Quantitative analysis of the binding affinity of poly(ADP-ribose) to specific binding proteins as a function of chain length

Poly(ADP-ribose) (PAR) is synthesized by poly(ADP-ribose) polymerases in response to genotoxic stress and interacts non-covalently with DNA damage checkpoint and repair proteins. Here, we present a variety of techniques to analyze this interaction in terms of selectivity and affinity. In vitro synthesized PAR was end-labeled using a carbonyl-reactive biotin analog. Binding of HPLC-fractionated PAR chains to the tumor suppressor protein p53 and to the nucleotide excision repair protein XPA was assessed using a novel electrophoretic mobility shift assay (EMSA). Long ADP-ribose chains (55-mer) promoted the formation of three specific complexes with p53. Short PAR chains (16-mer) were also able to bind p53, yet forming only one defined complex. In contrast, XPA did not interact with short polymer, but produced a single complex with long PAR chains (55-mer). In addition, we performed surface plasmon resonance with immobilized PAR chains, which allowed establishing binding constants and confirmed the results obtained by EMSA. Taken together, we developed several new protocols permitting the quantitative characterization of PAR–protein binding. Furthermore, we demonstrated that the affinity of the non-covalent PAR interactions with specific binding proteins (XPA, p53) can be very high (nanomolar range) and depends both on the PAR chain length and on the binding protein.     

Probing potential binding modes of the p53 tetramer to DNA based on the symmetries encoded in p53 response elements

Symmetries in the p53 response-element (p53RE) encode binding modes for p53 tetramer to recognize DNA. We investigated the molecular mechanisms and biological implications of the possible binding modes. The probabilities evaluated with molecular dynamics simulations and DNA sequence analyses were found to be correlated, indicating that p53 tetramer models studied here are able to read DNA sequence information. The traditionally believed mode with four p53 monomers binding at all four DNA quarter-sites does not cause linear DNA to bend. Alternatively, p53 tetramer can use only two monomers to recognize DNA sequence and induce DNA bending. With an arrangement of dimer of AB dimer observed in p53 trimer–DNA complex crystal, p53 can recognize supercoiled DNA sequence-specifically by binding to quarter-sites one and four (H14 mode) and recognize Holliday junction geometry-specifically. Examining R273H mutation and p53–DNA interactions, we found that at least three R273H monomers are needed to disable the p53 tetramer, consistent with experiments. But just one R273H monomer may greatly shift the binding mode probabilities. Our work suggests that p53 needs balanced binding modes to maintain genome stability. Inverse repeat p53REs favor the H14 mode and direct repeat p53REs may have high possibilities of other modes.     

Interactions between p53, hMSH2-hMSH6 and HMG I(Y) on Holliday junctions and bulged bases

The ability of the tumor suppressor protein, p53, to recognize certain types of DNA lesions may represent one of the mechanisms by which this protein modulates cellular response to DNA damage. p53 DNA binding properties are regulated by several factors, such as post-translational modifications including phosphorylation and acetylation, regulation by its own C-terminal domain and interactions with other cellular proteins. Substrates resembling Holliday junctions and extra base bulges were used to study the effect of three nuclear proteins, HMG-1, HMG I(Y) and hMSH2–hMSH6, on the lesion binding properties of p53. Gel retardation assays revealed that the three proteins had varying effects on p53 binding to these substrates. HMG-1 did not influence p53 binding to Holliday junctions or 3-cytosine bulges. HMG I(Y) rapidly dissociated p53 complexes with Holliday junctions but not 3-cytosine bulges. Finally, the mismatch repair protein complex, hMSH2–hMSH6, enhanced p53 binding to both substrates by 3–4-fold. Together, these results demonstrate that p53 DNA binding activity is highly influenced by the presence of other proteins, some having a dominant effect while others have a negative effect.     

NASP maintains histone H3–H4 homeostasis through two distinct H3 binding modes

Histone chaperones regulate all aspects of histone metabolism. NASP is a major histone chaperone for H3–H4 dimers critical for preventing histone degradation. Here, we identify two distinct histone binding modes of NASP and reveal how they cooperate to ensure histone H3–H4 supply. We determine the structures of a sNASP dimer, a complex of a sNASP dimer with two H3 α3 peptides, and the sNASP–H3–H4–ASF1b co-chaperone complex. This captures distinct functionalities of NASP and identifies two distinct binding modes involving the H3 α3 helix and the H3 αN region, respectively. Functional studies demonstrate the H3 αN-interaction represents the major binding mode of NASP in cells and shielding of the H3 αN region by NASP is essential in maintaining the H3–H4 histone soluble pool. In conclusion, our studies uncover the molecular basis of NASP as a major H3–H4 chaperone in guarding histone homeostasis.     

