P53 Transactivation Domain Research Articles

Pentamidine inhibit S100A4 - p53 interaction and decreases cell proliferation activity

Metastasis-associated S100A4 protein is a small calcium-binding protein typically overexpressed in several tumor forms, and it is widely accepted that S100A4 plays a significant role in the metastasis of cancer. Tumor suppressor p53 is one of the S100A4's main targets. Previous reports show that through p53, S100A4 regulates collagen expression and cell proliferation. When S100A4 interacts with p53, the S100A4 destabilizes wild type p53. In the current study, based on 1H–15N HSQC NMR experiments and HADDOCK results, S100A4 interacts with the intrinsically unstructured transactivation domain (TAD) of the protein p53 and the pentamidine molecules in the presence of calcium ions. Our results suggest that the p53 TAD and pentamidine molecules share similar binding sites on the S100A4 protein. This observation indicates that a competitive binding mechanism can interfere with the binding of S100A4-p53 and increase the level of p53. Also, we compare different aspects of p53 activity in the WST-1 test using MCF 7 cells. We found that the presence of a pentamidine molecule results in higher p53 activity, which is also reflected in less cell proliferation. Collectively, our results indicate that disrupting the S100A4-p53 interaction would prevent cancer progression, and thus S100A4-p53 inhibitors provide a new avenue for cancer therapy.

Changes in structure and flexibility of p53 TAD2 upon binding to p300 Taz2

p53 is a transcription factor with intrinsically disordered regions that plays an essential role in many cellular processes. As a tumor suppressor, the dysfunction of p53 causes various cancers. p53 can be activated by binding with cofactors in the cell due to stresses or DNA damages. The N-terminal transactivation domain (TAD) of p53 can regulate cell apoptosis by interacting with its binding partners, such as the transcriptional adaptor zinc-binding 2 (Taz2) domain of p300, and cofactors, i.e. cyclic-AMP response element-binding protein (CREB)-binding protein (CBP). The experimentally solved structure of p300 Taz2 and p53 TAD2 has provided insights into the interactions that potentially lead to the structural changes of p53 TAD2 and stabilize the complex. To explore the structural changes as well as the residues that lead to such changes from an isolated state to a bound state in p53 TAD2, we used all-atom molecular dynamics (MD) to simulate two different systems: (1) the p300 Taz2–p53 TAD2 complex and (2) isolated p53 TAD2 (residues 35–59). Although still largely unstructured, residues across the p53 TAD2 contribute significantly to stabilizing the binding between p300 Taz2 and p53 TAD2. Our results suggest that the binding affinity of the p300 Taz2–p53 TAD2 complex originates from hydrophobic and electrostatic interactions. The results are in agreement with previous reports and experimental data. By comparing the two simulated systems, our results not only demonstrate the structural changes of p53 TAD2 after binding with Taz2 but also identify the key residues leading to such changes. We also identify the critical residues that can provide insight into the interaction network between p300 Taz2 and p53 TAD2.

Structure Determination of the Transactivation Domain of p53 in Complex with S100A4 Using Annexin A2 as a Crystallization Chaperone

To fully understand the environmental factors that influence crystallization is an enormous task, therefore crystallographers are still forced to work "blindly" trying as many crystallizing conditions and mutations to improve crystal packing as possible. Numerous times these random attempts simply fail even when using state-of-the-art techniques. As an alternative, crystallization chaperones, having good crystal-forming properties, can be invoked. Today, the almost exclusively used such protein is the maltose-binding protein (MBP) and crystallographers need other widely applicable options. Here, we introduce annexin A2 (ANXA2), which has just as good, if not better, crystal-forming ability than the wild-type MBP. Using ANXA2 as heterologous fusion partner, we were able to solve the atomic resolution structure of a challenging crystallization target, the transactivation domain (TAD) of p53 in complex with the metastasis-associated protein S100A4. p53 TAD forms an asymmetric fuzzy complex with the symmetric S1004 and could interfere with its function.

