More Like this

1. Single molecule studies characterize the kinetic mechanism of tetrameric p53 binding to different native response elements

   https://doi.org/10.1371/journal.pone.0286193  

   Suwita, Johannes P.; Voong, Calvin K.; Ly, Elina; Goodrich, James A.; Kugel, Jennifer F. (August 2023, PLOS ONE)

   D’Auria, Sabato (Ed.)

   The transcriptional activator p53 is a tumor suppressor protein that controls cellular pathways important for cell fate decisions, including cell cycle arrest, senescence, and apoptosis. It functions as a tetramer by binding to specific DNA sequences known as response elements (REs) to control transcription via interactions with co-regulatory complexes. Despite its biological importance, the mechanism by which p53 binds REs remains unclear. To address this, we have used an in vitro single molecule fluorescence approach to quantify the dynamic binding of full-length human p53 to five native REs in real time under equilibrium conditions. Our approach enabled us to quantify the oligomeric state of DNA-bound p53. We found little evidence that dimer/DNA complexes form as intermediates en route to binding or dissociation of p53 tetramer/DNA complexes. Interestingly, however, at some REs dimers can rapidly exchange from tetramer/DNA complexes. Real time kinetic measurements enabled us to determine rate constants for association and dissociation at all five REs, which revealed two kinetically distinct populations of tetrameric p53/RE complexes. For the less stable population, the rate constants for dissociation were larger at REs closest to consensus, showing that the more favorable binding sequences form the least kinetically stable complexes. Together our single molecule measurements provide new insight into mechanisms by which tetrameric p53 forms complexes on different native REs. 

   more » « less
2. Single molecule studies reveal that p53 tetramers dynamically bind response elements containing one or two half sites

   https://doi.org/10.1038/s41598-020-73234-6  

   Ly, Elina; Kugel, Jennifer F.; Goodrich, James A. (September 2020, Scientific Reports)

   Abstract The tumor suppressor protein p53 is critical for cell fate decisions, including apoptosis, senescence, and cell cycle arrest. p53 is a tetrameric transcription factor that binds DNA response elements to regulate transcription of target genes. p53 response elements consist of two decameric half-sites, and data suggest one p53 dimer in the tetramer binds to each half-site. Despite a broad literature describing p53 binding DNA, unanswered questions remain, due partly to the need for more quantitative and structural studies with full length protein. Here we describe a single molecule fluorescence system to visualize full length p53 tetramers binding DNA in real time. The data revealed a dynamic interaction in which tetrameric p53/DNA complexes assembled and disassembled without a dimer/DNA intermediate. On a wild type DNA containing two half sites, p53/DNA complexes existed in two kinetically distinct populations. p53 tetramers bound response elements containing only one half site to form a single population of complexes with reduced kinetic stability. Altering the spacing and helical phasing between two half sites affected both the population distribution of p53/DNA complexes and their kinetic stability. Our real time single molecule measurements of full length p53 tetramers binding DNA reveal the parameters that define the stability of p53/DNA complexes, and provide insight into the pathways by which those complexes assemble. 

   more » « less
3. Multidimensional quantitative phenotypic and molecular analysis reveals neomorphic behaviors of p53 missense mutants

   https://doi.org/10.1038/s41523-023-00582-7  

   Pal, Anasuya; Gonzalez-Malerva, Laura; Eaton, Seron; Xu, Chenxi; Zhang, Yining; Grief, Dustin; Sakala, Lydia; Nwekwo, Lilian; Zeng, Jia; Christensen, Grant; et al (September 2023, npj Breast Cancer)

