scholarly journals A Quantitative Systems Approach to Define Novel Effects of Tumour p53 Mutations on Binding Oncoprotein MDM2

2021 ◽  
Vol 23 (1) ◽  
pp. 53
Author(s):  
Manuel Fuentes ◽  
Sanjeeva Srivastava ◽  
Angela M. Gronenborn ◽  
Joshua LaBaer
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Hot Spot ◽  
Point Mutations ◽  
Protein Microarrays ◽  
Binding Domain ◽  
Structural Domain ◽  
Dna Binding Domain ◽  
Label Free ◽  
Wide Range

Understanding transient protein interactions biochemically at the proteome scale remains a long-standing challenge. Current tools developed to study protein interactions in high-throughput measure stable protein complexes and provide binary readouts; they do not elucidate dynamic and weak protein interactions in a proteome. The majority of protein interactions are transient and cover a wide range of affinities. Nucleic acid programmable protein arrays (NAPPA) are self-assembling protein microarrays produced by freshly translating full-length proteins in situ on the array surface. Herein, we have coupled NAPPA to surface plasmon resonance imaging (SPRi) to produce a novel label-free platform that measures many protein interactions in real-time allowing the determination of the KDs and rate constants. The developed novel NAPPA-SPRi technique showed excellent ability to study protein-protein interactions of clinical mutants of p53 with its regulator MDM2. Furthermore, this method was employed to identify mutant p53 proteins insensitive to the drug nutlin-3, currently in clinical practice, which usually disrupts the p53-MDM2 interactions. Thus, significant differences in the interactions were observed for p53 mutants on the DNA binding domain (Arg-273-Cys, Arg-273-His, Arg-248-Glu, Arg-280-Lys), on the structural domain (His-179-Tyr, Cys-176-Phe), on hydrophobic moieties in the DNA binding domain (Arg-280-Thr, Pro-151-Ser, Cys-176-Phe) and hot spot mutants (Gly-245-Cys, Arg-273-Leu, Arg-248-Glu, Arg-248-Gly), which signifies the importance of point mutations on the MDM2 interaction and nutlin3 effect, even in molecular locations related to other protein activities.

scholarly journals Transactivation Functions of the N-Terminal Domains of Nuclear Hormone Receptors: Protein Folding and Coactivator Interactions

2003 ◽  
Vol 17 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Raj Kumar ◽  
E. Brad Thompson
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Hormone Receptors ◽  
Nuclear Hormone Receptor ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Protein Protein Interactions ◽  
Carboxy Terminal ◽  
And Function ◽  
Terminal Domains

Abstract The N-terminal domains (NTDs) of many members of the nuclear hormone receptor (NHR) family contain potent transcription-activating functions (AFs). Knowledge of the mechanisms of action of the NTD AFs has lagged, compared with that concerning other important domains of the NHRs. In part, this is because the NTD AFs appear to be unfolded when expressed as recombinant proteins. Recent studies have begun to shed light on the structure and function of the NTD AFs. Recombinant NTD AFs can be made to fold by application of certain osmolytes or when expressed in conjunction with a DNA-binding domain by binding that DNA-binding domain to a DNA response element. The sequence of the DNA binding site may affect the functional state of the AFs domain. If properly folded, NTD AFs can bind certain cofactors and primary transcription factors. Through these, and/or by direct interactions, the NTD AFs may interact with the AF2 domain in the ligand binding, carboxy-terminal portion of the NHRs. We propose models for the folding of the NTD AFs and their protein-protein interactions.

scholarly journals Substitutions at Nonconserved Rheostat Positions Modulate Function by Rewiring Long-Range, Dynamic Interactions

2020 ◽  
Vol 38 (1) ◽  
pp. 201-214 ◽  
Author(s):  
Paul Campitelli ◽  
Liskin Swint-Kruse ◽  
S Banu Ozkan
Keyword(s):  
Dna Binding ◽  
Long Range ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Dynamic Coupling ◽  
Long Distance ◽  
Repressor Protein ◽  
Functional Changes ◽  
Lactose Repressor ◽  
Wide Range

