The C‐terminal domain of transcription factor RfaH : Folding, fold switching and energy landscape

AbstractWe study the folding and fold switching of the C-terminal domain (CTD) of the transcription factor RfaH using a hybrid sequence-structure based model. We show that this model captures the essential thermodynamic behavior of this metamorphic domain, i.e., a switch in the global free energy minimum from an α-helical hairpin to a 5-stranded β-barrel upon separating the CTD from the rest of the protein. Using this model and Monte Carlo sampling techniques, we analyze the energy landscape of the CTD in terms of progress variables for folding towards the two folds. We find that, below the folding temperature, the energy landscape is characterized by a single, dominant funnel to the native β-barrel structure. The absence of a deep funnel to the α-helical hairpin state reflects a negligible population of this fold for the isolated CTD. We observe, however, a significantly higher α-helix structure content in the unfolded state compared to results from a similar but fold switch-incompetent version of our model. Moreover, in folding simulations started from an extended chain conformation we find transient α-helix structure that disappears as the chain progresses to the thermally stable β-barrel state.

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Multi-Funnel Landscape of the Fold-Switching Protein RfaH-CTD

10.1101/221143 ◽

2017 ◽

Author(s):

Nathan A. Bernhardt ◽

Ulrich H.E. Hansmann

Keyword(s):

Free Energy ◽

Transcription Factor ◽

Biological Function ◽

Double Helix ◽

Energy Landscape ◽

Three Dimensional ◽

Conversion Process ◽

Free Energy Landscape ◽

Enhanced Sampling ◽

Helix Bundle

AbstractProteins such as the transcription factor RfaH can change biological function by switching between distinct three-dimensional folds. RfaH regulates transcription if the C-terminal domain folds into a double helix bundle, and promotes translation when this domain assumes a β-barrel form. This fold-switch has been also observed for the isolated domain, dubbed by us RfaH-CTD, and is studied here with a variant of the RET approach recently introduced by us. We use the enhanced sampling properties of this technique to map the free energy landscape of RfaH-CTD and to propose a mechanism for the conversion process.TOC Image

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BTB Protein Keap1 Targets Antioxidant Transcription Factor Nrf2 for Ubiquitination by the Cullin 3-Roc1 Ligase

Molecular and Cellular Biology ◽

10.1128/mcb.25.1.162-171.2005 ◽

2005 ◽

Vol 25 (1) ◽

pp. 162-171 ◽

Cited By ~ 467

Author(s):

Manabu Furukawa ◽

Yue Xiong

Keyword(s):

Transcription Factor ◽

Ring Finger ◽

Negative Regulator ◽

Protein Accumulation ◽

Protein Motif ◽

Terminal Domain ◽

Catalytic Function ◽

Cullin 3

ABSTRACT The concentrations and functions of many eukaryotic proteins are regulated by the ubiquitin pathway, which consists of ubiquitin activation (E1), conjugation (E2), and ligation (E3). Cullins are a family of evolutionarily conserved proteins that assemble by far the largest family of E3 ligase complexes. Cullins, via a conserved C-terminal domain, bind with the RING finger protein Roc1 to recruit the catalytic function of E2. Via a distinct N-terminal domain, individual cullins bind to a protein motif present in multiple proteins to recruit specific substrates. Cullin 3 (Cul3), but not other cullins, binds directly with BTB domains to constitute a potentially large number of BTB-CUL3-ROC1 E3 ubiquitin ligases. Here we report that the human BTB-Kelch protein Keap1, a negative regulator of the antioxidative transcription factor Nrf2, binds to CUL3 and Nrf2 via its BTB and Kelch domains, respectively. The KEAP1-CUL3-ROC1 complex promoted NRF2 ubiquitination in vitro and knocking down Keap1 or CUL3 by short interfering RNA resulted in NRF2 protein accumulation in vivo. We suggest that Keap1 negatively regulates Nrf2 function in part by targeting Nrf2 for ubiquitination by the CUL3-ROC1 ligase and subsequent degradation by the proteasome. Blocking NRF2 degradation in cells expressing both KEAP1 and NRF2 by either inhibiting the proteasome activity or knocking down Cul3, resulted in NRF2 accumulation in the cytoplasm. These results may reconcile previously observed cytoplasmic sequestration of NRF2 by KEAP1 and suggest a possible regulatory step between KEAP1-NRF2 binding and NRF2 degradation.

