Physical and functional interactions between human mitochondrial single-stranded DNA-binding protein and tumour suppressor p53

Single-stranded DNA-binding proteins (SSB) form a class of proteins that bind preferentially single-stranded DNA with high affinity. They are involved in DNA metabolism in all organisms and serve a vital role in replication, recombination and repair of DNA. In this report, we identify human mitochondrial SSB (HmtSSB) as a novel protein-binding partner of tumour suppressor p53, in mitochondria. It binds to the transactivation domain (residues 1–61) of p53 via an extended binding interface, with dissociation constant of 12.7 (± 0.7) μM. Unlike most binding partners reported to date, HmtSSB interacts with both TAD1 (residues 1–40) and TAD2 (residues 41–61) subdomains of p53. HmtSSB enhances intrinsic 3′-5′ exonuclease activity of p53, particularly in hydrolysing 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) present at 3′-end of DNA. Taken together, our data suggest that p53 is involved in DNA repair within mitochondria during oxidative stress. In addition, we characterize HmtSSB binding to ssDNA and p53 N-terminal domain using various biophysical measurements and we propose binding models for both.     

NMR chemical shift and relaxation measurements provide evidence for the coupled folding and binding of the p53 transactivation domain

The interaction between the acidic transactivation domain of the human tumor suppressor protein p53 (p53TAD) and the 70 kDa subunit of human replication protein A (hRPA70) was investigated using heteronuclear magnetic resonance spectroscopy. A ¹H–¹⁵N heteronuclear single quantum coherence (HSQC) titration experiment was performed on a ¹⁵N-labeled fragment of hRPA70, containing the N-terminal 168 residues (hRPA70_1–168) and p53TAD. HRPA70_1–168 residues important for binding were identified and found to be localized to a prominent basic cleft. This binding site overlapped with a previously identified single-stranded DNA-binding site, suggesting that a competitive binding mechanism may regulate the formation of p53TAD–hRPA70 complex. The amide ¹H and ¹⁵N chemical shifts of an uniformly ¹⁵N-labeled sample of p53TAD were also monitored before and after the addition of unlabeled hRPA70_1–168. In the presence of unlabeled hRPA70_1–168, resonance lineshapes increased and corresponding intensity reductions were observed for specific p53TAD residues. The largest intensity reductions were observed for p53TAD residues 42–56. Minimal binding was observed between p53TAD and a mutant form of hRPA70_1–168, where the basic cleft residue R41 was changed to a glutamic acid (R41E), demonstrating that ionic interactions play an important role in specifying the binding interface. The region of p53TAD most affected by binding hRPA70_1–168 was found to have some residual alpha helical and beta strand structure; however, this structure was not stabilized by binding hRPA70_1–168. ¹⁵N relaxation experiments were performed to monitor changes in backbone dynamics of p53TAD when bound to hRPA70_1–168. Large changes in both the transverse (R₂) and rotating frame (R_1ρ) relaxation rates were observed for a subset of the p53TAD residues that had ¹H–¹⁵N HSQC resonance intensity reductions during the complex formation. The folding of p53TAD upon complex formation is suggested by the pattern of changes observed for both R₂ and R_1ρ. A model that couples the formation of a weak encounter complex between p53TAD and hRPA70_1–168 to the folding of p53TAD is discussed in the context of a functional role for the p53–hRPA70 complex in DNA repair.     

Regulation of ASPP2 Interaction with p53 Core Domain by an Intramolecular Autoinhibitory Mechanism

