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 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>

scholarly journals DNA-binding domain of human c-Myc produced in Escherichia coli.

1989 ◽  
Vol 9 (6) ◽  
pp. 2477-2486 ◽  
Author(s):  
C V Dang ◽  
H van Dam ◽  
M Buckmire ◽  
W M Lee
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Leucine Zipper ◽  
Protein A ◽  
Embryo Cell ◽  
Staphylococcal Protein A ◽  
Rat Fibroblasts ◽  
Type C ◽  
Basic Region

We have identified the domain of the human c-myc protein (c-Myc) produced in Escherichia coli that is responsible for the ability of the protein to bind sequence-nonspecific DNA. Using analysis of binding of DNA by proteins transferred to nitrocellulose, DNA-cellulose chromatography, and a nitrocellulose filter binding assay, we examined the binding properties of c-Myc peptides generated by cyanogen bromide cleavage, of mutant c-Myc, and of proteins that fuse portions of c-Myc to staphylococcal protein A. The results of these analyses indicated that c-Myc amino acids 265 to 318 were responsible for DNA binding and that other regions of the protein (including a highly conserved basic region and a region containing the leucine zipper motif) were not required. Some mutant c-Mycs that did not bind DNA maintained rat embryo cell-cotransforming activity, which indicated that the c-Myc property of in vitro DNA binding was not essential for this activity. These mutants, however, were unable to transform established rat fibroblasts (Rat-1a cells) that were susceptible to transformation by wild-type c-Myc, although this lack of activity may not have been due to their inability to bind DNA.

scholarly journals Highly conserved residues in the bZIP domain of yeast GCN4 are not essential for DNA binding.

1991 ◽  
Vol 11 (10) ◽  
pp. 4918-4926 ◽  
Author(s):  
W T Pu ◽  
K Struhl
Keyword(s):  
Dna Binding ◽  
Leucine Zipper ◽  
Alpha Helix ◽  
Conserved Residues ◽  
Sequence Recognition ◽  
Bzip Domain ◽  
Bzip Proteins ◽  
Basic Region

Yeast GCN4 and the Jun oncoprotein are transcriptional activators that bind DNA via a bZIP domain consisting of a leucine zipper dimerization element and an adjacent basic region that directly contacts DNA. Two highly conserved alanines (Ala-238 and Ala-239 in GCN4) and an invariant asparagine (Asn-235) in the basic region have been proposed to play important roles in DNA sequence recognition by bZIP proteins. Surprisingly, these conserved residues can be functionally replaced in GCN4 and in a derivative containing the Jun basic region (Jun-GCN4). The ability of an amino acid to functionally substitute for Asn-235 does not correlate with its preference for assuming the N-cap position of an alpha helix. This finding argues against the proposal of the scissors grip model that the invariant asparagine forms an N cap that permits the basic region to bend sharply and wrap around the DNA. In contrast to a prediction of the induced fork model, the pattern of functional substitutions of the conserved alanines together with the results of uracil interference experiments suggests that Ala-238 and Ala-239 do not make base-specific DNA contacts. Finally, the Jun-GCN4 chimeric proteins appear much more active in vivo than expected from their DNA-binding properties in vitro. The mechanistic and evolutionary implications of these results are discussed.

scholarly journals Highly conserved residues in the bZIP domain of yeast GCN4 are not essential for DNA binding

1991 ◽  
Vol 11 (10) ◽  
pp. 4918-4926
Author(s):  
W T Pu ◽  
K Struhl
Keyword(s):  
Dna Binding ◽  
Leucine Zipper ◽  
Alpha Helix ◽  
Conserved Residues ◽  
Sequence Recognition ◽  
Bzip Domain ◽  
Bzip Proteins ◽  
Basic Region

