Protein Determinants of Meiotic DNA Break Hot Spots

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Highlights? DSB hot spots for meiotic recombination are specified by largely unknown mechanisms ? Linear element proteins, required for most DSBs, are highly enriched at hot spots ? These proteins, the first known global hot spot determinants, bind without DSB formation ? Hot spot determination by these proteins provides a paradigm for crossover control     

Binding Hot Spots and Amantadine Orientation in the Influenza A Virus M2 Proton Channel

Structures of truncated versions of the influenza A virus M2 proton channel have been determined recently by x-ray crystallography in the open conformation of the channel, and by NMR in the closed state. The structures differ in the position of the bound inhibitors. The x-ray structure shows a single amantadine molecule in the middle of the channel, whereas in the NMR structure four drug molecules bind at the channel's outer surface. To study this controversy we applied computational solvent mapping, a technique developed for the identification of the most druggable binding hot spots of proteins. The method moves molecular probes—small organic molecules containing various functional groups—around the protein surface, finds favorable positions using empirical free energy functions, clusters the conformations, and ranks the clusters on the basis of the average free energy. The results of the mapping show that in both structures the primary hot spot is an internal cavity overlapping the amantadine binding site seen in the x-ray structure. However, both structures also have weaker hot spots at the exterior locations that bind rimantadine in the NMR structure, although these sites are partially due to the favorable interactions with the interfacial region of the lipid bilayer. As confirmed by docking calculations, the open channel binds amantadine at the more favorable internal site, in good agreement with the x-ray structure. In contrast, the NMR structure is based on a peptide/micelle construct that is able to accommodate the small molecular probes used for the mapping, but has a too narrow pore for the rimantadine to access the internal hot spot, and hence the drug can bind only at the exterior sites.     

Structure-Based Predictive Models for Allosteric Hot Spots

In allostery, a binding event at one site in a protein modulates the behavior of a distant site. Identifying residues that relay the signal between sites remains a challenge. We have developed predictive models using support-vector machines, a widely used machine-learning method. The training data set consisted of residues classified as either hotspots or non-hotspots based on experimental characterization of point mutations from a diverse set of allosteric proteins. Each residue had an associated set of calculated features. Two sets of features were used, one consisting of dynamical, structural, network, and informatic measures, and another of structural measures defined by Daily and Gray [1]. The resulting models performed well on an independent data set consisting of hotspots and non-hotspots from five allosteric proteins. For the independent data set, our top 10 models using Feature Set 1 recalled 68–81% of known hotspots, and among total hotspot predictions, 58–67% were actual hotspots. Hence, these models have precision P = 58–67% and recall R = 68–81%. The corresponding models for Feature Set 2 had P = 55–59% and R = 81–92%. We combined the features from each set that produced models with optimal predictive performance. The top 10 models using this hybrid feature set had R = 73–81% and P = 64–71%, the best overall performance of any of the sets of models. Our methods identified hotspots in structural regions of known allosteric significance. Moreover, our predicted hotspots form a network of contiguous residues in the interior of the structures, in agreement with previous work. In conclusion, we have developed models that discriminate between known allosteric hotspots and non-hotspots with high accuracy and sensitivity. Moreover, the pattern of predicted hotspots corresponds to known functional motifs implicated in allostery, and is consistent with previous work describing sparse networks of allosterically important residues.     

Hot Spots, Cold Spots, and the Geographic Mosaic Theory of Coevolution

Species interactions commonly coevolve as complex geographic mosaics of populations shaped by differences in local selection and gene flow. We use a haploid matching-alleles model for coevolution to evaluate how a pair of species coevolves when fitness interactions are reciprocal in some locations ("hot spots") but not in others ("cold spots"). Our analyses consider mutualistic and antagonistic interspecific interactions and a variety of gene flow patterns between hot and cold spots. We found that hot and cold spots together with gene flow influence coevolutionary dynamics in four important ways. First, hot spots need not be ubiquitous to have a global influence on evolution, although rare hot spots will not have a disproportionate impact unless selection is relatively strong there. Second, asymmetries in gene flow can influence local adaptation, sometimes creating stable equilibria at which species experience minimal fitness in hot spots and maximal fitness in cold spots, or vice versa. Third, asymmetries in gene flow are no more important than asymmetries in population regulation for determining the maintenance of local polymorphisms through coevolution. Fourth, intraspecific allele frequency differences among hot and cold spot populations evolve under some, but not all, conditions. That is, selection mosaics are indeed capable of producing spatially variable coevolutionary outcomes across the landscapes over which species interact. Altogether, our analyses indicate that coevolutionary trajectories can be strongly shaped by the geographic distribution of coevolutionary hot and cold spots, and by the pattern of gene flow among populations.     