ASPP2 is a key protein in regulating apoptosis both in p53-dependent and-independent pathways. The C-terminal part of ASPP2 contains four ankyrin repeats and an SH3 domain (Ank-SH3) that mediate the interactions of ASPP2 with apoptosis related proteins such as p53, Bcl-2 and the p65 subunit of NFκB. p53 core domain (p53CD) binds the n-src loop and the RT loop of ASPP2 SH3. ASPP2 contains a disordered proline rich domain (ASPP2 Pro) that forms an intramolecular autoinhibitory interaction with the Ank-SH3 domains. Here we show how this intramolecular interaction affects the intermolecular interactions of ASPP2 with p53, Bcl-2 and NFkB. We used biophysical methods to obtain better understanding of the relationship between ASPP2 and its partners for getting a comprehensive view on ASPP2 pathways. Fluorescence anisotropy competition experiments revealed that both ASPP2 Pro and p53CD competed for binding the n-src loop of the ASPP2 SH3, indicating regulation of p53CD binding to this loop by ASPP2 Pro. Peptides derived from the ASPP2-binding interface of Bcl-2 did not compete with p53CD or NFkB peptides for binding the ASPP2 n-src loop. However, p53CD displaced the NFκB peptide (residues 303–332) from its complex with ASPP2 Ank-SH3, indicating that NFκB 303–332 and p53CD bind a partly overlapping site in ASPP2 SH3, mostly in the RT loop. These results are in agreement with previous docking studies, which showed that ASPP2 Ank-SH3 binds Bcl-2 and NFκB mostly via distinct sites from p53. However they show some overlap between the binding sites of p53CD and NFkB in ASPP2 Ank-SH3. Our results provide experimental evidence that the intramolecular interaction in ASPP2 regulates its binding to p53CD and that ASPP2 Ank-SH3 binds Bcl-2 and NFκB via distinct sites.     

p53 and p73 display common and distinct requirements for sequence specific binding to DNA

Although p53 and p73 share considerable homology in their DNA-binding domains, there have been few studies examining their relative interactions with DNA as purified proteins. Comparing p53 and p73β proteins, our data show that zinc chelation by EDTA is significantly more detrimental to the ability of p73β than of p53 to bind DNA, most likely due to the greater effect that the loss of zinc has on the conformation of the DNA-binding domain of p73. Furthermore, prebinding to DNA strongly protects p73β but not p53 from chelation by EDTA suggesting that DNA renders the core domain of p73 less accessible to its environment. Further exploring these biochemical differences, a five-base sub-sequence was identified in the p53 consensus binding site that confers a greater DNA-binding stability on p73β than on full-length p53 in vitro. Surprisingly, p53 lacking its C-terminal non-specific DNA-binding domain (p53Δ30) demonstrates the same sequence discrimination as does p73β. In vivo, both p53 and p73β exhibit higher transactivation of a reporter with a binding site containing this sub-sequence, suggesting that lower in vitro dissociation translates to higher in vivo transactivation of sub-sequence-containing sites.     

Inhibition of nonsense-mediated decay rescues p53β/γ isoform expression and activates the p53 pathway in MDM2-overexpressing and select p53-mutant cancers

Inactivation of p53 is present in almost every tumor, and hence, p53-reactivation strategies are an important aspect of cancer therapy. Common mechanisms for p53 loss in cancer include expression of p53-negative regulators such as MDM2, which mediate the degradation of wildtype p53 (p53α), and inactivating mutations in the TP53 gene. Currently, approaches to overcome p53 deficiency in these cancers are limited. Here, using non–small cell lung cancer and glioblastoma multiforme cell line models, we show that two alternatively spliced, functional truncated isoforms of p53 (p53β and p53γ, comprising exons 1 to 9β or 9γ, respectively) and that lack the C-terminal MDM2-binding domain have markedly reduced susceptibility to MDM2-mediated degradation but are highly susceptible to nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. In cancer cells harboring MDM2 overexpression or TP53 mutations downstream of exon 9, NMD inhibition markedly upregulates p53β and p53γ and restores activation of the p53 pathway. Consistent with p53 pathway activation, NMD inhibition induces tumor suppressive activities such as apoptosis, reduced cell viability, and enhanced tumor radiosensitivity, in a relatively p53-dependent manner. In addition, NMD inhibition also inhibits tumor growth in a MDM2-overexpressing xenograft tumor model. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in p53-deficient tumors bearing MDM2 overexpression or p53 mutations downstream of exon 9, subgroups that comprise approximately 6% of all cancers.     

