Phosphopeptide Binding Research Articles

Abstract P4-10-01: Edgetic perturbation of BRCT-mediated interactions caused by the BRCA1 H1686Q sequence variant

Abstract Background: The ever-increasing number of variants of uncertain significance (VUS) that result from BRCA1/BRCA2 genetic testing poses a challenge to healthcare providers, thus impacting clinical decision-making. Though a quantitative posterior probability model, mostly reliant on genetic data, has been developed to estimate the clinical relevance of individual VUS, indirect evidence derived from functional tests continues to be a sought-after adjunct to achieve VUS categorization. Using a multimodal approach, we previously demonstrated the deleterious effect on protein localization and function of the Italian founder sequence alteration BRCA1 V1688del, and the neutral effect of a neighboring variant, BRCA1 V1687I, identified in a patient with early onset triple negative breast cancer. Disease-causality has been suggested for the adjacent BRCA1 H1686Q variant that occurs in the evolutionarily conserved THV motif of the first BRCT repeat and was described in a breast/ovarian cancer family (Giannini et al., 2008). We aimed at substantiating the predicted pathogenicity of BRCA1 H1686Q by assessing its biological significance. Methods: To investigate the structural and functional consequences of the BRCA1 H1686Q sequence alteration, we used a strategy that combined homology modeling with analysis of protein interactions (by immunoprecipitation and GST pull down techniques) and function (by a homologous recombination assay). Results: The three-dimensional model of the BRCA1 H1686Q BRCT domain suggested that the site and nature of the amino acid change could alter the interaction and relative orientation of secondary structure elements in the core region of the protein, thus possibly affecting the overall stability of the protein and triggering a long-range allosteric effect with repercussions on residues involved in phosphopeptide binding. Indeed, in transfected 293T cells, the recombinant BRCA1 H1686Q protein was less abundant than its wild-type counterpart. Moreover, while efficiently heterodimerizing, via its N-terminus, with the binding partner BARD1, the BRCA1 H1686Q protein displayed a peculiar behavior with regards to C-terminal, BRCT-mediated interactions as it bound BRIP1/FANCJ and not CtIP. These results, repeatedly observed in different cell lines, were confirmed by reciprocal immunoprecipitations as well as by GST pull down experiments using a recombinant BRCA1 H1686Q BRCT domain. Notably, the BRCA1 H1686Q protein displayed a homologous recombination defect similar to that observed for the clinically ascertained BRCA1 M1775R mutant protein. Conclusions: Our data suggest that the BRCA1 H1686Q variant, by affecting the BRCT ligand recognition ability, causes an edgetic perturbation (i.e. selective disruption/retention) of BRCT-mediated interactions, which is yet sufficient to impair the homologous recombination function of the encoded protein. These findings provide valuable structure/function insights that could help elucidate the link between BRCA1 DNA repair and tumor suppression functions and aid development of functional tests for VUS screening and classification. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-10-01.

Mechanistic Insights into Phosphopeptide–BRCT Domain Association: Preorganization, Flexibility, and Phosphate Recognition

Promiscuous proteins are commonly observed in biological systems, for example, in modular domains that recognize phosphopeptides during signal transduction. This promiscuous recognition is of fundamental interest in chemistry and biology but is challenging when designing phosphopeptides in silico for cell biology studies. To investigate promiscuous recognition and binding processes of phosphopeptides and the modular domain, we selected a domain essential in breast cancer-the breast-cancer-associated protein 1 (BRCA1) C-terminal (BRCT) repeats as our model system. We performed molecular dynamics simulations and detailed analyses of the dihedral space to study protein fluctuation and conformational changes with phosphopeptide binding. We also studied the association processes of phosphorylated and unphosphorylated peptides using Brownian dynamics with a coarse-grained model. We found that the BRCT domain is preorganized for phosphopeptide binding but has a moderate arrangement of side chains to form complexes with various types of phosphopeptides. Phosphopeptide binding restricts the system motion in general, while the nonpolar phosphopeptide becomes more flexible in the bound state. Our analysis found that the BRCT domain utilizes different mechanisms, usually termed lock and key, induced-fit, and population-shift/conformational-selection models, to recognize peptides with different features. Brownian dynamics simulations revealed that the charged phosphate group may not always accelerate peptide association processes, but it helps the phosphopeptide orient into binding pockets accurately and stabilizes the complex. This work provides insights into molecular recognition in the promiscuous protein system.