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A carboxy-terminal basic region controls RNA polymerase III transcription factor activity of human La protein.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.5823 ◽

1997 ◽

Vol 17 (10) ◽

pp. 5823-5832 ◽

Cited By ~ 51

Author(s):

J L Goodier ◽

H Fan ◽

R J Maraia

Keyword(s):

Transcription Factor ◽

Rna Polymerase ◽

Rna Binding ◽

Rna Polymerase Iii ◽

Transcription Factor Activity ◽

Factor Activity ◽

Terminal Domain ◽

La Protein ◽

Pol Iii ◽

Basic Region

Human La protein has been shown to serve as a transcription factor for RNA polymerase III (pol III) by facilitating transcription termination and recycling of transcription complexes. In addition, La binds to the 3' oligo(U) ends common to all nascent pol III transcripts, and in the case of B1-Alu RNA, protects it from 3'-end processing (R. J. Maraia, D. J. Kenan, and J. D. Keene, Mol. Cell. Biol. 14:2147-2158, 1994). Others have previously dissected the La protein into an N-terminal domain that binds RNA and a C-terminal domain that does not. Here, deletion and substitution mutants of La were examined for general RNA binding, RNA 3'-end protection, and transcription factor activity. Although some La mutants altered in a C-terminal basic region bind RNA in mobility shift assays, they are defective in RNA 3'-end protection and do not support transcription, while one C-terminal substitution mutant is defective only in transcription. Moreover, a C-terminal fragment lacking RNA binding activity appears able to support low levels of transcription by pol III. While efficient multiround transcription is supported only by mutants that bind RNA and contain a C-terminal basic region. These analyses indicate that RNA binding contributes to but is not sufficient for La transcription factor activity and that the C-terminal domain plays a role in transcription that is distinguishable from simple RNA binding. The transcription factor activity of La can be reversibly inhibited by RNA, suggesting the potential for feedback inhibition of pol III transcription.

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Structure of the N-terminal domain of the protein Expansion: an `Expansion' to the Smad MH2 fold

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004715001443 ◽

2015 ◽

Vol 71 (4) ◽

pp. 844-853 ◽

Cited By ~ 2

Author(s):

Mads Beich-Frandsen ◽

Eric Aragón ◽

Marta Llimargas ◽

Jordi Benach ◽

Antoni Riera ◽

...

Keyword(s):

Gene Expression ◽

Crystal Structure ◽

Transcription Factor ◽

Protein Interaction ◽

Signalling Pathway ◽

Interaction Site ◽

Terminal Domain ◽

Smad Proteins ◽

Helical Region

Gene-expression changes observed inDrosophilaembryos after inducing the transcription factor Tramtrack led to the identification of the protein Expansion. Expansion contains an N-terminal domain similar in sequence to the MH2 domain characteristic of Smad proteins, which are the central mediators of the effects of the TGF-β signalling pathway. Apart from Smads and Expansion, no other type of protein belonging to the known kingdoms of life contains MH2 domains. To compare the Expansion and Smad MH2 domains, the crystal structure of the Expansion domain was determined at 1.6 Å resolution, the first structure of a non-Smad MH2 domain to be characterized to date. The structure displays the main features of the canonical MH2 fold with two main differences: the addition of an α-helical region and the remodelling of a protein-interaction site that is conserved in the MH2 domain of Smads. Owing to these differences, to the new domain was referred to as Nα-MH2. Despite the presence of the Nα-MH2 domain, Expansion does not participate in TGF-β signalling; instead, it is required for other activities specific to the protostome phyla. Based on the structural similarities to the MH2 fold, it is proposed that the Nα-MH2 domain should be classified as a new member of the Smad/FHA superfamily.

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Structure of the C-terminal domain of transcription factor IIB from Trypanosoma brucei

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0904309106 ◽

2009 ◽

Vol 106 (32) ◽

pp. 13242-13247 ◽

Cited By ~ 13

Author(s):

B. S. Ibrahim ◽

N. Kanneganti ◽

G. E. Rieckhof ◽

A. Das ◽

D. V. Laurents ◽

...

Keyword(s):

Transcription Factor ◽

Trypanosoma Brucei ◽

Terminal Domain ◽

Transcription Factor Iib

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E2F4’s cytoplasmic role in multiciliogenesis is mediated via an N-terminal domain that binds two components of the centriole replication machinery, Deup1 and SAS6

Molecular Biology of the Cell ◽

10.1091/mbc.e21-01-0039 ◽

2021 ◽

pp. mbc.E21-01-0039

Author(s):

Renin Hazan ◽

Munemasa Mori ◽

Paul S. Danielian ◽

Vincent J. Guen ◽

Seth M. Rubin ◽

...

Keyword(s):

Transcription Factor ◽

Reproductive Organs ◽

Transcriptional Program ◽

Replication Machinery ◽

Terminal Domain ◽

Multiciliated Cells ◽

N Terminus ◽

Shared Roles

Multiciliated cells play critical roles in the airway, reproductive organs and brain. Generation of multiple cilia requires both activation of a specialized transcriptional program and subsequent massive amplification of centrioles within the cytoplasm. The E2F4 transcription factor is required for both roles, and consequently for multiciliogenesis. Here, we establish that E2F4 associates with two distinct components of the centriole replication machinery, Deup1 and SAS6, targeting non-homologous domains in these proteins. We map Deup1 and SAS6 binding to E2F4’s N-terminus, and show that this domain is sufficient to mediate E2F4’s cytoplasmic role in multiciliogenesis. This sequence is highly conserved across the E2F family, but the ability to bind Deup1 and SAS6 is specific to E2F4 and E2F5, consistent with their shared roles in multiciliogenesis. By generating E2F4/E2F1 chimeras, we identify a six-residue motif that is critical for Deup1 and SAS6 binding. We propose that the ability of E2F4 and E2F5 to recruit Deup1 and/or SAS6, and enable centriole replication, contributes to their cytoplasmic roles in multiciliogenesis.

