Intrinsically Disordered Regions of the DNA-Binding Domain of Human FoxP1 Facilitate Domain Swapping

The human transcription factor FOXO3 (a member of the ‘forkhead’ family of transcription factors) controls a variety of cellular functions that make it a highly relevant target for intervention in anti-cancer and anti-aging therapies. FOXO3 is a mostly intrinsically disordered protein (IDP). Absence of knowledge of its structural properties outside the DNA-binding domain constitutes a considerable obstacle to a better understanding of structure/function relationships. Here, I present extensive molecular dynamics (MD) simulation data based on implicit solvation models of the entire FOXO3/DNA complex, and accelerated MD simulations under explicit solvent conditions of a central region of particular structural interest (FOXO3120–530). A new graphical tool for studying and visualizing the structural diversity of IDPs, the Local Compaction Plot (LCP), is introduced. The simulations confirm the highly disordered nature of FOXO3 and distinguish various degrees of folding propensity. Unexpectedly, two ‘linker’ regions immediately adjacent to the DNA-binding domain are present in a highly extended conformation. This extended conformation is not due to their amino acid composition, but rather is caused by electrostatic repulsion of the domains connected by the linkers. FOXO3 is thus an IDP present in an unusually extended conformation to facilitate interaction with molecular interaction partners.

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Rewiring bacterial two-component systems by modular DNA-binding domain swapping

Nature Chemical Biology ◽

10.1038/s41589-019-0286-6 ◽

2019 ◽

Vol 15 (7) ◽

pp. 690-698 ◽

Cited By ~ 21

Author(s):

Sebastian R. Schmidl ◽

Felix Ekness ◽

Katri Sofjan ◽

Kristina N.-M. Daeffler ◽

Kathryn R. Brink ◽

...

Keyword(s):

Dna Binding ◽

Binding Domain ◽

Domain Swapping ◽

Dna Binding Domain ◽

Component Systems ◽

Two Component ◽

Two Component Systems

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DNA-binding regulates site-specific ubiquitination of IRF-1

Biochemical Journal ◽

10.1042/bj20121076 ◽

2013 ◽

Vol 449 (3) ◽

pp. 707-717 ◽

Cited By ~ 16

Author(s):

Vivien Landré ◽

Emmanuelle Pion ◽

Vikram Narayan ◽

Dimitris P. Xirodimas ◽

Kathryn L. Ball

Keyword(s):

Dna Binding ◽

E3 Ligase ◽

Binding Domain ◽

Dna Binding Domain ◽

Docking Site ◽

Interacting Protein ◽

Site Specific ◽

E3 Ligases ◽

Intrinsically Disordered ◽

C Terminus

Understanding the determinants for site-specific ubiquitination by E3 ligase components of the ubiquitin machinery is proving to be a challenge. In the present study we investigate the role of an E3 ligase docking site (Mf2 domain) in an intrinsically disordered domain of IRF-1 [IFN (interferon) regulatory factor-1], a short-lived IFNγ-regulated transcription factor, in ubiquitination of the protein. Ubiquitin modification of full-length IRF-1 by E3 ligases such as CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and MDM2 (murine double minute 2), which dock to the Mf2 domain, was specific for lysine residues found predominantly in loop structures that extend from the DNA-binding domain, whereas no modification was detected in the more conformationally flexible C-terminal half of the protein. The E3 docking site was not available when IRF-1 was in its DNA-bound conformation and cognate DNA-binding sequences strongly suppressed ubiquitination, highlighting a strict relationship between ligase binding and site-specific modification at residues in the DNA-binding domain. Hyperubiquitination of a non-DNA-binding mutant supports a mechanism where an active DNA-bound pool of IRF-1 is protected from polyubiquitination and degradation.

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Solution structure and backbone dynamics of the DNA-binding domain of FOXP1: Insight into its domain swapping and DNA binding

Protein Science ◽

10.1002/pro.626 ◽

2011 ◽

Vol 20 (5) ◽

pp. 908-924 ◽

Cited By ~ 21

Author(s):

Yuan-Ping Chu ◽

Chia-Hao Chang ◽

Jia-Hau Shiu ◽

Yao-Tsung Chang ◽

Chiu-Yueh Chen ◽

...