Structural flexibility regulates phosphopeptide-binding activity of the tyrosine kinase binding domain of Cbl-c

Through their ubiquitin ligase activity, Cbl-family proteins suppress signalling mediated by protein-tyrosine kinases (PTKs), but can also function as adaptor proteins to positively regulate signalling. The tyrosine kinase binding (TKB) domain of this family is critical for binding with tyrosine-phosphorylated target proteins. Here, we analysed the crystal structure of the TKB domain of Cbl-c/Cbl-3 (Cbl-c TKB), which is a distinct member of the mammalian Cbl-family. In comparison with Cbl TKB, Cbl-c TKB showed restricted structural flexibility upon phosphopeptide binding. A mutation in Cbl-c TKB augmenting this flexibility enhanced its binding to target phosphoproteins. These results suggest that proteins, post-translational modifications or mutations that alter structural flexibility of the TKB domain of Cbl-family proteins could regulate their binding to target phosphoproteins and thereby, affect PTK-mediated signalling.

Counteracting Effects Operating on Src Homology 2 Domain-containing Protein-tyrosine Phosphatase 2 (SHP2) Function Drive Selection of the Recurrent Y62D and Y63C Substitutions in Noonan Syndrome*♦

Activating mutations in PTPN11 cause Noonan syndrome, the most common nonchromosomal disorder affecting development and growth. PTPN11 encodes SHP2, an Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase that positively modulates RAS function. Here, we characterized functionally all possible amino acid substitutions arising from single-base changes affecting codons 62 and 63 to explore the molecular mechanisms lying behind the largely invariant occurrence of the Y62D and Y63C substitutions recurring in Noonan syndrome. We provide structural and biochemical data indicating that the autoinhibitory interaction between the N-SH2 and protein-tyrosine phosphatase (PTP) domains is perturbed in both mutants as a result of an extensive structural rearrangement of the N-SH2 domain. Most mutations affecting Tyr(63) exerted an unpredicted disrupting effect on the structure of the N-SH2 phosphopeptide-binding cleft mediating the interaction of SHP2 with signaling partners. Among all the amino acid changes affecting that codon, the disease-causing mutation was the only substitution that perturbed the stability of the inactive conformation of SHP2 without severely impairing proper phosphopeptide binding of N-SH2. On the other hand, the disruptive effect of the Y62D change on the autoinhibited conformation of the protein was balanced, in part, by less efficient binding properties of the mutant. Overall, our data demonstrate that the selection-by-function mechanism acting as driving force for PTPN11 mutations affecting codons 62 and 63 implies balancing of counteracting effects operating on the allosteric control of the function of SHP2.

BRCA1 tumor suppressor network: focusing on its tail

Germline mutations of the BRCA1 tumor suppressor gene are a major cause of familial breast and ovarian cancer. BRCA1 plays critical roles in the DNA damage response that regulates activities of multiple repair and checkpoint pathways for maintaining genome stability. The BRCT domains of BRCA1 constitute a phospho-peptide binding domain recognizing a phospho-SPxF motif (S, serine; P, proline; × varies; F, phenylalanine). The BRCT domains are frequently targeted by clinically important mutations and most of these mutations disrupt the binding surface of the BRCT domains to phosphorylated peptides. The BRCT domain and its capability to bind phosphorylated protein is required for the tumor suppressor function of BRCA1. Through its BRCT phospho-binding ability BRCA1 forms at least three mutually exclusive complexes by binding to phosphorylated proteins Abraxas, Bach1 and CTIP. The A, B and C complexes, at lease partially undertake BRCA1's role in mechanisms of cell cycle checkpoint and DNA repair that maintain genome stability, thus may play important roles in BRCA1's tumor suppressor function.