Abstract Amino acid substitutions at nonconserved protein positions can have noncanonical and “long-distance” outcomes on protein function. Such outcomes might arise from changes in the internal protein communication network, which is often accompanied by changes in structural flexibility. To test this, we calculated flexibilities and dynamic coupling for positions in the linker region of the lactose repressor protein. This region contains nonconserved positions for which substitutions alter DNA-binding affinity. We first chose to study 11 substitutions at position 52. In computations, substitutions showed long-range effects on flexibilities of DNA-binding positions, and the degree of flexibility change correlated with experimentally measured changes in DNA binding. Substitutions also altered dynamic coupling to DNA-binding positions in a manner that captured other experimentally determined functional changes. Next, we broadened calculations to consider the dynamic coupling between 17 linker positions and the DNA-binding domain. Experimentally, these linker positions exhibited a wide range of substitution outcomes: Four conserved positions tolerated hardly any substitutions (“toggle”), ten nonconserved positions showed progressive changes from a range of substitutions (“rheostat”), and three nonconserved positions tolerated almost all substitutions (“neutral”). In computations with wild-type lactose repressor protein, the dynamic couplings between the DNA-binding domain and these linker positions showed varied degrees of asymmetry that correlated with the observed toggle/rheostat/neutral substitution outcomes. Thus, we propose that long-range and noncanonical substitutions outcomes at nonconserved positions arise from rewiring long-range communication among functionally important positions. Such calculations might enable predictions for substitution outcomes at a range of nonconserved positions.

PU.1 is a suppressor of myeloid leukemia, inactivated in mice by gene deletion and mutation of its DNA binding domain

Blood ◽  
2004 ◽  
Vol 104 (12) ◽  
pp. 3437-3444 ◽  
Author(s):  
Wendy D. Cook ◽  
Benjamin J. McCaw ◽  
Christopher Herring ◽  
Deborah L. John ◽  
Simon J. Foote ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Dna Binding ◽  
Myeloid Leukemia ◽  
Gene Deletion ◽  
Hot Spot ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Chromosome 2 ◽  
Monocytic Differentiation ◽  
Minimal Growth

In most myeloid leukemias induced in mice by γ-radiation, one copy of chromosome 2 has suffered a deletion. To search for a potential tumor suppressor gene in that region, we have delineated the deletions in a panel of these tumors. A commonly deleted region of 2 megabase pairs (Mbp) includes the gene encoding the PU.1 transcription factor, a powerful inducer of granulocytic/monocytic differentiation. Significantly, in 87% of these tumors the remaining PU.1 allele exhibited point mutations in the PU.1 DNA binding domain. Surprisingly, 86% of these mutations altered a single CpG, implicating deamination of deoxycytidine, a common mutational mechanism, as the origin of this lesion. The “hot spot” resides in the codon for a contact residue essential for DNA binding by PU.1. In keeping with a tumor suppressor role for PU.1, enforced expression of wild-type PU.1 in the promyelocytic leukemia cells inhibited their clonogenic growth, induced monocytic differentiation, and elicited apoptosis. The mutant PU.1 found in tumors retained only minimal growth suppressive function. The results suggest that PU.1 normally suppresses development of myeloid leukemia by promoting differentiation and that the combination of gene deletion and a point mutation that impairs its ability to bind DNA is particularly leukemogenic.

scholarly journals Overproduction of v-Myc in the nucleus and its excess over Max are not required for avian fibroblast transformation

1993 ◽  
Vol 13 (6) ◽  
pp. 3623-3631
Author(s):  
A T Tikhonenko ◽  
A R Hartman ◽  
M L Linial
Keyword(s):  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Point Mutations ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Localization Signal ◽  
Infected Cells ◽  
Fibroblast Transformation

The cellular proto-oncogene c-myc can acquire transforming potential by a number of different means, including retroviral transduction. The transduced allele generally contains point mutations relative to c-myc and is overexpressed in infected cells, usually as a v-Gag-Myc fusion protein. Upon synthesis, v-Gag-Myc enters the nucleus, forms complexes with its heterodimeric partner Max, and in this complex binds to DNA in a sequence-specific manner. To delineate the role for each of these events in fibroblast transformation, we introduced several mutations into the myc gene of the avian retrovirus MC29. We observed that Gag-Myc with a mutated nuclear localization signal is confined predominantly in the cytoplasm and only about 5% of the protein could be detected in the nucleus (less than the amount of endogenous c-Myc). Consequently, only a small fraction of Max is associated with Myc. However, cells infected with this mutant exhibit a completely transformed phenotype in vitro, suggesting that production of enough v-Gag-Myc to tie up all cellular Max is not needed for transformation. While the nuclear localization signal is dispensable for transformation, minimal changes in the v-Gag-Myc DNA-binding domain completely abolish its transforming potential, consistent with a role of Myc as a transcriptional regulator. One of its potential targets might be the endogenous c-myc, which is repressed in wild-type MC29-infected cells. Our experiments with MC29 mutants demonstrate that c-myc down-regulation depends on the integrity of the v-Myc DNA-binding domain and occurs at the RNA level. Hence, it is conceivable that v-Gag-Myc, either directly or circuitously, regulates c-myc transcription.

scholarly journals Molecular and genetic analysis of the toxic effect of RAP1 overexpression in yeast.