Molecular basis for Gcn5/PCAF histone acetyltransferase selectivity for histone and nonhistone substrates

Histone acetyltransferase (HAT) proteins often exhibit a high degree of specificity for lysine-bearing protein substrates. We have previously reported on the structure of the Tetrahymena Gcn5 HAT protein (tGcn5) bound to its preferred histone H3 substrate, revealing the mode of substrate binding by the Gcn5/PCAF family of HAT proteins. Interestingly, the Gcn5/PCAF HAT family has a remarkable ability to acetylate lysine residues within diverse cognate sites such as those found around lysines 14, 8, and 320 of histones H3, H4, and p53, respectively. To investigate the molecular basis for this, we now report on the crystal structures of tGcn5 bound to 19-residue histone H4 and p53 peptides. A comparison of these structures with tGcn5 bound to histone H3 reveals that the Gcn5/PCAF HATs can accommodate divergent substrates by utilizing analogous interactions with the lysine target and two C-terminal residues with a related chemical nature, suggesting that these interactions play a general role in Gcn5/PCAF substrate binding selectivity. In contrast, while the histone H3 complex shows extensive interactions with tGcn5 and peptide residues N-terminal to the target lysine, the corresponding residues in histone H4 and p53 are disordered, suggesting that the N-terminal substrate region plays an important role in the enhanced affinity of the Gcn5/PCAF HAT proteins for histone H3. Together, these studies provide a framework for understanding the substrate selectivity of HAT proteins.     

Phosphoproteomic analysis of thrombin- and p38 MAPK-regulated signaling networks in endothelial cells

Endothelial dysfunction is a hallmark of inflammation and is mediated by inflammatory factors that signal through G protein–coupled receptors including protease-activated receptor-1 (PAR1). PAR1, a receptor for thrombin, signals via the small GTPase RhoA and myosin light chain intermediates to facilitate endothelial barrier permeability. PAR1 also induces endothelial barrier disruption through a p38 mitogen-activated protein kinase–dependent pathway, which does not integrate into the RhoA/MLC pathway; however, the PAR1-p38 signaling pathways that promote endothelial dysfunction remain poorly defined. To identify effectors of this pathway, we performed a global phosphoproteome analysis of thrombin signaling regulated by p38 in human cultured endothelial cells using multiplexed quantitative mass spectrometry. We identified 5491 unique phosphopeptides and 2317 phosphoproteins, four distinct dynamic phosphoproteome profiles of thrombin-p38 signaling, and an enrichment of biological functions associated with endothelial dysfunction, including modulators of endothelial barrier disruption and a subset of kinases predicted to regulate p38-dependent thrombin signaling. Using available antibodies to detect identified phosphosites of key p38-regulated proteins, we discovered that inhibition of p38 activity and siRNA-targeted depletion of the p38α isoform increased basal phosphorylation of extracellular signal–regulated protein kinase 1/2, resulting in amplified thrombin-stimulated extracellular signal–regulated protein kinase 1/2 phosphorylation that was dependent on PAR1. We also discovered a role for p38 in the phosphorylation of α-catenin, a component of adherens junctions, suggesting that this phosphorylation may function as an important regulatory process. Taken together, these studies define a rich array of thrombin- and p38-regulated candidate proteins that may serve important roles in endothelial dysfunction.     

Why structure and chain length matter: on the biological significance underlying the structural heterogeneity of poly(ADP-ribose)

Poly(ADP-ribosyl)ation (PARylation) is a multifaceted post-translational modification, carried out by poly(ADP-ribosyl)transferases (poly-ARTs, PARPs), which play essential roles in (patho-) physiology, as well as cancer therapy. Using NAD⁺ as a substrate, acceptors, such as proteins and nucleic acids, can be modified with either single ADP-ribose units or polymers, varying considerably in length and branching. Recently, the importance of PAR structural heterogeneity with regards to chain length and branching came into focus. Here, we provide a concise overview on the current knowledge of the biochemical and physiological significance of such differently structured PAR. There is increasing evidence revealing that PAR’s structural diversity influences the binding characteristics of its readers, PAR catabolism, and the dynamics of biomolecular condensates. Thereby, it shapes various cellular processes, such as DNA damage response and cell cycle regulation. Contrary to the knowledge on the consequences of PAR’s structural diversity, insight into its determinants is just emerging, pointing to specific roles of different PARP members and accessory factors. In the future, it will be interesting to study the interplay with other post-translational modifications, the contribution of natural PARP variants, and the regulatory role of accessory molecules. This has the exciting potential for new therapeutic approaches, with the targeted modulation and tuning of PARPs’ enzymatic functions, rather than their complete inhibition, as a central premise.     