BRCA1 tumor suppressor network: focusing on its tail

Germline mutations of the BRCA1 tumor suppressor gene are a major cause of familial breast and ovarian cancer. BRCA1 plays critical roles in the DNA damage response that regulates activities of multiple repair and checkpoint pathways for maintaining genome stability. The BRCT domains of BRCA1 constitute a phospho-peptide binding domain recognizing a phospho-SPxF motif (S, serine; P, proline; × varies; F, phenylalanine). The BRCT domains are frequently targeted by clinically important mutations and most of these mutations disrupt the binding surface of the BRCT domains to phosphorylated peptides. The BRCT domain and its capability to bind phosphorylated protein is required for the tumor suppressor function of BRCA1. Through its BRCT phospho-binding ability BRCA1 forms at least three mutually exclusive complexes by binding to phosphorylated proteins Abraxas, Bach1 and CTIP. The A, B and C complexes, at lease partially undertake BRCA1's role in mechanisms of cell cycle checkpoint and DNA repair that maintain genome stability, thus may play important roles in BRCA1's tumor suppressor function.

Specific recognition of phosphorylated tail of H2AX by the tandem BRCT domains of MCPH1 revealed by complex structure

MCPH1 is especially important for linking chromatin remodeling to DNA damage response. It contains three BRCT (BRCA1-carboxyl terminal) domains. The N-terminal region directly binds with chromatin remodeling complex SWI-SNF, and the C-terminal BRCT2-BRCT3 domains (tandem BRCT domains) are involved in cellular DNA damage response. The MCPH1 gene associates with evolution of brain size, and its variation can cause primary microcephaly. In this study we solve the crystal structures of MCPH1 natural variant (A761) C-terminal tandem BRCT domains alone as well as in complex with γH2AX tail. Compared with other structures of tandem BRCT domains, the most significant differences lie in phosphopeptide binding pocket. Additionally, fluorescence polarization assays demonstrate that MCPH1 tandem BRCT domains show a binding selectivity on pSer +3 and prefer to bind phosphopeptide with free COOH-terminus. Taken together, our research provides new structural insights into BRCT-phosphopeptide recognition mechanism.

Regulation of lipid binding underlies the activation mechanism of class IA PI3-kinases

Somatic missense mutations in PIK3CA, which encodes the p110α catalytic subunit of phosphoinositide 3-kinases (PI3Ks), occur frequently in human cancers. Activating mutations spread across multiple domains, some of which are located at inhibitory contact sites formed with the regulatory subunit p85α. PIK3R1, which encodes p85α, also has activating somatic mutations. We find a strong correlation between lipid kinase and lipid binding activities, for both wild-type (WT) and a representative set of oncogenic mutant complexes of p110α/p85α. Lipid binding involves both electrostatic and hydrophobic interactions. Activation caused by a phosphorylated receptor tyrosine kinase (RTK) peptide binding to the p85α N-terminal SH2 domain (nSH2) induces lipid binding. This depends on the polybasic activation loop as well as a conserved hydrophobic motif in the C-terminal region of the kinase domain. The hotspot E545K mutant largely mimics the activated WT p110α. It shows the highest basal activity and lipid binding, and is not significantly activated by an RTK phosphopeptide. Both the hotspot H1047R mutant and rare mutations (C420R, M1043I, H1047L, G1049R and p85α-N564D) also show increased basal kinase activities and lipid binding. However, their activities are further enhanced by an RTK phosphopeptide to levels markedly exceeding that of activated WT p110α. Phosphopeptide binding to p110β/p85α and p110δ/p85α complexes also induces their lipid binding. We present a crystal structure of WT p110α complexed with the p85α inter-SH2 domain (iSH2) and the inhibitor PIK-108. Additional to the ATP-binding pocket, an unexpected, second PIK-108 binding site is observed in the kinase C-lobe. We show a global conformational change in p110α consistent with allosteric regulation of the kinase domain by nSH2. These findings broaden our understanding of the differential biological outputs exhibited by distinct types of mutations regarding growth factor dependence, and suggest a two-tier classification scheme relating p110α and p85α mutations with signalling potential.