Yeast GCN4 and the Jun oncoprotein are transcriptional activators that bind DNA via a bZIP domain consisting of a leucine zipper dimerization element and an adjacent basic region that directly contacts DNA. Two highly conserved alanines (Ala-238 and Ala-239 in GCN4) and an invariant asparagine (Asn-235) in the basic region have been proposed to play important roles in DNA sequence recognition by bZIP proteins. Surprisingly, these conserved residues can be functionally replaced in GCN4 and in a derivative containing the Jun basic region (Jun-GCN4). The ability of an amino acid to functionally substitute for Asn-235 does not correlate with its preference for assuming the N-cap position of an alpha helix. This finding argues against the proposal of the scissors grip model that the invariant asparagine forms an N cap that permits the basic region to bend sharply and wrap around the DNA. In contrast to a prediction of the induced fork model, the pattern of functional substitutions of the conserved alanines together with the results of uracil interference experiments suggests that Ala-238 and Ala-239 do not make base-specific DNA contacts. Finally, the Jun-GCN4 chimeric proteins appear much more active in vivo than expected from their DNA-binding properties in vitro. The mechanistic and evolutionary implications of these results are discussed.

DNA-binding domain of human c-Myc produced in Escherichia coli

1989 ◽  
Vol 9 (6) ◽  
pp. 2477-2486
Author(s):  
C V Dang ◽  
H van Dam ◽  
M Buckmire ◽  
W M Lee
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Leucine Zipper ◽  
Protein A ◽  
Embryo Cell ◽  
Staphylococcal Protein A ◽  
Rat Fibroblasts ◽  
Type C ◽  
Basic Region

We have identified the domain of the human c-myc protein (c-Myc) produced in Escherichia coli that is responsible for the ability of the protein to bind sequence-nonspecific DNA. Using analysis of binding of DNA by proteins transferred to nitrocellulose, DNA-cellulose chromatography, and a nitrocellulose filter binding assay, we examined the binding properties of c-Myc peptides generated by cyanogen bromide cleavage, of mutant c-Myc, and of proteins that fuse portions of c-Myc to staphylococcal protein A. The results of these analyses indicated that c-Myc amino acids 265 to 318 were responsible for DNA binding and that other regions of the protein (including a highly conserved basic region and a region containing the leucine zipper motif) were not required. Some mutant c-Mycs that did not bind DNA maintained rat embryo cell-cotransforming activity, which indicated that the c-Myc property of in vitro DNA binding was not essential for this activity. These mutants, however, were unable to transform established rat fibroblasts (Rat-1a cells) that were susceptible to transformation by wild-type c-Myc, although this lack of activity may not have been due to their inability to bind DNA.

scholarly journals Multicomponent differentiation-regulated transcription factors in F9 embryonal carcinoma stem cells.

1991 ◽  
Vol 11 (3) ◽  
pp. 1686-1695 ◽  
Author(s):  
M K Shivji ◽  
N B La Thangue
Keyword(s):  
Stem Cells ◽  
Transcription Factors ◽  
Dna Binding ◽  
Embryonal Carcinoma ◽  
Regulatory Sequence ◽  
Dependent Manner ◽  
Sequence Specificity ◽  
Dna Sequence Specificity ◽  
Cell Extracts

Murine F9 embryonal carcinoma (F9 EC) stem cells have an E1a-like transcription activity that is down-regulated as these cells differentiate to parietal endoderm. For the adenovirus E2A promoter, this activity requires at least two sequence-specific transcription factors, one that binds the cyclic AMP-responsive element (CRE) and the other, DRTF1, the DNA-binding activity of which is down-regulated as F9 EC cells differentiate. Here we report the characterization of several binding activities in F9 EC cell extracts, referred to as DRTF 1a, 1b and 1c, that recognize the DRTF1 cis-regulatory sequence (-70 to -50 region). These activities can be chromatographically separated but are not distinguishable by DNA sequence specificity. Activity 1a is a detergent-sensitive complex in which DNA binding is regulated by phosphorylation. In contrast, activities 1b and 1c are unaffected by these treatments but exist as multicomponent protein complexes even before DNA binding. Two sets of DNA-binding polypeptides, p50DR and p30DR, affinity purified from F9 EC cell extracts produce complexes 1b and 1c. Both polypeptides appear to be present in the same DNA-bound protein complex and both directly contact DNA. These affinity-purified polypeptides activate transcription in vitro in a binding-site-dependent manner. These data indicate the in F9 EC stem cells, multicomponent differentiation-regulated transcription factors contribute to the cellular E1a-like activity.