Keyword(s):

Dna Binding ◽

Solution Structure ◽

Binding Domain ◽

Domain Swapping ◽

Backbone Dynamics ◽

Dna Binding Domain ◽

Insight Into

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Long-range regulation of p53 DNA binding by its intrinsically disordered N-terminal transactivation domain

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814051115 ◽

2018 ◽

Vol 115 (48) ◽

pp. E11302-E11310 ◽

Cited By ~ 17

Author(s):

Alexander S. Krois ◽

H. Jane Dyson ◽

Peter E. Wright

Keyword(s):

Dna Binding ◽

Electrostatic Interactions ◽

Target Genes ◽

Structural Characteristics ◽

Full Length ◽

Transactivation Domain ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

N Terminus ◽

Disordered Regions

Atomic resolution characterization of the full-length p53 tetramer has been hampered by its size and the presence of extensive intrinsically disordered regions at both the N and C termini. As a consequence, the structural characteristics and dynamics of the disordered regions are poorly understood within the context of the intact p53 tetramer. Here we apply trans-intein splicing to generate segmentally 15N-labeled full-length p53 constructs in which only the resonances of the N-terminal transactivation domain (NTAD) are visible in NMR spectra, allowing us to observe this region of p53 with unprecedented detail within the tetramer. The N-terminal region is dynamically disordered in the full-length p53 tetramer, fluctuating between states in which it is free and fully exposed to solvent and states in which it makes transient contacts with the DNA-binding domain (DBD). Chemical-shift changes and paramagnetic spin-labeling experiments reveal that the amphipathic AD1 and AD2 motifs of the NTAD interact with the DNA-binding surface of the DBD through primarily electrostatic interactions. Importantly, this interaction inhibits binding of nonspecific DNA to the DBD while having no effect on binding to a specific p53 recognition element. We conclude that the NTAD:DBD interaction functions to enhance selectivity toward target genes by inhibiting binding to nonspecific sites in genomic DNA. This work provides some of the highest-resolution data on the disordered N terminus of the nearly 180-kDa full-length p53 tetramer and demonstrates a regulatory mechanism by which the N terminus of p53 transiently interacts with the DBD to enhance target site discrimination.

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Dynamic Consequences of Specificity within the Cytidine Repressor DNA-Binding Domain

10.1101/2021.02.28.433298 ◽

2021 ◽

Author(s):

Colleen L Moody ◽

Jenaro Soto ◽

Vira Tretyachenko-Ladokhina ◽

Donald F Senear ◽

Melanie J Cocco

Keyword(s):

Dna Binding ◽

Hydrogen Exchange ◽

Intrinsically Disordered Protein ◽

Structural Features ◽

Binding Domain ◽

Free State ◽

Dna Binding Domain ◽

Intrinsically Disordered ◽

Cytidine Repressor ◽

Half Site

The E. coli cytidine repressor (CytR) is a member of the LacR family of bacterial repressors that regulates nine operons with distinct spacing and orientations of recognition sites. Understanding the structural features of the CytR DNA-binding domain (DBD) when bound to DNA is critical to understanding differential mechanisms of gene regulation. We previously reported the structure of the CytR DBD monomer bound specifically to half-site DNA and found that the DBD exists as a three-helix bundle containing a canonical helix-turn-helix motif, similar to other proteins that interact with DNA [Moody, et al (2011), Biochemistry 50:6622-32]. We also studied the free state of the monomer and found that since NMR spectra show it populates up to four distinct conformations, the free state exists as an intrinsically disordered protein (IDP). Here, we present further analysis of the DBD structure and dynamics in the context of full-site operator or nonspecific DNA. DBDs bound to full-site DNA show one set of NMR signals, consistent with fast exchange between the two binding sites. When bound to full-length DNA, we observed only slight changes in structure compared to the monomer structure and no folding of the hinge helix. Notably, the CytR DBD behaves quite differently when bound to nonspecific DNA compared to LacR. A dearth of NOEs and complete lack of protection from hydrogen exchange are consistent with the protein populating a flexible, molten state when associated with DNA nonspecifically, similar to fuzzy complexes. The CytR DBD structure is significantly more stable when bound specifically to the udp half-site substrate. For CytR, the transition from nonspecific association to specific recognition results in substantial changes in protein mobility that are coupled to structural rearrangements. These effects are more pronounced in the CytR DBD compared to other LacR family members.

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