scholarly journals The influence of various regions of the FOXP2 sequence on its structure and DNA binding function

2020 ◽  
Author(s):  
Monare Thulo ◽  
Megan A Rabie ◽  
Naadira Pahad ◽  
Heather L Donald ◽  
Ashleigh A Blane ◽  
...  
Keyword(s):  
Dna Binding ◽  
Leucine Zipper ◽  
Helical Structure ◽  
Leucine Zipper Motif ◽  
Binding Function ◽  
Forkhead Domain ◽  
Binding Event ◽  
Specificity Of Binding ◽  
The Stability ◽  
Unstructured Regions

FOX proteins are a superfamily of transcription factors which share a DNA binding domain referred to as the forkhead domain. Our focus is on the FOXP subfamily members, which are involved in language and cognition amongst other things. The FOXP proteins contain a conserved zinc finger and a leucine zipper motif in addition to the forkhead domain. The remainder of the sequence is predicted to be unstructured and includes an acidic C-terminal tail. In this study we aim to investigate how both the structured and unstructured regions of the sequence cooperate so as to enable FOXP proteins to perform their function. We do this by studying the effect of these regions on both oligomerisation and DNA binding. Structurally, the FOXP proteins appear to be comparatively globular with a high proportion of helical structure. The proteins multimerise via the leucine zipper and the stability of the multimers is controlled by the unstructured interlinking sequence including the acid rich tail. FOXP2 is more compact than FOXP1, has a greater propensity to form higher order oligomers, and binds DNA with stronger affinity. We conclude that while the forkhead domain is necessary for DNA binding, the affinity of the binding event is attributable to the leucine zipper, and the unstructured regions play a significant role in the specificity of binding. The acid rich tail forms specific contacts with the forkhead domain which may influence oligomerisation and DNA binding and therefore the acid rich tail may play an important regulatory role in FOXP transcription. 

Design of DNA-binding peptides based on the leucine zipper motif

Science ◽  
1990 ◽  
Vol 249 (4970) ◽  
pp. 774-778 ◽  
Author(s):  
K. O'Neil ◽  
R. Hoess ◽  
W. DeGrado
Keyword(s):  
Dna Binding ◽  
Leucine Zipper ◽  
Leucine Zipper Motif ◽  
Binding Peptides

scholarly journals Combining rational design and continuous evolution on minimalist proteins that target DNA

2020 ◽  
Author(s):  
Ichiro Inamoto ◽  
Inder Sheoran ◽  
Serban C. Popa ◽  
Montdher Hussain ◽  
Jumi A. Shin
Keyword(s):  
Dna Binding ◽  
Rational Design ◽  
Leucine Zipper ◽  
Sequence Specificity ◽  
Continuous Evolution ◽  
Binding Function ◽  
E Box ◽  
Functional Features ◽  
Basic Region

ABSTRACTWe designed MEF to mimic the basic region/helix-loop-helix/leucine zipper (bHLHZ) domain of transcription factors Max and Myc, which bind with high DNA sequence specificity and affinity to the E-box motif (enhancer box, CACGTG). To make MEF, we started with our rationally designed ME47, a hybrid of the Max basic region and E47 HLH, that effectively inhibited tumor growth in a mouse model of breast cancer. ME47, however, displays propensity for instability and misfolding. We therefore sought to improve ME47’s structural and functional features. We used phage-assisted continuous evolution (PACE) to uncover “nonrational” changes to complement our rational design. PACE mutated Arg12 that contacts the DNA phosphodiester backbone. We would not have rationally made such a change, but this mutation improved ME47’s stability with little change in DNA-binding function. We mutated Cys29 to Ser and Ala in ME47’s HLH to eliminate undesired disulfide formation; these mutations reduced E-box binding activity. To compensate, we fused the designed FosW leucine zipper to ME47 to increase the dimerization interface and improve protein stability and E-box targeting activity. This “franken-protein” MEF comprises the Max basic region, E47 HLH, and FosW leucine zipper—plus mutations that arose during PACE and rational design—and is a tractable, reliable protein in vivo and in vitro. Compared with ME47, MEF gives three-fold stronger binding to E-box with four-fold increased specificity for E-box over nonspecific DNA. Generation of MEF demonstrates that combining rational design and continuous evolution can be a powerful tool for designing proteins with robust structure and strong DNA-binding function.