Proteomic Screening Method for Phosphopeptide Motif Binding Proteins Using Peptide Libraries

Phosphopeptide binding domains mediate the directed and localized assembly of protein complexes essential to intracellular kinase signaling. To identify phosphopeptide binding proteins, we developed a proteomic screening method using immobilized partially degenerate phosphopeptide mixtures combined with SILAC and microcapillary LC-MS/MS. The method was used to identify proteins that specifically bound to phosphorylated peptide library affinity matrices, including pTyr, and the motifs pSer/pThr-Pro, pSer/pThr-X-X-X-pSer/pThr, pSer/pThr-Glu/Asp, or pSer/pThr-pSer/pThr in degenerate sequence contexts. Heavy and light SILAC lysates were applied to columns containing these phosphorylated and nonphosphorylated (control) peptide libraries respectively, and bound proteins were eluted, combined, digested, and analyzed by LC-MS/MS using a hybrid quadrupole-TOF mass spectrometer. Heavy/light peptide ion ratios were calculated, and peptides that yielded ratios greater than ∼3:1 were considered as being from potential phosphopeptide binding proteins since this ratio represents the lowest ratio from a known positive control. Many of those identified were known phosphopeptide-binding proteins, including the SH2 domain containing p85 subunit of PI3K bound to pTyr, 14-3-3 bound to pSer/pThr-Asp/Glu, polo-box domain containing PLK1 and Pin1 bound to pSer/pThr-Pro, and pyruvate kinase M2 binding to pTyr. Approximately half of the hits identified by the peptide library screens were novel. Protein domain enrichment analysis revealed that most pTyr hits contain SH2 domains, as expected, and to a lesser extent SH3, C1, STAT, Tyr phosphatase, Pkinase, C2, and PH domains; however, pSer/pThr motifs did not reveal enriched domains across hits.

Enhancing the Identification of Phosphopeptides from Putative Basophilic Kinase Substrates Using Ti (IV) Based IMAC Enrichment

Metal and metal oxide chelating-based phosphopeptide enrichment technologies provide powerful tools for the in-depth profiling of phosphoproteomes. One weakness inherent to current enrichment strategies is poor binding of phosphopeptides containing multiple basic residues. The problem is exacerbated when strong cation exchange (SCX) is used for pre-fractionation, as under low pH SCX conditions phosphorylated peptides with multiple basic residues elute with the bulk of the tryptic digest and therefore require more stringent enrichment. Here, we report a systematic evaluation of the characteristics of a novel phosphopeptide enrichment approach based on a combination of low pH SCX and Ti(4+)-immobilized metal ion affinity chromatography (IMAC) comparing it one-to-one with the well established low pH SCX-TiO(2) enrichment method. We also examined the effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFP), trifluoroacetic acid (TFA), or 2,5-dihydroxybenzoic acid (DHB) in the loading buffer, as it has been hypothesized that high levels of TFA and the perfluorinated solvent HFP improve the enrichment of phosphopeptides containing multiple basic residues. We found that Ti(4+)-IMAC in combination with TFA in the loading buffer, outperformed all other methods tested, enabling the identification of around 5000 unique phosphopeptides containing multiple basic residues from 400 μg of a HeLa cell lysate digest. In comparison, ∼ 2000 unique phosphopeptides could be identified by Ti(4+)-IMAC with HFP and close to 3000 by TiO(2). We confirmed, by motif analysis, the basic phosphopeptides enrich the number of putative basophilic kinases substrates. In addition, we performed an experiment using the SCX/Ti(4+)-IMAC methodology alongside the use of collision-induced dissociation (CID), higher energy collision induced dissociation (HCD) and electron transfer dissociation with supplementary activation (ETD) on considerably more complex sample, consisting of a total of 400 μg of triple dimethyl labeled MCF-7 digest. This analysis led to the identification of over 9,000 unique phosphorylation sites. The use of three peptide activation methods confirmed that ETD is best capable of sequencing multiply charged peptides. Collectively, our data show that the combination of SCX and Ti(4+)-IMAC is particularly advantageous for phosphopeptides with multiple basic residues.