RNA length has a non-trivial effect in the stability of biomolecular condensates formed by RNA-binding proteins

Biomolecular condensates formed via liquid–liquid phase separation (LLPS) play a crucial role in the spatiotemporal organization of the cell material. Nucleic acids can act as critical modulators in the stability of these protein condensates. To unveil the role of RNA length in regulating the stability of RNA binding protein (RBP) condensates, we present a multiscale computational strategy that exploits the advantages of a sequence-dependent coarse-grained representation of proteins and a minimal coarse-grained model wherein proteins are described as patchy colloids. We find that for a constant nucleotide/protein ratio, the protein fused in sarcoma (FUS), which can phase separate on its own—i.e., via homotypic interactions—only exhibits a mild dependency on the RNA strand length. In contrast, the 25-repeat proline-arginine peptide (PR₂₅), which does not undergo LLPS on its own at physiological conditions but instead exhibits complex coacervation with RNA—i.e., via heterotypic interactions—shows a strong dependence on the length of the RNA strands. Our minimal patchy particle simulations suggest that the strikingly different effect of RNA length on homotypic LLPS versus RBP–RNA complex coacervation is general. Phase separation is RNA-length dependent whenever the relative contribution of heterotypic interactions sustaining LLPS is comparable or higher than those stemming from protein homotypic interactions. Taken together, our results contribute to illuminate the intricate physicochemical mechanisms that influence the stability of RBP condensates through RNA inclusion.     

Histone H2A and H4 N-terminal Tails Are Positioned by the MEP50 WD Repeat Protein for Efficient Methylation by the PRMT5 Arginine Methyltransferase

The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1–20) and H4(1–20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate K_m, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site.     

Ubiquitin plays an atypical role in GPCR-induced p38 MAP kinase activation on endosomes

Protease-activated receptor 1 (PAR1) is a G protein–coupled receptor (GPCR) for thrombin and promotes inflammatory responses through multiple pathways including p38 mitogen-activated protein kinase signaling. The mechanisms that govern PAR1-induced p38 activation remain unclear. Here, we define an atypical ubiquitin-dependent pathway for p38 activation used by PAR1 that regulates endothelial barrier permeability. Activated PAR1 K63-linked ubiquitination is mediated by the NEDD4-2 E3 ubiquitin ligase and initiated recruitment of transforming growth factor-β–activated protein kinase-1 binding protein-2 (TAB2). The ubiquitin-binding domain of TAB2 was essential for recruitment to PAR1-containing endosomes. TAB2 associated with TAB1, which induced p38 activation independent of MKK3 and MKK6. The P2Y₁ purinergic GPCR also stimulated p38 activation via NEDD4-2–mediated ubiquitination and TAB1–TAB2. TAB1–TAB2-dependent p38 activation was critical for PAR1-promoted endothelial barrier permeability in vitro, and p38 signaling was required for PAR1-induced vascular leakage in vivo. These studies define an atypical ubiquitin-mediated signaling pathway used by a subset of GPCRs that regulates endosomal p38 signaling and endothelial barrier disruption.     

The checkpoint Saccharomyces cerevisiae Rad9 protein contains a tandem tudor domain that recognizes DNA

DNA damage checkpoints are signal transduction pathways that are activated after genotoxic insults to protect genomic integrity. At the site of DNA damage, ‘mediator’ proteins are in charge of recruiting ‘signal transducers’ to molecules ‘sensing’ the damage. Budding yeast Rad9, fission yeast Crb2 and metazoan 53BP1 are presented as mediators involved in the activation of checkpoint kinases. Here we show that, despite low sequence conservation, Rad9 exhibits a tandem tudor domain structurally close to those found in human/mouse 53BP1 and fission yeast Crb2. Moreover, this region is important for the resistance of Saccharomyces cerevisiae to different genotoxic stresses. It does not mediate direct binding to a histone H3 peptide dimethylated on K79, nor to a histone H4 peptide dimethylated on lysine 20, as was demonstrated for 53BP1. However, the tandem tudor region of Rad9 directly interacts with single-stranded DNA and double-stranded DNAs of various lengths and sequences through a positively charged region absent from 53BP1 and Crb2 but present in several yeast Rad9 homologs. Our results argue that the tandem tudor domains of Rad9, Crb2 and 53BP1 mediate chromatin binding next to double-strand breaks. However, their modes of chromatin recognition are different, suggesting that the corresponding interactions are differently regulated.     