Molecular simulation oF MDM2 and E6AP proteins as P53 regulator in cervical cancer

Cervical cancer is a type of cancer characterized by abnormal cell growth in the cervical area. One of the main events that happen during tumorigenesis is the inactivation or degradation of the genome guardian, P53. Under normal circumstances, P53 is regulated by MDM2 protein. MDM2 can transfer ubiquitin to the transactivation domain of P53, targeting it for degradation by proteasomes. However, in the presence of HPV, HPV-E6 can utilize cellular E6AP to perform P53 degradation through the ubiquitin ligase mechanism. We validated both interactions through molecular docking in ClusPro. We also checked their normalized expression level in The Cancer Genomic Atlas (TCGA) using TCGA-Assembler. We found out both interactions are highly likely due to the spontaneity of the reaction indicated by the low free energy. MDM2 is overexpressed in cervical cancer cells, but E6AP expression is relatively constant. This indicates that the MDM2-mediated pathway is still sustained in cervical cancer cells. But since E6AP-mediated pathway is finally activated due to the presence of HPV-E6, both pathways may happen simultaneously. Further research is needed to confirm both pathways existence in cervical cancer cells and how they may coincide and affect each other.

Dual regulation of p53 by the ribosome maturation factor SBDS

The Shwachman-Bodian Diamond syndrome (SBDS)-associated gene, SBDS, is involved in rRNA synthesis and ribosome maturation, but the role of SBDS in cancer is largely elusive. In this study, we found that SBDS is often overexpressed or amplified in human cancers, and high level of endogenous SBDS is significantly associated with unfavorable prognosis. Conversely, knockdown of SBDS leads to p53 stabilization and activation through the ribosomal stress-RPL5/RPL11-MDM2 pathway, resulting in the repression of cancer cell proliferation and invasion. Interestingly, ectopic SBDS in the nucleoplasm also suppresses tumor cell growth and proliferation in vitro and in vivo. Mechanistically, ectopically expressed SBDS triggered by, for example, ribosomal stress binds to the transactivation domain of p53 and perturbs the MDM2–p53 interaction, consequently leading to impaired p53 ubiquitination and proteasomal degradation. Altogether, our finding for the first time demonstrates the dual functions of SBDS in cancer development by coordinating ribosome biogenesis and p53 activity.

Conformational Flexibility of p53 Transactivation Domain Controls DNA Binding Specificity and Promoter Selectivity

Mutations in the tumor suppressor p53 affect its transactivation of genes responsible for cell differentiation, cell cycle progression, and apoptosis. A majority of p53 mutations found in cancer cells are found in its DNA Binding Domain (DBD) at sites termed “hotspots.” Some of these hotspots are gain-of-function mutations that lead to inappropriate gene activation. Binding specificity and promoter selectivity of p53 are partially controlled by a high affinity DBD and C-terminal regulatory domain. It is well established the disordered N-terminal transactivation domain (p53TAD) is necessary for the recruitment of preinitiation complex components but whether it plays any role in DNA binding and promoter selectivity was unclear until our group demonstrated that an intramolecular interaction between DBD and two P53TAD subdomains modifies DBD's association with DNA. The addition of these subdomains, TAD2 and the proline rich region (PRR), decreases the affinity of DBD for DNA. However, p53TAD decreases the affinity of DBD for nonpromoter DNA sequences more than it decreases the affinity for p53 response element DNA, leading to an increase in DNA selectivity. To further investigate this intramolecular interaction and its effects on DNA binding, we have inserted disordered, flexible linkers between TAD2 and PRR or between PRR and DBD to increase conformational flexibility between the domains to uncouple their interaction with DBD. We will measure binding affinity of these mutants to DNA and compare it with that of DBD with and without p53TAD in vitro. Mutants that effectively disrupt the intramolecular interaction will be used in vivo to determine their effect on promoter selectivity and on their transcriptional activation ability. These studies will give insight into how the p53TAD contributes to specific cell fates and into how DBD hotspot mutations may lead to gain of function.