Structural mechanism associated with domain opening in gain‐of‐function mutations in SHP2 phosphatase

The SHP2 phosphatase plays a central role in a number of signaling pathways were it dephosphorylates various substrate proteins. Regulation of SHP2 activity is, in part, achieved by an intramolecular interaction between the PTP domain of the protein, which contains the catalytic site, and the N-SH2 domain leading to a "closed" protein conformation and autoinhibition. Accordingly, "opening" of the N-SH2 and PTP domains is required for the protein to become active. Binding of phosphopeptides to the N-SH2 domain is known to induce the opening event, while a number of gain-of-function (GOF) mutants, implicated in Noonan's Syndrome and childhood leukemias, are thought to facilitate opening. In the present study, a combination of computational and experimental methods are used to investigate the structural mechanism of opening of SHP2 and the impact of three GOF mutants, D61G, E76K, and N308D, on the opening mechanism. Calculated free energies of opening indicate that opening must be facilitated by effector molecules, possibly the protein substrates themselves, as the calculated free energies preclude spontaneous opening. Simulations of both wild type (WT) SHP2 and GOF mutants in the closed state indicate GOF activity to involve increased solvent exposure of selected residues, most notably Arg362, which in turn may enhance interactions of SHP2 with its substrate proteins and thereby aid opening. In addition, GOF mutations cause structural changes in the phosphopeptide-binding region of the N-SH2 domain leading to conformations that mimic the bound state. Such conformational changes are suggested to enhance binding of phosphopeptides and/or decrease interactions between the PTP and N-SH2 domains thereby facilitating opening. Experimental assays of the impact of effector molecules on SHP2 phosphatase activity against both small molecule and peptide substrates support the hypothesized mechanism of GOF mutant action. The present calculations also suggest a role for the C-SH2 domain of SHP2 in stabilizing the overall conformation of the protein in the open state, thereby aiding conformational switching between the open active and closed inactive states.

FHA Domain pThr Binding Specificity: It's All about Me

The FHA domain is a phospho-peptide binding module involved in a wide range of cellular pathways, with a striking specificity for phospho-threonine over phospho-serine binding partners. Biochemical, structural, and dynamic simulations analysis allowed Pennell and colleagues to unravel the molecular basis of FHA domain phospho-threonine specificity.

PTMScout, a Web Resource for Analysis of High Throughput Post-translational Proteomics Studies

The rate of discovery of post-translational modification (PTM) sites is increasing rapidly and is significantly outpacing our biological understanding of the function and regulation of those modifications. To help meet this challenge, we have created PTMScout, a web-based interface for viewing, manipulating, and analyzing high throughput experimental measurements of PTMs in an effort to facilitate biological understanding of protein modifications in signaling networks. PTMScout is constructed around a custom database of PTM experiments and contains information from external protein and post-translational resources, including gene ontology annotations, Pfam domains, and Scansite predictions of kinase and phosphopeptide binding domain interactions. PTMScout functionality comprises data set comparison tools, data set summary views, and tools for protein assignments of peptides identified by mass spectrometry. Analysis tools in PTMScout focus on informed subset selection via common criteria and on automated hypothesis generation through subset labeling derived from identification of statistically significant enrichment of other annotations in the experiment. Subset selection can be applied through the PTMScout flexible query interface available for quantitative data measurements and data annotations as well as an interface for importing data set groupings by external means, such as unsupervised learning. We exemplify the various functions of PTMScout in application to data sets that contain relative quantitative measurements as well as data sets lacking quantitative measurements, producing a set of interesting biological hypotheses. PTMScout is designed to be a widely accessible tool, enabling generation of multiple types of biological hypotheses from high throughput PTM experiments and advancing functional assignment of novel PTM sites. PTMScout is available at http://ptmscout.mit.edu.

Protein-tyrosine Phosphatase SHP2 Contributes to GDNF Neurotrophic Activity through Direct Binding to Phospho-Tyr687 in the RET Receptor Tyrosine Kinase

The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr(687) in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr(687) and association with components of the Tyr(1062) signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser(696), a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr(687) as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions.