Interaction of Saccharomyces Cdc13p with Pol1p, Imp4p, Sir4p and Zds2p is involved in telomere replication, telomere maintenance and cell growth control

Telomeres are the physical ends of eukaryotic chromosomes. They are important for maintaining the integrity of chromosomes and this function is mediated through a number of protein factors. In Saccharomyces cerevisiae, Cdc13p binds to telomeres and affects telomere maintenance, telomere position effects and cell cycle progression through G₂/M phase. We identified four genes encoding Pol1p, Sir4p, Zds2p and Imp4p that interact with amino acids 1–252 of Cdc13p using a yeast two-hybrid screening system. Interactions of these four proteins with Cdc13p were through direct protein–protein interactions as judged by in vitro pull-down assays. Direct protein–protein interactions were also observed between Pol1p–Imp4p, Pol1p–Sir4p and Sir4p–Zds2p, whereas no interaction was detected between Imp4p–Sir4p and Zds2p–Imp4p, suggesting that protein interactions were specific in the complex. Pol1p was shown to interact with Cdc13p. Here we show that Zds2p and Imp4p also form a stable complex with Cdc13p in yeast cells, because Zds2p and Imp4p co-immunoprecipitate with Cdc13p, whereas Sir4p does not. The function of the N-terminal 1–252 region of Cdc13p was also analyzed. Expressing Cdc13(252–924)p, which lacks amino acids 1–252 of Cdc13p, causes defects in progressive cell growth and eventually arrested in the G₂/M phase of the cell cycle. These growth defects were not caused by progressive shortening of telomeres because telomeres in these cells were long. Point mutants in the amino acids 1–252 region of Cdc13p that reduced the interaction between Cdc13p and its binding proteins resulted in varying level of defects in cell growth and telomeres. These results indicate that the interactions between Cdc13(1–252)p and its binding proteins are important for the function of Cdc13p in telomere regulation and cell growth. Together, our results provide evidence for the formation of a Cdc13p-mediated telosome complex through its N-terminal region that is involved in telomere maintenance, telomere length regulation and cell growth control.     

Molecular interactions of ASPP1 and ASPP2 with the p53 protein family and the apoptotic promoters PUMA and Bax

The apoptosis stimulating p53 proteins, ASPP1 and ASPP2, are the first two common activators of the p53 protein family that selectively enable the latter to regulate specific apoptotic target genes, which facilitates yes yet unknown mechanisms for discrimination between cell cycle arrest and apoptosis. To better understand the interplay between ASPP- and p53-family of proteins we investigated the molecular interactions between them using biochemical methods and structure-based homology modelling. The data demonstrate that: (i) the binding of ASPP1 and ASPP2 to p53, p63 and p73 is direct; (ii) the C-termini of ASPP1 and ASPP2 interact with the DNA-binding domains of p53 protein family with dissociation constants, K_d, in the lower micro-molar range; (iii) the stoichiometry of binding is 1:1; (iv) the DNA-binding domains of p53 family members are sufficient for these protein–protein interactions; (v) EMSA titrations revealed that while tri-complex formation between ASPPs, p53 family of proteins and PUMA/Bax is mutually exclusive, ASPP2 (but not ASPP1) formed a complex with PUMA (but not Bax) and displaced p53 and p73. The structure-based homology modelling revealed subtle differences between ASPP2 and ASPP1 and together with the experimental data provide novel mechanistic insights.     

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.     

The linker histone homolog Hho1p from Saccharomyces cerevisiae represents a winged helix-turn-helix fold as determined by NMR spectroscopy

Hho1p is assumed to serve as a linker histone in Saccharomyces cerevisiae and, notably, it possesses two putative globular domains, designated HD1 (residues 41–118) and HD2 (residues 171–252), that are homologous to histone H5 from chicken erythrocytes. We have determined the three-dimensional structure of globular domain HD1 with high precision by heteronuclear magnetic resonance spectroscopy. The structure had a winged helix–turn–helix motif composed of an αβααββ fold and closely resembled the structure of the globular domain of histone H5. Interestingly, the second globular domain, HD2, in Hho1p was unstructured under physiological conditions. Gel mobility assay demonstrated that Hho1p preferentially binds to supercoiled DNA over linearized DNA. Furthermore, NMR analysis of the complex of a deletion mutant protein (residues 1–118) of Hho1p with a linear DNA duplex revealed that four regions within the globular domain HD1 are involved in the DNA binding. The above results suggested that Hho1p possesses properties similar to those of linker histones in higher eukaryotes in terms of the structure and binding preference towards supercoiled DNA.