The RING domain of mitochondrial E3 ubiquitin ligase 1 and its complex with Ube2D2: crystallization and X-ray diffraction.

Mitochondrial E3 ubiquitin ligase 1 (MUL1) is located in the mitochondrial outer membrane and regulates various biological processes, including apoptosis, cell growth, mitophagy and mitochondrial dynamics. The C-terminal region of MUL1 faces the cytoplasm and contains the RING domain (MUL1-RING) where the Ub~E2 thioester binds. Unlike most RING-type E3 enzymes, MUL1-RING alone does not have an additional region that recruits a substrate protein, yet is still able to ubiquitylate the substrate, the p53 protein. Nevertheless, the exact mechanism of the ubiquitylation of p53 by MUL1-RING has not yet been elucidated. In order to understand this novel ubiquitylation mechanism, it is necessary to determine the three-dimensional structures of MUL1-RING and of its complex with the cognate E2 enzyme. Here, Ube2D2 was validated as a functional E2 enzyme for the ubiquitylation of the p53 transactivation domain (p53-TAD) by MUL1-RING, and purification and crystallization processes for MUL1-RING and the MUL1-RING-Ube2D2 complex are reported.

Structure Determination of the Transactivation Domain of P53 in Complex with S100A4 Using Annexin A2 as a Crystallization Chaperone

In spite of the fact that structure solving methods are constantly improving, the biggest challenge of protein crystallography remains the production of well diffracting single protein crystals. Full understanding the environmental factors that influence crystal packing would be an enormous task, therefore crystallographers are still forced to work “blindly” trying as many crystallizing conditions as possible. Numerous times the random attempts simply fail even when using crystallization robots. As an alternative option in these cases, crystallization chaperones can be used. These proteins have a unique property, namely they easily form protein crystals, which can be exploited by using them as a heterologous fusion partner to promote crystal contact formation. Today, the most frequently used crystallization chaperone is the maltose-binding protein (MBP) and crystallographers are in need of other options. Our previous results showed the outstanding crystallization properties of a non-EF- hand calcium-binding protein annexin A2 (ANXA2). Here, we compared ANXA2 with the wild type MBP and found that ANXA2 is just as good, if not a better crystallization chaperone. Using ANXA2 for this purpose, we were able to solve the atomic resolution structure of a challenging crystallization target, the transactivation domain (TAD) of p53 in complex with S100A4, an EF-hand calcium-binding protein associated with metastatic tumors. The full-length TAD forms an asymmetric fuzzy complex with S100A4 and could interfere with its function.

Residual Structures and Transient Long-Range Interactions of p53 Transactivation Domain: Assessment of Explicit Solvent Protein Force Fields.

Molecular dynamics simulations using physics-based atomistic force fields have been increasingly used to characterize the heterogeneous structural ensembles of intrinsically disordered proteins (IDPs). To evaluate the accuracy of the latest atomistic explicit-solvent force fields in modeling larger IDPs with nontrivial structural features, we focus on the 61-residue N-terminal transactivation domain (TAD) of tumor suppressor p53, an important protein in cancer biology that has been extensively studied, and abundant experimental data is available for evaluation of simulated ensembles. We performed extensive replica exchange with solute tempering simulations, in excess of 1.0 μs/replica, to generate disordered structural ensembles of p53-TAD using six latest explicit solvent protein force fields. Multiple local and long-range structural properties, including chain dimension, residual secondary structures, and transient long-range contacts, were analyzed and compared against available experimental data. The results show that IDPs such as p53-TAD remain highly challenging for atomistic simulations due to conformational complexity and difficulty in achieving adequate convergence. Structural ensembles of p53-TAD generated using various force fields differ significantly from each other. The a99SB-disp force field demonstrates the best agreement with experimental data at all levels and proves to be suitable for simulating unbound p53-TAD and how its conformational properties may be modulated by phosphorylation and other cellular signals or cancer-associated mutations. Feasibility of such detailed structural characterization is a key step toward establishing the sequence-disordered ensemble-function-disease relationship of p53 and other biologically important IDPs.

