confirmed that FOXO4-DBD assumes the characteristic forkhead topology shared by these types of transcription factors, but its binding mode differs significantly from those of other members of the family. The results showed that the binding interaction stabilized regions that were rather flexible and disordered in the unbound form. Surprisingly, the conformational effects were not limited only to the interface between bound components, but extended also to distal regions that may be essential to recruiting additional factors to the transcription machinery. In addition to providing valuable new insights into the binding mechanism, this project provided an excellent evaluation of the merits of structural proteomics approaches in the investigation of systems that are not directly amenable to traditional high-resolution techniques.
Highlights
A key role in the regulatory of expression machinery is covered by transcription factors (TFs), proteins that recognize target DNA sequences called response elements and establish specific interactions with additional factors to activate or inhibit the transcription process [1,2,3,4]
The crystal structure available for the Forkhead box protein O4 (FOXO4)-DBDDBE complex does not cover the entire sequence of the DNA binding domain [48], which spans only the 82–207 section of FOXO4 and omits flanking regions that have been hypothesized to promote the recruiting of additional components of the transcription machinery
These samples were submitted to hydrogen-deuterium exchange, quantitative protein-protein cross-linking, protein-DNA cross-linking, and docking experiments to obtain complementary information on their mutual interactions and spatial arrangement
Summary
A key role in the regulatory of expression machinery is covered by transcription factors (TFs), proteins that recognize target DNA sequences called response elements and establish specific interactions with additional factors to activate or inhibit the transcription process [1,2,3,4]. The Protein Data Bank contains the high-resolution structures of at least 483 TFs from different species [7], which include less than 10% of all predicted human TFs [6]. Of such structures, only one third include the cognate DNA response element, and only one fifth are available in both bound and unbound states (Figure 1). The natural interactions established in vivo, which are responsible for stabilizing well-defined functional conformations, cannot be properly replicated in vitro These challenges explain the chronic lack of comprehensive information on full-fledged TF structures, which still hampers the elucidation of their mechanism of action at the molecular level
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