scholarly journals Combining Rational Design and Continuous Evolution on Minimalist Proteins That Target DNA

2020 ◽  
Author(s):  
Jumi Shin ◽  
Ichiro Inamoto ◽  
Inder Sheoran ◽  
Serban Popa ◽  
Montdher Hussain
Keyword(s):  
Dna Binding ◽  
Rational Design ◽  
Leucine Zipper ◽  
Sequence Specificity ◽  
Continuous Evolution ◽  
Binding Function ◽  
E Box ◽  
Functional Features ◽  
Basic Region

We designed MEF to mimic the basic region/helix-loop-helix/leucine zipper (bHLHZ) domain of transcription factors Max and Myc, which bind with high DNA sequence specificity and affinity to the E-box motif (enhancer box, CACGTG). To make MEF, we started with our rationally designed ME47, a hybrid of the Max basic region and E47 HLH, that effectively inhibited tumor growth in a mouse model of breast cancer. ME47, however, displays propensity for instability and misfolding. We therefore sought to improve ME47's structural and functional features. We used phage-assisted continuous evolution (PACE) to uncover "nonrational" changes to complement our rational design. PACE mutated Arg12 that contacts the DNA phosphodiester backbone. We would not have rationally made such a change, but this mutation improved ME47's stability with little change in DNA-binding function. We mutated Cys29 to Ser and Ala in ME47's HLH to eliminate undesired disulfide formation; these mutations reduced E-box binding activity. To compensate, we fused the designed FosW leucine zipper to ME47 to increase the dimerization interface and improve protein stability and E-box targeting activity. This "franken-protein" MEF comprises the Max basic region, E47 HLH, and FosW leucine zipper—plus mutations that arose during PACE and rational design—and is a tractable, reliable protein in vivo and in vitro. Compared with ME47, MEF gives three-fold stronger binding to Ebox with four-fold increased specificity for E-box over nonspecific DNA. Generation of MEF demonstrates that combining rational design and continuous evolution can be a powerful tool for designing proteins with robust structure and strong DNA-binding function. <br>

Distinct roles for the two cGATA-1 finger domains

1992 ◽  
Vol 12 (10) ◽  
pp. 4562-4570
Author(s):  
H Y Yang ◽  
T Evans
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Dna Binding Domain ◽  
Mouse Homolog ◽  
Cis Element ◽  
Functional Assays ◽  
Binding Function ◽  
The Family ◽  
The Stability ◽  
Basic Region

We have generated and analyzed by functional assays mutations of the chicken erythroid transcription factor GATA-1. The cGATA-1 protein contains two related finger domains highly conserved across species and characteristic of the family of GATA-binding factors. We find that mutations in the C-terminal finger or adjacent basic region abolish sequence-specific DNA binding, confirming that this region constitutes a novel DNA-binding domain sufficient to recognize the consensus WGATAR motif. At least three separate regions outside of this finger II domain contribute in a cooperative manner to the trans-activation potential of the protein. As expected from previous results analyzing the mouse homolog, we find that the N-terminal finger plays a role in DNA binding by affecting the stability of the DNA-protein complex. In addition, we find mutations of finger I subtly altered in DNA-binding function which greatly diminish trans-activation. Our results support the notion that the GATA-1 protein must be positioned precisely on the GATA cis element to enable the activation of target genes.

scholarly journals Distinct roles for the two cGATA-1 finger domains.

1992 ◽  
Vol 12 (10) ◽  
pp. 4562-4570 ◽  
Author(s):  
H Y Yang ◽  
T Evans
Keyword(s):  
Dna Binding ◽  
Target Genes ◽  
Dna Binding Domain ◽  
Mouse Homolog ◽  
Cis Element ◽  
Functional Assays ◽  
Binding Function ◽  
The Family ◽  
The Stability ◽  
Basic Region