Solution structure of MUL1-RING domain and its interaction with p53 transactivation domain

Mitochondrial E3 ubiquitin ligase 1 (MUL1) is a multifunctional mitochondrial protein involved in various biological processes such as mitochondrial dynamics, cell growth, apoptosis, and mitophagy. MUL1 mediates the ubiquitylation of mitochondrial p53 for proteasomal degradation. Although the interaction of MUL1-RING domain with its substrate, p53, is a unique mechanism in RING-mediated ubiquitylation, the molecular basis of this process remains unknown. In this study, we determined the solution structure of the MUL1-RING domain and characterized its interaction with the p53 transactivation domain (p53-TAD) by nuclear magnetic resonance (NMR) spectroscopy. The overall structure of the MUL1-RING domain is similar to those of RING domains of other E3 ubiquitinases. The MUL1-RING domain adopts a ββαβ fold with three anti-parallel β-strands and one α-helix, containing a canonical cross-brace motif for the ligation of two zinc ions. Through NMR chemical shift perturbation experiments, we determined the p53-TAD-binding site in the MUL1-RING domain and showed that the MUL1-RING domain interacts mainly with the p53-TAD2 subdomain composed of residues 39–57. Taken together, our results provide a molecular basis for the novel recognition mechanism of the p53-TAD substrate by the MUL1-RING domain.

Loss of the p53 transactivation domain results in high amyloid aggregation of the Δ40p53 isoform in endometrial carcinoma cells

Dysfunctional p53 formation and activity can result from aberrant expression and subcellular localization of distinct p53 isoforms or aggregates. Endometrial carcinoma (EC) is a cancer type in which p53 status is correlated with prognosis, and TP53 mutations are a frequent genetic modification. Here we aimed to evaluate the expression patterns of different p53 isoforms and their contributions to the formation and subcellular localization of p53 amyloid aggregates in both EC and endometrial nontumor cell lines. We found that full-length (fl) p53 and a truncated p53 isoform, Δ40p53, resulting from alternative splicing of exon 2 or alternative initiation of translation at ATG-40, are the predominantly expressed p53 variants in EC cells. However, Δ40p53 was the major p53 isoform in endometrial nontumor cells. Immunofluorescence assays revealed that Δ40p53 is mainly localized to cytoplasmic punctate structures of EC cells, resembling solid-phase structures similar to those found in neurodegenerative pathologies. Using light-scattering kinetics, CD, and transmission EM, we noted that the p53 N-terminal transactivation domain significantly reduces aggregation of the WT p53 DNA-binding domain, confirming the higher aggregation tendency of Δ40p53, which lacks this domain. This is the first report of cytoplasmic Δ40p53 in EC cells being a major component of amyloid aggregates. The differential aggregation properties of p53 isoforms in EC cells may open up new avenues in the development of therapeutic strategies that preferentially target specific p53 isoforms to prevent p53 amyloid aggregate formation.

P53 Phosphomimetics Preserve Transient Secondary Structure but Reduce Binding to Mdm2 and MdmX.