Almost half of the RTX domain is dispensable for complement receptor 3 binding and cell-invasive activity of the Bordetella adenylate cyclase toxin

The whooping cough agent Bordetella pertussis secretes an adenylate cyclase toxin (CyaA) that through its large carboxy-proximal Repeat-in-ToXin (RTX) domain binds the complement receptor 3 (CR3). The RTX domain consists of five blocks (I–V) of characteristic glycine and aspartate-rich nonapeptides that fold into five Ca²⁺-loaded parallel β-rolls. Previous work indicated that the CR3-binding structure comprises the interface of β-rolls II and III. To test if further portions of the RTX domain contribute to CR3 binding, we generated a construct with the RTX block II/III interface (CyaA residues 1132–1294) linked directly to the C-terminal block V fragment bearing the folding scaffold (CyaA residues 1562–1681). Despite deletion of 267 internal residues of the RTX domain, the Ca²⁺-driven folding of the hybrid block III/V β-roll still supported formation of the CR3-binding structure at the interface of β-rolls II and III. Moreover, upon stabilization by N- and C-terminal flanking segments, the block III/V hybrid-comprising constructs competed with CyaA for CR3 binding and induced formation of CyaA toxin-neutralizing antibodies in mice. Finally, a truncated CyaAΔ_1295-1561 toxin bound and penetrated erythrocytes and CR3-expressing cells, showing that the deleted portions of RTX blocks III, IV, and V (residues 1295–1561) were dispensable for CR3 binding and for toxin translocation across the target cell membrane. This suggests that almost a half of the RTX domain of CyaA is not involved in target cell interaction and rather serves the purpose of toxin secretion.     

Domain specific interaction in the XRCC1-DNA polymerase beta complex

XRCC1 (X-ray cross-complementing group 1) is a DNA repair protein that forms complexes with DNA polymerase β (β-Pol), DNA ligase III and poly-ADP-ribose polymerase in the repair of DNA single strand breaks. The domains in XRCC1 have been determined, and characterization of the domain–domain interaction in the XRCC1-β-Pol complex has provided information on the specificity and mechanism of binding. The domain structure of XRCC1, determined using limited proteolysis, was found to include an N-terminal domain (NTD), a central BRCT-I (breast cancer susceptibility protein-1) domain and a C-terminal BRCT-II domain. The BRCT-I–linker–BRCT-II C-terminal fragment and the linker–BRCT-II C-terminal fragment were relatively stable to proteolysis suggestive of a non-random conformation of the linker. A predicted inner domain was found not to be stable to proteolysis. Using cross-linking experiments, XRCC1 was found to bind intact β-Pol and the β-Pol 31 kDa domain. The XRCC1-NTD_1–183 (residues 1–183) was found to bind β-Pol, the β-Pol 31 kDa domain and the β-Pol C-terminal palm-thumb (residues 140–335), and the interaction was further localized to XRCC1-NTD_1–157 (residues 1–157). The XRCC1-NTD_1–183-β-Pol 31 kDa domain complex was stable at high salt (1 M NaCl) indicative of a hydrophobic contribution. Using a yeast two-hybrid screen, polypeptides expressed from two XRCC1 constructs, which included residues 36–355 and residues 1–159, were found to interact with β-Pol, the β-Pol 31 kDa domain, and the β-Pol C-terminal thumb-only domain polypeptides expressed from the respective β-Pol constructs. Neither the XRCC1-NTD_1–159, nor the XRCC1_36–355 polypeptide was found to interact with a β-Pol thumbless polypeptide. A third XRCC1 polypeptide (residues 75–212) showed no interaction with β-Pol. In quantitative gel filtration and analytical ultracentrifugation experiments, the XRCC1-NTD_1–183 was found to bind β-Pol and its 31 kDa domain in a 1:1 complex with high affinity (K_d of 0.4–2.4 µM). The combined results indicate a thumb-domain specific 1:1 interaction between the XRCC1-NTD_1–159 and β-Pol that is of an affinity comparable to other binding interactions involving β-Pol.     

p53 isoforms differentially impact on the POLι dependent DNA damage tolerance pathway