We have generated and analyzed by functional assays mutations of the chicken erythroid transcription factor GATA-1. The cGATA-1 protein contains two related finger domains highly conserved across species and characteristic of the family of GATA-binding factors. We find that mutations in the C-terminal finger or adjacent basic region abolish sequence-specific DNA binding, confirming that this region constitutes a novel DNA-binding domain sufficient to recognize the consensus WGATAR motif. At least three separate regions outside of this finger II domain contribute in a cooperative manner to the trans-activation potential of the protein. As expected from previous results analyzing the mouse homolog, we find that the N-terminal finger plays a role in DNA binding by affecting the stability of the DNA-protein complex. In addition, we find mutations of finger I subtly altered in DNA-binding function which greatly diminish trans-activation. Our results support the notion that the GATA-1 protein must be positioned precisely on the GATA cis element to enable the activation of target genes.

scholarly journals Combining Rational Design and Continuous Evolution on Minimalist Proteins That Target DNA

2020 ◽  
Author(s):  
Jumi Shin ◽  
Ichiro Inamoto ◽  
Inder Sheoran ◽  
Serban Popa ◽  
Montdher Hussain
Keyword(s):  
Dna Binding ◽  
Rational Design ◽  
Leucine Zipper ◽  
Sequence Specificity ◽  
Continuous Evolution ◽  
Binding Function ◽  
E Box ◽  
Functional Features ◽  
Basic Region

We designed MEF to mimic the basic region/helix-loop-helix/leucine zipper (bHLHZ) domain of transcription factors Max and Myc, which bind with high DNA sequence specificity and affinity to the E-box motif (enhancer box, CACGTG). To make MEF, we started with our rationally designed ME47, a hybrid of the Max basic region and E47 HLH, that effectively inhibited tumor growth in a mouse model of breast cancer. ME47, however, displays propensity for instability and misfolding. We therefore sought to improve ME47's structural and functional features. We used phage-assisted continuous evolution (PACE) to uncover "nonrational" changes to complement our rational design. PACE mutated Arg12 that contacts the DNA phosphodiester backbone. We would not have rationally made such a change, but this mutation improved ME47's stability with little change in DNA-binding function. We mutated Cys29 to Ser and Ala in ME47's HLH to eliminate undesired disulfide formation; these mutations reduced E-box binding activity. To compensate, we fused the designed FosW leucine zipper to ME47 to increase the dimerization interface and improve protein stability and E-box targeting activity. This "franken-protein" MEF comprises the Max basic region, E47 HLH, and FosW leucine zipper—plus mutations that arose during PACE and rational design—and is a tractable, reliable protein in vivo and in vitro. Compared with ME47, MEF gives three-fold stronger binding to Ebox with four-fold increased specificity for E-box over nonspecific DNA. Generation of MEF demonstrates that combining rational design and continuous evolution can be a powerful tool for designing proteins with robust structure and strong DNA-binding function. <br>

scholarly journals Minimalist proteins: Design of new molecular recognition scaffolds

2004 ◽  
Vol 76 (7-8) ◽  
pp. 1579-1590 ◽  
Author(s):  
J. A. Shin
Keyword(s):  
Dna Binding ◽  
Molecular Recognition ◽  
Leucine Zipper ◽  
Alpha Helix ◽  
Helical Structure ◽  
Binding Specificity ◽  
Mutant Proteins ◽  
Coulombic Interactions ◽  
Design And Synthesis ◽  
Binding Specificity And Affinity

We hypothesize that we can exploit what Nature has already evolved by manipulating the alpha-helix molecular recognition scaffold. Therefore, minimalist proteins capable of sequence-specific, high-affinity binding of DNA were generated to probe how proteins are used and can be used to recognize DNA. The already minimal basic region/leucine zipper motif (bZIP) of GCN4 was reduced to an even more simplified structure by substitution with alanine residues —hence, a generic, Ala-based, helical scaffold. The proteins generated, wt bZIP, 4A,11A, and 18A, contain 0, 4, 11, and 18 alanine mutations in their DNA-binding basic regions, respectively. All alanine mutants still retain alpha-helical structure and DNA-bind- ing function, despite loss of virtually all Coulombic protein-DNA interactions. Mass spectrometry allowed characterization of proteins and post-translational modifications. Fluorescence anisotropy and DNase I footprinting were used to measure in situ binding of these mutant proteins to DNA duplexes containing target sites AP-1 (5'-TGACTCA-3'), ATF/CREB (5'-TGACGTCA-3'),or nonspecific DNA. The roles of van der Waals and Coulombic interactions toward binding specificity and affinity are being investigated. Thus, both DNA-binding specificity and affinity are maintained in all our bZIP derivatives. This Ala-rich scaffold may be useful in design and synthesis of small, alpha-helical proteins with desired DNA-recognition properties.