   Abstract Mutations in theTP53tumor suppressor gene occur in >80% of the triple-negative or basal-like breast cancer. To test whether neomorphic functions of specificTP53missense mutations contribute to phenotypic heterogeneity, we characterized phenotypes of non-transformed MCF10A-derived cell lines expressing the ten most common missense mutant p53 proteins and observed a wide spectrum of phenotypic changes in cell survival, resistance to apoptosis and anoikis, cell migration, invasion and 3D mammosphere architecture. The p53 mutants R248W, R273C, R248Q, and Y220C are the most aggressive while G245S and Y234C are the least, which correlates with survival rates of basal-like breast cancer patients. Interestingly, a crucial amino acid difference at one position—R273C vs. R273H—has drastic changes on cellular phenotype. RNA-Seq and ChIP-Seq analyses show distinct DNA binding properties of different p53 mutants, yielding heterogeneous transcriptomics profiles, and MD simulation provided structural basis of differential DNA binding of different p53 mutants. Integrative statistical and machine-learning-based pathway analysis on gene expression profiles with phenotype vectors across the mutant cell lines identifies quantitative association of multiple pathways including the Hippo/YAP/TAZ pathway with phenotypic aggressiveness. Further, comparative analyses of large transcriptomics datasets on breast cancer cell lines and tumors suggest that dysregulation of the Hippo/YAP/TAZ pathway plays a key role in driving the cellular phenotypes towards basal-like in the presence of more aggressive p53 mutants. Overall, our study describes distinct gain-of-function impacts on protein functions, transcriptional profiles, and cellular behaviors of different p53 missense mutants, which contribute to clinical phenotypic heterogeneity of triple-negative breast tumors. 

   more » « less
4. Network Theory Analysis of Allosteric Drug-Rescue Mechanisms in the Tumor Suppressor Protein p53 Y220C Mutant

   https://doi.org/10.3390/ijms26146884  

   Cowan, Benjamin S; Thayer, Kelly M (July 2025, International Journal of Molecular Sciences)

   Network theory analysis has emerged as a powerful approach for investigating the complex behavior of dynamic and interactive systems, including proteomic systems. One key application of these methods is the study of long-range signaling dynamics in proteins, a phenomenon known as allostery. In this study, we applied computational models using network theory analysis to explore long-range electrostatic interactions and allosteric drug rescue mechanisms in the DNA-binding domain (DBD) of the p53 protein, a critical tumor suppressor whose dysfunction, often caused by missense mutations, is implicated in over 50% of human cancers. Using heat kernel and Wasserstein distance-based analyses, we explored the allosteric behavior of p53-DBD constructs with the Y220C mutation in the presence or absence of allosteric effector drugs. Our results demonstrated that these network theory-based protocols effectively detected the differential efficacies of small molecule allosteric effector drug compounds in restoring long-range electrostatic dynamics in the Y220C mutant. Furthermore, our approach identified key long-range electrostatic interactions critical to both the nominal and drug-rescued functionality of the p53-DBD, providing valuable insights into allosteric modulation and its therapeutic potential. 

   more » « less
5. Two distinct nucleic acid binding surfaces of Cdc5 regulate development

   https://doi.org/10.1042/BCJ20190502  

   Wang, Chao; Li, Mu; Li, Guorui; Liu, Xinsen; Zhao, Wensheng; Yu, Bin; Liu, Junfeng; Yang, Jun; Peng, You-Liang (November 2019, Biochemical Journal)

   Cell division cycle 5 (Cdc5) is a highly conserved nucleic acid binding protein among eukaryotes and plays critical roles in development. Cdc5 can simultaneously bind to DNA and RNA by its N-terminal DNA-binding domain (DBD), but molecular mechanisms describing its nucleic acid recognition and the regulation of development through its nucleic acid binding remain unclear. Herein, we present a crystal structure of the N-terminal DBD of MoCdc5 (MoCdc5-DBD) from the rice blast fungus Magnaporthe oryzae. Residue K100 of MoCdc5 is on the periphery of a positively charged groove that is formed by K42, K45, R47, and N92 and is evolutionally conserved. Mutation of K100 significantly reduces the affinity of MoCdc5-DBD to a Cdc5-binding element but not to a conventional myeloblastosis (Myb) domain-binding element, suggesting that K100 is a key residue of the high binding affinity to Cdc5-binding element. Another conserved residue (R31) is located close to the U6 RNA in the structure of the spliceosome, and its mutation dramatically reduces the binding capacity of MoCdc5-DBD for U6 RNA. Importantly, mutations in these key residues, including R31, K42, and K100 in AtCDC5, an Arabidopsis thaliana ortholog of MoCdc5, greatly impair the functions of AtCDC5, resulting in pleiotropic development defects and reduced levels of primary microRNA transcripts. Taken together, our findings suggest that Cdc5-DBD binds nucleic acids with two distinct binding surfaces, one for DNA and another for RNA, which together contribute to establishing the regulation mechanism of Cdc5 on development through nucleic acid binding. 

   more » « less