The recently discovered p53-dependent DNA damage tolerance (DDT) pathway relies on its biochemical activities in DNA-binding, oligomerization, as well as complex formation with the translesion synthesis (TLS) polymerase iota (POLι). These p53-POLι complexes slow down nascent DNA synthesis for safe, homology-directed bypass of DNA replication barriers. In this study, we demonstrate that the alternative p53-isoforms p53β, p53γ, Δ40p53α, Δ133p53α, and Δ160p53α differentially affect this p53-POLι-dependent DDT pathway originally described for canonical p53α. We show that the C-terminal isoforms p53β and p53γ, comprising a truncated oligomerization domain (OD), bind PCNA. Conversely, N-terminally truncated isoforms have a reduced capacity to engage in this interaction. Regardless of the specific loss of biochemical activities required for this DDT pathway, all alternative isoforms were impaired in promoting POLι recruitment to PCNA in the chromatin and in decelerating DNA replication under conditions of enforced replication stress after Mitomycin C (MMC) treatment. Consistent with this, all alternative p53-isoforms no longer stimulated recombination, i.e., bypass of endogenous replication barriers. Different from the other isoforms, Δ133p53α and Δ160p53α caused a severe DNA replication problem, namely fork stalling even in untreated cells. Co-expression of each alternative p53-isoform together with p53α exacerbated the DDT pathway defects, unveiling impaired POLι recruitment and replication deceleration already under unperturbed conditions. Such an inhibitory effect on p53α was particularly pronounced in cells co-expressing Δ133p53α or Δ160p53α. Notably, this effect became evident after the expression of the isoforms in tumor cells, as well as after the knockdown of endogenous isoforms in human hematopoietic stem and progenitor cells. In summary, mimicking the situation found to be associated with many cancer types and stem cells, i.e., co-expression of alternative p53-isoforms with p53α, carved out interference with p53α functions in the p53-POLι-dependent DDT pathway.Subject terms: Mechanisms of disease, Tumour-suppressor proteins     

Casein Kinase 1α Regulates an MDMX Intramolecular Interaction To Stimulate p53 Binding

MDMX is an important regulator of p53 during embryonic development and malignant transformation. Previous studies showed that casein kinase 1α (CK1α) stably associates with MDMX, stimulates MDMX-p53 binding, and cooperates with MDMX to inactivate p53. However, the mechanism by which CK1α stimulates MDMX-p53 interaction remains unknown. Here, we present evidence that p53 binding by the MDMX N-terminal domain is inhibited by the central acidic region through an intramolecular interaction that competes for the p53 binding pocket. CK1α binding to the MDMX central domain and phosphorylation of S289 disrupts the intramolecular interaction, allowing the N terminus to bind p53 with increased affinity. After DNA damage, the MDMX-CK1α complex is disrupted by Chk2-mediated phosphorylation of MDMX at S367, leading to reduced MDMX-p53 binding. Therefore, CK1α is an important functional partner of MDMX. DNA damage activates p53 in part by disrupting CK1α-MDMX interaction and reducing MDMX-p53 binding affinity.     

Cooperative binding of DNA and CBFbeta to the Runt domain of the CBFalpha studied via MD simulations

The Runt domain (RD) is the DNA-binding region of the Runx genes. A related protein, known as core binding factor β (CBFβ) also binds to the RD to enhance RD–DNA interaction by 6- to 10-fold. Here, we report results from molecular dynamics (MD) simulations of RD alone, as a dimer in complexes with DNA and CBFβ and in a ternary complex with DNA and CBFβ. Consistent with the experimental findings, in the presence of CBFβ the estimated free energy of binding of RD to the DNA is more favorable, which is shown to be due to more favorable intermolecular interactions and desolvation contributions. Also contributing to the enhanced binding are favorable intramolecular interactions between the ‘wing’ residues (RD residues 139–145) and the ‘wing1’ residues (RD residues 104–116). The simulation studies also indicate that the RD–CBFβ binding is more favorable in the presence of DNA due to a more favorable RD–CBFβ interaction energy. In addition, it is predicted that long-range interactions involving ionic residues contribute to binding cooperativity. Results from the MD calculations are used to interpret a variety of experimental mutagenesis data. A novel role for RD Glu116 to the RD–CBFβ interaction is predicted.     