The disordered p53 transactivation domain (p53TAD) contains specific levels of transient helical secondary structure that are necessary for its binding to the negative regulators, mouse double minute 2 (Mdm2) and MdmX. The interactions of p53 with Mdm2 and MdmX are also modulated by posttranslational modifications (PTMs) of p53TAD including phosphorylation at S15, T18 and S20 that inhibits p53-Mdm2 binding. It is unclear whether the levels of transient secondary structure in p53TAD are changed by phosphorylation or other PTMs. We used phosphomimetic mutants to determine if adding a negative charge at positions 15 and 18 has any effect on the transient secondary structure of p53TAD and protein-protein binding. Using a combination of biophysical and structural methods, we investigated the effects of single and multisite phosphomimetics on the transient secondary structure of p53TAD and its interaction with Mdm2, MdmX, and the KIX domain. The phosphomimetics reduced Mdm2 and MdmX binding affinity by 3–5-fold, but resulted in minimal changes in transient secondary structure, suggesting that the destabilizing effect of phosphorylation on the p53TAD-Mdm2 interaction is primarily electrostatic. Phosphomimetics had no effect on the p53-KIX interaction, suggesting that increased binding of phosphorylated p53 to KIX may be influenced by decreased competition with its negative regulators.

Three Color Single-Molecule FRET of Fast IDP Binding and Folding

Molecular interactions of intrinsically disordered proteins (IDPs) are different from those between folded molecules because IDP binding involves large convormational changes. To understand the mechanism of IDP binding, three-color Förster resonance energy transfer (FRET) will be very useful because both the interaction between two molecules and conformational changes can be probed simultaneously. In this work, we investigated binding of a disordered protein, the transactivation domain (TAD) of p53 and one of its binding partners, nuclear coactivator binding domain (NCBD) of CBP. TAD labeled with Alexa488 (donor) and Alexa594 (acceptor 1, A1) site-specifically was immobilized on a glass surface and incubated with CF680R (acceptor 2, A2)-labeled NCBD in solution. To determine all three FRET efficiencies, alternating excitation of the donor and A1 is typically used. However, in binding experiments, direct excitation of A2 attached to NCBD in solution by A1 excitation laser produces a high background signal. Therefore, we devised a method using a single donor excitation that determines all three FRET efficiencies by utilizing incomplete labeling of A2. When TAD binds to unlabled NCBD, the FRET efficiency between the donor and A1 (E1) can be determined. Using this information, it is possible to determine the other two FRET efficiencies in principle. However, due to the fast binding and dissociation kinetics, the bound and unbound states are not well separated in binned FRET efficiency trajectories (1 ms bin time), which prevents pre-determination of E1. We employed the maximum likelihood method developed by Gopich and Szabo that analyzes photon trajectories directly without binning the data. By globally analyzing the data using a kinetic model that includes binding to both labeled and unlabled NCBD, rate coefficients of the sub-millisecond kinetics and all three FRET efficiencies were determined accurately.

Kinetic and thermodynamic effects of phosphorylation on p53 binding to MDM2

p53 is frequently mutated in human cancers. Its levels are tightly regulated by the E3 ubiquitin ligase MDM2. The complex between MDM2 and p53 is largely formed by the interaction between the N-terminal domain of MDM2 and the N-terminal transactivation (TA) domain of p53 (residues 15–29). We investigated the kinetic and thermodynamic basis of the MDM2/p53 interaction by using wild-type and mutant variants of the TA domain. We focus on the effects of phosphorylation at positions Thr18 and Ser20 including their substitution with phosphomimetics. Conformational propensities of the isolated peptides were investigated using in silico methods and experimentally by circular dichroism and 1H-NMR in aqueous solution. Both experimental and computational analyses indicate that the p53 peptides are mainly disordered in aqueous solution, with evidence of nascent helix around the Ser20-Leu25 region. Both phosphorylation and the phosphomimetics at Thr18 result in a decrease in the binding affinity by ten- to twenty-fold when compared to the wild-type. Phosphorylation and phosphomimetics at Ser20 result in a smaller decrease in the affinity. Mutation of Lys24 and Leu25 also disrupts the interaction. Our results may be useful for further development of peptide-based drugs targeting the MDM2/p53 interaction.

Single-molecule dynamics and genome-wide transcriptomics reveal that NF-kB (p65)-DNA binding times can be decoupled from transcriptional activation.

