MS-Based Approaches Enable the Structural Characterization of Transcription Factor/DNA Response Element Complex

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.

Developments in Hydrogen/Deuterium Exchange Mass Spectrometry

“…Copyright 2017, with permission from Elsevier. (B) Peptides maps for digestion of the FOXO4 protein (from ref ) show that digestion with nepenthesin-1 alone, pepsin alone, or the combination of pepsin+nepenthesin-1 lead to different coverage ().…”

“…We wanted to highlight just a few of the methodological combinations with HDX MS, publications which describe more the combination of the tools rather than the outcome of combining them toward study of a particular protein or system. Many exciting details are to be found in these reports including HDX MS combined with size-exclusion chromatography and modeling, combined with SEC-SAXS, combined with circular dichroism, combined with differential scanning fluorimetry, and combined with cross-linking and molecular docking and modeling. , …”

Host-Pathogen Adhesion as the Basis of Innovative Diagnostics for Emerging Pathogens

“…CryoEM and, to a lesser extent, X-ray crystallography are the best methods for higher-order assemblies. Furthermore, cross-linking MS (XL–MS) and hydrogen-deuterium exchange MS (HDX–MS) are being increasingly used to determine structural constraints between interacting proteins, protein complexes, and their binding interfaces [ 77 , 78 , 79 , 80 , 81 ]. Such constraints can facilitate binding optimization, improvement of ligand design, and thus improved capture for pre-analytical diagnostic steps [ 82 , 83 , 84 , 85 , 86 , 87 , 88 ].…”

Targeting ERK-Hippo Interplay in Cancer Therapy

“…MST kinase regulates glucose metabolism in T-ALL, laryngeal squamous and breast cancer cells through inhibition of the TEAD transcription activity and expression of GLUT (glucose transporter) [76,78,79]. MST kinase induces apoptosis through expression of pro-apoptotic protein NOXA in several cancer cells through phosphorylation and activation of FOXO (forkhead box O) transcription factors [80][81][82]. MST kinase directly phosphorylates and activates pro-apoptotic protein BIM, caspase-3, -9 and apoptosis in pancreatic beta-cells [83].…”