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CBP/p300-Interacting Transactivator, with Glu/Asp-Rich C-Terminal Domain, 2, and Pre-B-Cell Leukemia Transcription Factor 1 in Human Adrenal Development and Disease

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2008-1064 ◽

2009 ◽

Vol 94 (2) ◽

pp. 678-683 ◽

Cited By ~ 20

Author(s):

Bruno Ferraz-de-Souza ◽

Franziska Martin ◽

Delphine Mallet ◽

Rebecca E. Hudson-Davies ◽

Patricia Cogram ◽

...

Keyword(s):

Transcription Factor ◽

B Cell ◽

Mutational Analysis ◽

Dependent Manner ◽

Cell Leukemia ◽

Positive Interaction ◽

Terminal Domain ◽

B Cell Leukemia ◽

And Function ◽

Transcription Factor 1

Abstract Context: Disorders of adrenal development result in significant morbidity and mortality. However, the molecular basis of human adrenal development, and many forms of disease, is still poorly understood. Objectives: We evaluated the role of two new candidate genes, CBP/p300-interacting transactivator, with Glu/Asp-rich C-terminal domain, 2 (CITED2), and pre-B-cell leukemia transcription factor 1 (PBX1), in human adrenal development and disease. Design: CITED2 and PBX1 expression in early human fetal adrenal development was assessed using RT-PCR and in situ hybridization. The regulation of CITED2 and PBX1 by steroidogenic factor-1 (SF-1) and dosage-sensitive sex reversal, adrenal hypoplasia congenital, critical region on the X chromosome, gene-1 (DAX1) was evaluated in NCI-H295R human adrenocortical tumor cells by studying promoter regulation. Finally, mutational analysis of CITED2 and PBX1 was performed in patients with primary adrenal disorders. Results: CITED2 and PBX1 are expressed in the human fetal adrenal gland during early development. Both genes are activated by SF-1 in a dose-dependent manner in NCI-H295R cells, and, surprisingly, PBX1 is synergistically activated by SF-1 and DAX1. Mutational analysis failed to reveal significant coding sequence changes in individuals with primary adrenal disorders. Conclusions: CITED2 and PBX1 are likely to be important mediators of adrenal development and function in humans, but mutations in these genes are not common causes of adrenal failure in patients in whom a molecular diagnosis remains unknown. The positive interaction between DAX1 and SF-1 in regulating PBX1 may be an important mechanism in this process.

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Phosphatase activity of small C-terminal domain phosphatase 1 (SCP1) controls the stability of the key neuronal regulator RE1-silencing transcription factor (REST)

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.004722 ◽

2018 ◽

Vol 293 (43) ◽

pp. 16851-16861 ◽

Cited By ~ 5

Author(s):

Nathaniel Tate Burkholder ◽

Joshua E. Mayfield ◽

Xiaohua Yu ◽

Seema Irani ◽

Daniel K. Arce ◽

...

Keyword(s):

Transcription Factor ◽

Gene Silencing ◽

Phosphatase Activity ◽

Neurological Diseases ◽

Regulatory Protein ◽

Neuronal Cells ◽

Hek293 Cells ◽

Terminal Domain ◽

Neuronal Gene

The RE1-silencing transcription factor (REST) is the major scaffold protein for assembly of neuronal gene silencing complexes that suppress gene transcription through regulating the surrounding chromatin structure. REST represses neuronal gene expression in stem cells and non-neuronal cells, but it is minimally expressed in neuronal cells to ensure proper neuronal development. Dysregulation of REST function has been implicated in several cancers and neurological diseases. Modulating REST gene silencing is challenging because cellular and developmental differences can affect its activity. We therefore considered the possibility of modulating REST activity through its regulatory proteins. The human small C-terminal domain phosphatase 1 (SCP1) regulates the phosphorylation state of REST at sites that function as REST degradation checkpoints. Using kinetic analysis and direct visualization with X-ray crystallography, we show that SCP1 dephosphorylates two degron phosphosites of REST with a clear preference for phosphoserine 861 (pSer-861). Furthermore, we show that SCP1 stabilizes REST protein levels, which sustains REST's gene silencing function in HEK293 cells. In summary, our findings strongly suggest that REST is a bona fide substrate for SCP1 in vivo and that SCP1 phosphatase activity protects REST against degradation. These observations indicate that targeting REST via its regulatory protein SCP1 can modulate its activity and alter signaling in this essential developmental pathway.

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