Transcription factors (TFs) regulate gene expression in both prokaryotes and eukaryotes by recognizing and binding to specific DNA promoter sequences. In higher eukaryotes, it remains unclear how the duration of TF binding to DNA relates to downstream transcriptional output. Here, we address this question for the transcriptional activator NF-κB (p65), by live-cell single molecule imaging of TF-DNA binding kinetics and genome-wide quantification of p65-mediated transcription. We used mutants of p65, perturbing either the DNA binding domain (DBD) or the protein-protein transactivation domain (TAD). We found that p65-DNA binding time was predominantly determined by its DBD and directly correlated with its transcriptional output as long as the TAD is intact. Surprisingly, mutation or deletion of the TAD did not modify p65-DNA binding stability, suggesting that the p65 TAD generally contributes neither to the assembly of an “enhanceosome,” nor to the active removal of p65 from putative specific binding sites. However, TAD removal did reduce p65-mediated transcriptional activation, indicating that protein-protein interactions act to translate the long-lived p65-DNA binding into productive transcription.

Chemical synthesis of transactivation domain (TAD) of tumor suppressor protein p53 by native chemical ligation of three peptide segments

Chemical composition of tumor suppressor protein p53 is altered via multiple post-translational modifications which modulate its cellular lifetime and interactions with other biomolecules. Here we report total chemical synthesis of a 61-residue form of transactivation domain (TAD) of p53 based on native chemical ligation of three peptide segments. The experiments to characterize its binding to nuclear co-activator binding domain (NCBD) of CREB-binding protein confirmed native-like induced folding upon binding to NCBD. Thus, the synthetic approach described herein can be useful for the preparation of various post-translationally modified analogues of TAD-p53 for further functional biochemical and biophysical studies.

Structural Basis for the Interaction between p53 Transactivation Domain and the Mediator Subunit MED25.

Eukaryotic transcription initiation is mediated by interactions between transcriptional activators and the mediator coactivator complex. Molecular interaction of p53 transcription factor with mediator complex subunit 25 (MED25) is essential for its target gene transcription. In this study, we characterized the molecular interaction between p53 transactivation domain (p53TAD) and activator interaction domain (ACID) of MED25 using nuclear magnetic resonance (NMR) spectroscopy. The NMR chemical shift perturbation and isothermal titration calorimetry (ITC) data showed that p53TAD interacted with MED25 ACID mainly through the p53TAD2 sequence motif. Taken together with the mutagenesis data, the refined structural model of MED25 ACID/p53TAD2 peptide complex showed that an amphipathic α-helix of p53TAD2 peptide bound an elongated hydrophobic groove of MED25 ACID. Furthermore, our results revealed the highly conserved mechanism of MED25 interaction with intrinsically unfolded acidic TADs from the transcriptional activators p53, ERM (Ets-related molecule), and herpes simplex virus protein 16 (VP16).

Binding Kinetics of the Intrinsically Disordered p53 Family Transactivation Domains and MDM2.

Because of their prominent roles in cell-cycle regulation and cancer, the interaction between MDM2 and the intrinsically disordered transactivation domain (TAD) of p53 is exceptionally well-studied. However, although there are numerous computational studies on the interaction mechanism, there is a paucity of experimental data regarding the kinetics and mechanism. We have used stopped flow fluorescence to investigate the binding reaction between MDM2 and TAD from p53 as well as from its paralogs p63 and p73, and in particular, focused on the salt dependence of the interaction. The observed kinetics are consistent with a two-state mechanism within the time frame of the stopped flow methodology; thus, any conformational changes including the previously identified MDM2 lid dynamics must occur on a time scale <5 ms at 10 °C. The association rate constants are similar for the three TADs, and differences in the dissociation rate constants determine the various affinities with MDM2. In contrast to previous studies, we found a relatively small ionic-strength dependence for all three interactions, highlighting the large variation in the role of electrostatics among binding reactions of intrinsically disordered proteins (IDPs). The basal association rate constants in the absence of electrostatic interactions were relatively high (≥2 × 106 M-1 s-1 at 10 °C), suggesting that a large number of initial contacts may lead to a productive complex. Our findings support an emerging picture of "conformational funneling" occurring in the initial stages of interactions involving IDPs and that these early binding events can rely on hydrophobic as well as charge-charge interactions.

Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells.

The role of p53 transactivation domain (p53-TAD), a multifunctional and dynamic domain, on DNA repair and retaining DNA integrity in human induced pluripotent stem cells (hiPSCs) has never been studied. p53-TAD was knocked out in iPSCs using CRISPR/Cas9 and was confirmed by DNA sequencing. p53-TAD knockout (KO) cells were characterized by accelerated proliferation, decreased population doubling time, and unaltered Bcl-2, Bcl-2-binding component 3, insulin-like growth factor 1 receptor, and Bax and altered Mdm2, p21, and p53-induced death domain transcript expression. In p53-TAD KO cells, the p53-regulated DNA repair proteins xeroderma pigmentosum group A, DNA polymerase H, and DNA-binding protein 2 expression were found to be reduced compared with p53 wild-type cells. Exposure to a low dose of doxorubicin (Doxo) induced similar DNA damage and DNA damage response (DDR) as measured by RAD50 and MRE11 expression, checkpoint kinase 2 activation, and γH2A.X recruitment at DNA strand breaks in both cell groups, indicating that silence of p53-TAD does not affect the DDR mechanism upstream of p53. After removal of Doxo, p53 wild-type hiPSCs underwent DNA repair, corrected their damaged DNA, and restored DNA integrity. Conversely, p53-TAD KO hiPSCs did not undergo complete DNA repair and failed to restore DNA integrity. More importantly, continuous culture of p53-TAD KO hiPSCs underwent G2/M cell cycle arrest and expressed the cellular senescent marker p16INK4a. Our data clearly show that silence of the TAD of p53 did not affect DDR but affected the DNA repair process, implying the crucial role of p53-TAD in maintaining DNA integrity. Therefore, activating p53-TAD domain using small molecules may promote DNA repair and integrity of cells and prevent cellular senescence.

In Situ Monitoring of p53 Protein and MDM2 Protein Interaction in Single Living Cells Using Single-Molecule Fluorescence Spectroscopy.

Protein-protein interactions play a central role in signal transduction, transcription regulations, enzymatic activity, and protein synthesis. The p53 protein is a key transcription factor, and its activity is precisely regulated by the p53-MDM2 interaction. Although the p53-MDM2 interaction has been studied, it is still not clear how p53 structures and external factors influence the p53-MDM2 interaction in living cells. Here, we developed a direct method for monitoring the p53-MDM2 interaction in single living cells using single-molecule fluorescence cross-correlation spectroscopy with a microfluidic chip. First, we labeled p53 and MDM2 proteins with enhanced green fluorescent protein (EGFP) and mCherry, respectively, using lentivirus infection. We then designed various mutants covering the three main domains of p53 (tetramerization, transactivation, and DNA-binding domains) and systematically studied effects of p53 protein primary, secondary, and quaternary structures on p53-MDM2 binding affinity in single living cells. We found that p53 dimers and tetramers can bind to MDM2, that the binding affinity of p53 tetramers is higher than that of p53 dimers, and that the affinity is closely correlated to the helicity of the p53 transactivation domain. The hot-spot mutation R175H in the DNA-binding domain reduced the binding of p53 to MDM2. Finally, we studied effects of inhibitors on p53-MDM2 interactions and dissociation dynamics of p53-MDM2 complexes in single living cells. We found that inhibitors Nutlin 3α and MI773 efficiently inhibited the p53-MDM2 interaction, but RITA did not work in living cells. This study provides a direct way for quantifying the relationship between protein structure and protein-protein interactions and evaluation of inhibitors in living cells.