Transcriptional regulation by Fos and Jun in vitro: interaction among multiple activator and regulatory domains

1991 ◽  
Vol 11 (7) ◽  
pp. 3624-3632
Author(s):  
C Abate ◽  
D Luk ◽  
T Curran
Keyword(s):  
Transcriptional Regulation ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Leucine Zipper ◽  
Regulatory Function ◽  
Activation Domain ◽  
Regulatory Domains ◽  
Heterodimeric Complex ◽  
Transcriptional Stimulation

The proteins encoded by the proto-oncogenes c-fos and c-jun (Fos and Jun, respectively) form a heterodimeric complex that regulates transcription by interacting with the DNA-regulatory element known as the activator protein 1 (AP-1) binding site. Fos and Jun are members of a family of related transcription factors that dimerize via a leucine zipper structure and interact with DNA through a bipartite domain formed between regions of each protein that are rich in basic amino acids. Here we have defined other domains in the Fos-Jun heterodimer that contribute to transcriptional function in vitro. Although DNA-binding specificity is mediated by the leucine zipper and basic regions, Jun also contains a proline- and glutamine-rich region that functions as an ancillary DNA-binding domain but does not contribute directly to transcriptional activation. Transcriptional stimulation in vitro was associated with two regions in Fos and a single N-terminal activation domain in Jun. These activator regions were capable of operating independently; however, they appear to function cooperatively in the heterodimeric complex. The activity of these domains was modulated by inhibitory regions in Fos and Jun that repressed transcription in vitro. In the context of the heterodimer, the Jun activation domain was the major contributor to transcriptional stimulation and the inhibitory regions in Fos were the major contributors to transcriptional repression in vitro. Potentially, the inhibitory domains could serve a regulatory function in vivo. Thus, transcriptional regulation by the Fos-Jun heterodimer results from a complex integration of multiple activator and regulatory domains.

Transformation by Fos proteins requires a C-terminal transactivation domain

1993 ◽  
Vol 13 (12) ◽  
pp. 7429-7438
Author(s):  
R Wisdon ◽  
I M Verma
Keyword(s):  
Dna Binding ◽  
Leucine Zipper ◽  
Fibroblast Cell ◽  
Binding Activity ◽  
Sequence Specificity ◽  
Transactivation Domain ◽  
Functional Requirements ◽  
Dna Binding Activity ◽  
The Difference ◽  
Transforming Proteins

The Fos family of proteins now includes seven members: the retroviral proteins FBR-v-Fos and FBJ-v-Fos and the cellular proteins c-Fos, FosB, FosB2, Fra1, and Fra2. Four proteins (FBR-v-Fos, FBJ-v-Fos, c-Fos, and FosB) transform established rodent fibroblast cell lines, while three (FosB2, Fra1, and Fra2) do not. As all family members display sequence-specific DNA-binding activity as part of a heterodimeric complex with Jun proteins, other features must account for the differences in transforming potential. We demonstrate here that all transforming members have a C-terminal transactivation domain that is lacking in nontransforming members. The nontransforming proteins Fra1 and Fra2 can be converted to transforming proteins by fusion of a transactivation domain from either FosB or VP16. We also demonstrate that differences in the basic region-leucine zipper domain affecting either the affinity or sequence specificity of DNA binding are not determinants of the difference in transforming potential among members of the Fos family. The results further define the functional requirements for transformation by Fos proteins and suggest that the subunit composition of AP1 complexes is an important determinant of mitogenic signalling capability.

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