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

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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Molecular Dynamics Simulations of Human FOXO3 Reveal Intrinsically Disordered Regions Spread Spatially by Intramolecular Electrostatic Repulsion

Biomolecules ◽

10.3390/biom11060856 ◽

2021 ◽

Vol 11 (6) ◽

pp. 856

Author(s):

Robert O.J. Weinzierl

Keyword(s):

Molecular Dynamics ◽

Dna Binding ◽

Intrinsically Disordered Protein ◽

Binding Domain ◽

Electrostatic Repulsion ◽

Dna Binding Domain ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Extended Conformation ◽

Graphical Tool

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

Journal of Molecular Biology ◽

10.1016/j.jmb.2020.07.017 ◽

2020 ◽

Vol 432 (19) ◽

pp. 5411-5429 ◽

Cited By ~ 1

Author(s):

Exequiel Medina ◽

Pablo Villalobos ◽

George L. Hamilton ◽

Elizabeth A. Komives ◽

Hugo Sanabria ◽

...

Keyword(s):

Dna Binding ◽

Binding Domain ◽

Domain Swapping ◽

Dna Binding Domain ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Disordered Regions

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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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Structures of p63 DNA binding domain in complexes with half-site and with spacer-containing full response elements

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1013657108 ◽

2011 ◽

Vol 108 (16) ◽

pp. 6456-6461 ◽

Cited By ~ 31

Author(s):

C. Chen ◽

N. Gorlatova ◽

Z. Kelman ◽

O. Herzberg

Keyword(s):

Dna Binding ◽

Binding Domain ◽

Dna Binding Domain ◽

Response Elements ◽

Half Site

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Hydrogen Exchange of Individual Amide Protons in the E. ColilacRepressor DNA-binding Domain: A Nuclear Magnetic Resonance Study (29)

Journal of Biomolecular Structure and Dynamics ◽

10.1080/07391102.1985.10508416 ◽

1985 ◽

Vol 3 (2) ◽

pp. 269-280 ◽

Cited By ~ 14

Author(s):

R. Boelens ◽

P. Gros ◽

R. M. Scheek ◽

J. A. Verpoorte ◽

R. Kaptein

Keyword(s):

Nuclear Magnetic Resonance ◽

Magnetic Resonance ◽

Dna Binding ◽

Hydrogen Exchange ◽

Nuclear Magnetic Resonance Study ◽

Binding Domain ◽

Dna Binding Domain ◽

Magnetic Resonance Study ◽

Nuclear Magnetic ◽

Amide Protons

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Transcription Activation by the DNA-Binding Domain of the AraC Family Protein RhaS in the Absence of Its Effector-Binding Domain

Journal of Bacteriology ◽

10.1128/jb.00530-07 ◽

2007 ◽

Vol 189 (14) ◽

pp. 4984-4993 ◽

Cited By ~ 23

Author(s):

Jason R. Wickstrum ◽

Jeff M. Skredenske ◽

Ana Kolin ◽

Ding J. Jin ◽

Jianwen Fang ◽

...

Keyword(s):

Dna Binding ◽

Transcription Activation ◽

In Vitro Transcription ◽

Binding Domain ◽

Receptor Protein ◽

Dna Binding Domain ◽

Dimerization Domain ◽

Half Site

ABSTRACT The Escherichia coli l-rhamnose-responsive transcription activators RhaS and RhaR both consist of two domains, a C-terminal DNA-binding domain and an N-terminal dimerization domain. Both function as dimers and only activate transcription in the presence of l-rhamnose. Here, we examined the ability of the DNA-binding domains of RhaS (RhaS-CTD) and RhaR (RhaR-CTD) to bind to DNA and activate transcription. RhaS-CTD and RhaR-CTD were both shown by DNase I footprinting to be capable of binding specifically to the appropriate DNA sites. In vivo as well as in vitro transcription assays showed that RhaS-CTD could activate transcription to high levels, whereas RhaR-CTD was capable of only very low levels of transcription activation. As expected, RhaS-CTD did not require the presence of l-rhamnose to activate transcription. The upstream half-site at rhaBAD and the downstream half-site at rhaT were found to be the strongest of the known RhaS half-sites, and a new putative RhaS half-site with comparable strength to known sites was identified. Given that cyclic AMP receptor protein (CRP), the second activator required for full rhaBAD expression, cannot activate rhaBAD expression in a ΔrhaS strain, it was of interest to test whether CRP could activate transcription in combination with RhaS-CTD. We found that RhaS-CTD allowed significant activation by CRP, both in vivo and in vitro, although full-length RhaS allowed somewhat greater CRP activation. We conclude that RhaS-CTD contains all of the determinants necessary for transcription activation by RhaS.

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