Crystal structure of the N-terminal region of human Topoisomerase IIβ binding protein 1

Human DNA Topoisomerase IIβ binding protein 1 (TopBP1) is a modulating protein that plays an essential role in the response to DNA damage. The N-terminal region of TopBP1, which contains predicted BRCA1-carboxy terminal (BRCT) domains 1 and 2, binds to Rad9, a component of the cell cycle checkpoint clamp Rad9-Hus1-Rad1 complex. Here, we report the crystal structure of the TopBP1 N-terminal region (residues 1–290) at 2.4 Å resolution. Interestingly, in addition to the predicted tandem BRCT1–2 repeats (residues 103–284), residues 7–98 form a previously unreported BRCT domain (here, BRCT0). In contrast to both BRCT1 and BRCT2, which possess the conventional phosphopeptide binding residues within a surface pocket, the corresponding pocket in BRCT0 is largely hydrophobic. Structural comparisons together with peptide binding studies indicate that the tandem BRCT1–2 domains are the binding region for phosphorylated Ser387 in Rad9.

Computational Analysis of Phosphopeptide Binding to the Polo-Box Domain of the Mitotic Kinase PLK1 Using Molecular Dynamics Simulation

The Polo-Like Kinase 1 (PLK1) acts as a central regulator of mitosis and is over-expressed in a wide range of human tumours where high levels of expression correlate with a poor prognosis. PLK1 comprises two structural elements, a kinase domain and a polo-box domain (PBD). The PBD binds phosphorylated substrates to control substrate phosphorylation by the kinase domain. Although the PBD preferentially binds to phosphopeptides, it has a relatively broad sequence specificity in comparison with other phosphopeptide binding domains. We analysed the molecular determinants of recognition by performing molecular dynamics simulations of the PBD with one of its natural substrates, CDC25c. Predicted binding free energies were calculated using a molecular mechanics, Poisson-Boltzmann surface area approach. We calculated the per-residue contributions to the binding free energy change, showing that the phosphothreonine residue and the mainchain account for the vast majority of the interaction energy. This explains the very broad sequence specificity with respect to other sidechain residues. Finally, we considered the key role of bridging water molecules at the binding interface. We employed inhomogeneous fluid solvation theory to consider the free energy of water molecules on the protein surface with respect to bulk water molecules. Such an analysis highlights binding hotspots created by elimination of water molecules from hydrophobic surfaces. It also predicts that a number of water molecules are stabilized by the presence of the charged phosphate group, and that this will have a significant effect on the binding affinity. Our findings suggest a molecular rationale for the promiscuous binding of the PBD and highlight a role for bridging water molecules at the interface. We expect that this method of analysis will be very useful for probing other protein surfaces to identify binding hotspots for natural binding partners and small molecule inhibitors.

Structure of the DNA-bound BRCA1 C-terminal Region from Human Replication Factor C p140 and Model of the Protein-DNA Complex

BRCA1 C-terminal domain (BRCT)-containing proteins are found widely throughout the animal and bacteria kingdoms where they are exclusively involved in cell cycle regulation and DNA metabolism. Whereas most BRCT domains are involved in protein-protein interactions, a small subset has bona fide DNA binding activity. Here, we present the solution structure of the BRCT region of the large subunit of replication factor C bound to DNA and a model of the structure-specific complex with 5'-phosphorylated double-stranded DNA. The replication factor C BRCT domain possesses a large basic patch on one face, which includes residues that are structurally conserved and ligate the phosphate in phosphopeptide binding BRCT domains. An extra alpha-helix at the N terminus, which is required for DNA binding, inserts into the major groove and makes extensive contacts to the DNA backbone. The model of the protein-DNA complex suggests 5'-phosphate recognition by the BRCT domains of bacterial NAD(+)-dependent ligases and a nonclamp loading role for the replication factor C complex in DNA transactions.