Crystal structure determination and site-directed mutagenesis of the Pyrococcus abyssi aCBF5-aNOP10 complex reveal crucial roles of the C-terminal domains of both proteins in H/ACA sRNP activity

In archaeal rRNAs, the isomerization of uridine into pseudouridine (Ψ) is achieved by the H/ACA sRNPs and the minimal set of proteins required for RNA:Ψ-synthase activity is the aCBF5–aNOP10 protein pair. The crystal structure of the aCBF5–aNOP10 heterodimer from Pyrococcus abyssi was solved at 2.1 Å resolution. In this structure, protein aNOP10 has an extended shape, with a zinc-binding motif at the N-terminus and an α-helix at the C-terminus. Both motifs contact the aCBF5 catalytic domain. Although less efficiently as does the full-length aNOP10, the aNOP10 C-terminal domain binds aCBF5 and stimulates the RNA-guided activity. We show that the C-terminal domain of aCBF5 (the PUA domain), which is wrapped by an N-terminal extension of aCBF5, plays a crucial role for aCBF5 binding to the guide sRNA. Addition of this domain in trans partially complement particles assembled with an aCBF5ΔPUA truncated protein. In the crystal structure, the aCBF5–aNOP10 complex forms two kinds of heterotetramers with parallel and perpendicular orientations of the aNOP10 terminal α-helices, respectively. By gel filtration assay, we showed that aNOP10 can dimerize in solution. As both residues Y41 and L48 were needed for dimerization, the dimerization likely takes place by interaction of parallel α-helices.     

Structure of the DNA-binding domain of NgTRF1 reveals unique features of plant telomere-binding proteins

Telomeres are protein–DNA elements that are located at the ends of linear eukaryotic chromosomes. In concert with various telomere-binding proteins, they play an essential role in genome stability. We determined the structure of the DNA-binding domain of NgTRF1, a double-stranded telomere-binding protein of tobacco, using multidimensional NMR spectroscopy and X-ray crystallography. The DNA-binding domain of NgTRF1 contained the Myb-like domain and C-terminal Myb-extension that is characteristic of plant double-stranded telomere-binding proteins. It encompassed amino acids 561–681 (NgTRF1^561–681), and was composed of 4 α-helices. We also determined the structure of NgTRF1^561–681 bound to plant telomeric DNA. We identified several amino acid residues that interacted directly with DNA, and confirmed their role in the binding of NgTRF1 to telomere using site-directed mutagenesis. Based on a structural comparison of the DNA-binding domains of NgTRF1 and human TRF1 (hTRF1), NgTRF1 has both common and unique DNA-binding properties. Interaction of Myb-like domain with telomeric sequences is almost identical in NgTRF1^561–681 with the DNA-binding domain of hTRF1. The interaction of Arg-638 with the telomeric DNA, which is unique in NgTRF1^561–681, may provide the structural explanation for the specificity of NgTRF1 to the plant telomere sequences, (TTTAGGG)_n.     

Pin1 modulates p63α protein stability in regulation of cell survival,proliferation and tumor formation

The homolog of p53 gene, p63, encodes multiple p63 protein isoforms. TAp63 proteins contain an N-terminal transactivation domain similar to that of p53 and function as tumor suppressors; whereas ΔNp63 isoforms, which lack the intact N-terminal transactivation domain, are associated with human tumorigenesis. Accumulating evidence demonstrating the important roles of p63 in development and cancer development, the regulation of p63 proteins, however, is not fully understood. In this study, we show that peptidyl-prolyl isomerase Pin1 directly binds to and stabilizes TAp63α and ΔNp63α via inhibiting the proteasomal degradation mediated by E3 ligase WWP1. We further show that Pin1 specifically interacts with T₅₃₈P which is adjacent to the P₅₅₀PxY₅₄₃ motif, and disrupts p63α–WWP1 interaction. In addition, while Pin1 enhances TAp63α-mediated apoptosis, it promotes ΔNp63α-induced cell proliferation. Furthermore, knockdown of Pin1 in FaDu cells inhibits tumor formation in nude mice, which is rescued by simultaneous knockdown of WWP1 or ectopic expression of ΔNp63α. Moreover, overexpression of Pin1 correlates with increased expression of ΔNp63α in human oral squamous cell carcinoma samples. Together, these results suggest that Pin1-mediated modulation of ΔNp63α may have a causative role in tumorigenesis.