Reciprocal effects of C/EBPα and PKCδ on JunB expression and monocytic differentiation depend upon the C/EBPα basic region

Blood ◽  
2003 ◽  
Vol 101 (10) ◽  
pp. 3885-3892 ◽  
Author(s):  
Huaitian Liu ◽  
Jeffrey R. Keefer ◽  
Qian-fei Wang ◽  
Alan D. Friedman
Keyword(s):  
Protein Kinase C ◽  
Dna Binding ◽  
Leucine Zipper ◽  
Reciprocal Effects ◽  
Monocytic Differentiation ◽  
Transactivation Domains ◽  
Kinase C ◽  
Enhancer Binding Protein ◽  
The Stability ◽  
Basic Region

Abstract Monocytic differentiation of 32DPKCδ cells in response to activation of protein kinase C δ (PKCδ) by phorbol 12-myristate 13-acetate (PMA) was inhibited by exogenous CCAAT/enhancer binding protein α–estradiol receptor (C/EBPα-ER), which impeded morphologic maturation and induction of macrosialin mRNA. Inhibition of monopoiesis was also evident in 32DPKCδ subclones expressing C/EBPαLeu12Val-ER, which cannot dimerize or bind DNA because of mutation of the leucine zipper, C/EBPαGZ-ER, in which the leucine zipper has been replaced by the GCN4 zipper, or C/EBPαΔ3-8-ER, lacking the C/EBPα transactivation domains. In contrast, C/EBPαBR3-ER, containing a mutant basic region, did not inhibit monocytic differentiation. C/EBPα-ER strongly inhibited endogenous AP-1 DNA-binding. Supershift analysis revealed that the major AP-1 complex contains JunB. Activation of C/EBPα-ER specifically reduced endogenous JunB RNA and protein and exogenous JunB levels without affecting endogenous or exogenous c-Jun. The stability of PMA-induced JunB was not affected. Thus, C/EBPα-ER suppresses both JunB transcription and posttranscriptional protein generation or induction. PU.1 levels and activity were increased. The Leu12Val, GZ, and Δ3-8 mutants also inhibited JunB expression, whereas the BR3 mutant was ineffective, indicating that inhibition of JunB expression and monocytic differentiation by C/EBPα-ER depends upon an interaction mediated by its basic region. Exogenous JunB restored AP-1 DNA-binding but did not prevent inhibition of macrosialin expression by C/EBPα-ER, indicating that JunB is not the only target relevant to inhibition of monopoiesis by C/EBPα.

scholarly journals Mating Type in Chlamydomonas Is Specified by mid, the Minus-Dominance Gene

Genetics ◽  
1997 ◽  
Vol 146 (3) ◽  
pp. 859-869 ◽  
Author(s):  
Patrick J Ferris ◽  
Ursula W Goodenough
Keyword(s):  
Transcription Factor ◽  
Mating Type ◽  
Codon Bias ◽  
Leucine Zipper ◽  
Laboratory Strain ◽  
Leucine Zipper Motif ◽  
Type Locus ◽  
Diploid Cells ◽  
Necessary And Sufficient

Diploid cells of Chlamydomonas reinhardtii that are heterozygous at the mating-type locus (mt  +/mt  –) differentiate as minus gametes, a phenomenon known as minus dominance. We report the cloning and characterization of a gene that is necessary and sufficient to exert this minus dominance over the plus differentiation program. The gene, called mid, is located in the rearranged (R) domain of the mt  – locus, and has duplicated and transposed to an autosome in a laboratory strain. The imp11 mt  – mutant, which differentiates as a fusion-incompetent plus gamete, carries a point mutation in mid. Like the fus1 gene in the mt  + locus, mid displays low codon bias compared with other nuclear genes. The mid sequence carries a putative leucine zipper motif, suggesting that it functions as a transcription factor to switch on the minus program and switch off the plus program of gametic differentiation. This is the first sex-determination gene to be characterized in a green organism.

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