Tomato 14-3-3 Protein 7 Positively Regulates Immunity-Associated Programmed Cell Death by Enhancing Protein Abundance and Signaling Ability of MAPKKK α

Programmed cell death (PCD) is triggered when Pto, a Ser-Thr protein kinase, recognizes either the AvrPto or AvrPtoB effector from Pseudomonas syringae pv tomato. This PCD requires mitogen-activated protein kinase kinase kinase (MAPKKK alpha ) as a positive regulator in tomato (Solanum lycopersicum) and Nicotiana benthamiana. To examine how PCD-eliciting activity of the tomato MAPKKK alpha protein is regulated, we screened for MAPKKK alpha -interacting proteins in tomato and identified a 14-3-3 protein, TFT7. Virus-induced gene silencing using the TFT7 gene in N. benthamiana compromised both Pto- and MAPKKK alpha -mediated PCD, and coexpression of TFT7 with tomato MAPKKK alpha enhanced MAPKKK alpha -mediated PCD. TFT7 was also required for PCD associated with several other disease resistance proteins and contributed to resistance against P. syringae pv tomato. Coexpression of TFT7 with MAPKKK alpha in vivo caused increased accumulation of the kinase and enhanced phosphorylation of two MAP kinases. TFT7 protein contains a phosphopeptide binding motif that is present in human 14-3-3 epsilon, and substitutions in this motif abolished interaction with MAPKKK alpha in vivo and also the PCD-enhancing activity of TFT7. A 14-3-3 binding motif, including a putative phosphorylated Ser-535, is present in the C-terminal region of MAPKKK alpha. An S535A substitution in MAPKKK alpha reduced interaction with TFT7 and both PCD-eliciting ability and stability of MAPKKK alpha. Our results provide new insights into a role for 14-3-3 proteins in regulating immunity-associated PCD pathways in plants.

Insights from the crystal structure of the sixth BRCT domain of topoisomerase IIβ binding protein 1

Topoisomerase IIbeta binding protein 1 (TopBP1) is a major player in the DNA damage response and interacts with a number of protein partners via its eight BRCA1 carboxy-terminal (BRCT) domains. In particular, the sixth BRCT domain of TopBP1 has been implicated in binding to the phosphorylated transcription factor, E2F1, and poly(ADP-ribose) polymerase 1 (PARP-1), where the latter interaction is responsible for the poly(ADP-ribosyl)ation of TopBP1. To gain a better understanding of the nature of TopBP1 BRCT6 interactions, we solved the crystal structure of BRCT6 to 1.34 A. The crystal structure reveals a degenerate phospho-peptide binding pocket and lacks conserved hydrophobic residues involved in packing of tandem BRCT repeats, which, together with results from phospho-peptide binding studies, strongly suggest that TopBP1 BRCT6 independently does not function as a phospho-peptide binding domain. We further provide insight into poly(ADP-ribose) binding and sites of potential modification by PARP-1.

A Supramodular FHA/BRCT-Repeat Architecture Mediates Nbs1 Adaptor Function in Response to DNA Damage

The Mre11/Rad50/Nbs1 protein complex plays central enzymatic and signaling roles in the DNA-damage response. Nuclease (Mre11) and scaffolding (Rad50) components of MRN have been extensively characterized, but the molecular basis of Nbs1 function has remained elusive. Here, we present a 2.3A crystal structure of the N-terminal region of fission yeast Nbs1, revealing an unusual but conserved architecture in which the FHA- and BRCT-repeat domains structurally coalesce. We demonstrate that diphosphorylated pSer-Asp-pThr-Asp motifs, recently identified as multicopy docking sites within Mdc1, are evolutionarily conserved Nbs1 binding targets. Furthermore, we show that similar phosphomotifs within Ctp1, the fission yeast ortholog of human CtIP, promote interactions with the Nbs1 FHA domain that are necessary for Ctp1-dependent resistance to DNA damage. Finally, we establish that human Nbs1 interactions with Mdc1 occur through both its FHA- and BRCT-repeat domains, suggesting how their structural and functional interdependence underpins Nbs1 adaptor functions in the DNA-damage response.