Genetics ◽  
1995 ◽  
Vol 141 (4) ◽  
pp. 1253-1262 ◽  
Author(s):  
K Freeman ◽  
M Gwadz ◽  
D Shore
Keyword(s):  
Dna Binding ◽  
Genetic Analysis ◽  
Toxic Effect ◽  
Transcriptional Activation ◽  
Ribosomal Proteins ◽  
Regulatory Protein ◽  
Point Mutations ◽  
Binding Domain ◽  
Dna Binding Domain

Abstract Rap1p is a context-dependent regulatory protein in yeast that functions as a transcriptional activator of many essential genes, including those encoding ribosomal proteins and glycolytic enzymes. Rap1p also participates in transcriptional silencing at HM mating-type loci and telomeres. Overexpression of RAP1 strongly inhibits cell growth, perhaps by interfering with essential transcriptional activation functions within the cell. Here we report a molecular and genetic analysis of the toxic effect of RAP1 overexpression. We show that toxicity does not require the previously defined Rap1p activation and silencing domains, but instead is dependent upon the DNA-binding domain and an adjacent region of unknown function. Point mutations were identified in the DNA-binding domain that relieve the toxic effect of overexpression. Two of these mutations can complement a RAP1 deletion yet cause growth defects and altered DNA-binding properties in vitro. However, a small deletion of the adjacent (downstream) region that abolishes overexpression toxicity has, by itself, no apparent effect on growth or DNA binding. SKO1/ACR1, which encodes a CREB-like repressor protein in yeast, was isolated as a high copy suppressor of the toxicity caused by RAP1 overexpression. Models related to the regulation of Rap1p activity are discussed.

Precise Coating of a Wide Range of DNA Templates by a Protein Polymer with a DNA Binding Domain

ACS Nano ◽  
2016 ◽  
Vol 11 (1) ◽  
pp. 144-152 ◽  
Author(s):  
Armando Hernandez-Garcia ◽  
Nicole A. Estrich ◽  
Marc W. T. Werten ◽  
Johan R. C. Van Der Maarel ◽  
Thomas H. LaBean ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Dna Templates ◽  
Protein Polymer ◽  
Wide Range

scholarly journals A consensus motif in the RFX DNA binding domain and binding domain mutants with altered specificity.

1996 ◽  
Vol 16 (8) ◽  
pp. 4486-4494 ◽  
Author(s):  
P Emery ◽  
M Strubin ◽  
K Hofmann ◽  
P Bucher ◽  
B Mach ◽  
...  
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
Selection Procedure ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Consensus Binding Site ◽  
Consensus Motif

The RFX DNA binding domain is a novel motif that has been conserved in a growing number of dimeric DNA-binding proteins, having diverse regulatory functions, in eukaryotic organisms ranging from yeasts to humans. To characterize this novel motif, we have performed a detailed dissection of the site-specific DNA binding activity of RFX1, a prototypical member of the RFX family. First, we have performed a site selection procedure to define the consensus binding site of RFX1. Second, we have developed a new mutagenesis-selection procedure to derive a precise consensus motif, and to test the accuracy of a secondary structure prediction, for the RFX domain. Third, a modification of this procedure has allowed us to isolate altered-specificity RFX1 mutants. These results should facilitate the identification both of additional candidate genes controlled by RFX1 and of new members of the RFX family. Moreover, the altered-specificity RFX1 mutants represent valuable tools that will permit the function of RFX1 to be analyzed in vivo without interference from the ubiquitously expressed endogenous protein. Finally, the simplicity, efficiency, and versatility of the selection procedure we have developed make it of general value for the determination of consensus motifs, and for the isolation of mutants exhibiting altered functional properties, for large protein domains involved in protein-DNA as well as protein-protein interactions.

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