Preparation and Characterization of Monoclonal Antibodies to Serotonin Binding Protein

Serotonin binding protein (SBP) is a constituent of the synaptic vesicles of serotonergic neurons. Two types of SBP, with molecular masses of 45 kDa and 56 kDa, have been purified. To determine whether there are shared epitopes between the two forms of SBP, we raised and tested for cross-reactivity monoclonal antibodies (MAbs) against each form of SBP. We obtained 12 MAbs, all of which recognize both forms of SBP. Hybridoma clones were produced by fusing P3 X 63Ag8.653 mouse myeloma cells with spleen cells from a mouse that had been immunized with 45-kDa or 56-kDa SBP. Culture supernatants were screened for the presence of anti-SBP antibodies. MAb isotypes were determined by immunodiffusion, using immunoglobulin type-specific antisera. Each antibody to SBP consisted of only a single subclass of immunoglobulin (IgM). We obtained 12 MAbs, each of which interacted with both forms of SBP, as judged by enzyme-linked immunosorbent assay and immunoblot analysis. Ascites fluid to one clone (44-10) was obtained and affinity-purified. In the presence of goat anti-mouse IgM, the partially purified 44-10 antibodies quantitatively immunoprecipitated SBP from crude brain extracts. Immunoblotting revealed two major bands corresponding to 45 kDa and 56 kDa and a minor band corresponding to 68 kDa. MAb 44-10 blocked the binding of [3H]serotonin ([3H]5-HT) to 45-kDa and 56-kDa SBP in a concentration-dependent manner. The 68-kDa protein was found to bind [3H]5-HT. Sites reacting with MAB 44-10 were located immunocytochemically in sections of rat brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

豌豆核酮糖1,5-二磷酸羧化酶/加氧酶亚基结合蛋白的纯化及特性

用~(35)S-Met在照光下与豌豆完整叶绿体保温,显示新合成的标记的RubisCO大亚基与结合蛋白形成一复合物,经ATP处理后解离为结合蛋白亚基,同时释放出的标记的RubisCO大亚基参与了RubisCO的装配。豌豆叶片提取液经热处理,硫酸铵分部,DEAE-Sepharose fast flow和Sephacryl S-300柱层析在ND-PAGE,SDS-PAGE上显示为一条带,估计纯度达90％以上,得率比以前报道的高12倍。纯化的结合蛋白表面巯基数经测定为12±1个,总巯基数为36±1个。远紫外CD光谱具有典型的α-螺旋结构的光谱特性,α-螺旋度为39％。此外,以纯化的豌豆结合蛋白制备了多克隆抗体。琼脂糖双扩散实验显示,结合蛋白的抗体与结合蛋白产生一条沉淀线,而与豌豆的RubisCO无沉淀反应,这表明所得到的抗体是高度专一的。     

Characterization of a Sulfoglucuronyl Carbohydrate Binding Protein in the Developing Nervous System

Abstract: The developmentally regulated and stage-specifically expressed HNK-1 carbohydrate found on sulfoglucuronylglycolipids (SGGLs) and certain glycoproteins has been proposed to be involved in neural cell adhesion and recognition processes through its interaction with protein "receptors." We have isolated and purified a ∼30-kDa SGGL-binding protein (SBP-1) from neonatal rat brain. SBP-1 specifically bound to SGGLs and sulfatide both in solid-phase immunobinding and high-performance thin-layer chromatography-immunooverlay assays. N-terminal sequence analysis showed that SBP-1 is similar to an adhesive neurite outgrowth promoting protein amphoterin. Desulfation of SGGLs resulted in abolition of SBP-1 binding. However, chemical modification of glucuronic acid moiety by either esterification or reduction of the carboxyl group had no effect, suggesting requirement of the carbohydrate-linked sulfate group for SBP-1 binding. The binding of SBP-1 to SGGLs was specifically inhibited by HNK-1 antibody but not by other IgM antibodies. The binding of SBP-1 to sulfatide, however, was not inhibited by HNK-1 antibody. Heparin, fucoidan, and dextran sulfate (50K) also inhibited the binding of SBP-1 to SGGLs. During development of the rat cerebral cortex, the level of SBP-1 decreased after embryonic day 18 to an almost undetectable level by postnatal day 10; whereas in the cerebellum, the expression of SBP-1 was maximal at postnatal day 7. SBP-1 also bound specifically to the HNK-1 glycoproteins isolated from rat brain by HNK-1 immunoaffinity chromatography. Proteins without HNK-1 carbohydrate did not bind SBP-1. The binding to HNK-1 glycoproteins was inhibited by HNK-1 antibody, but not by other IgM antibodies, indicating that the binding was mediated through the HNK-1 carbohydrate moiety of the proteins. The interaction and coexpression of SBP-1 with SGGLs and HNK-1 glycoproteins, during the perinatal brain development, suggest a functional role for this protein.     

Molecular Determinants Underlying Binding Specificities of the ABL Kinase Inhibitors: Combining Alanine Scanning of Binding Hot Spots with Network Analysis of Residue Interactions and Coevolution

Quantifying binding specificity and drug resistance of protein kinase inhibitors is of fundamental importance and remains highly challenging due to complex interplay of structural and thermodynamic factors. In this work, molecular simulations and computational alanine scanning are combined with the network-based approaches to characterize molecular determinants underlying binding specificities of the ABL kinase inhibitors. The proposed theoretical framework unveiled a relationship between ligand binding and inhibitor-mediated changes in the residue interaction networks. By using topological parameters, we have described the organization of the residue interaction networks and networks of coevolving residues in the ABL kinase structures. This analysis has shown that functionally critical regulatory residues can simultaneously embody strong coevolutionary signal and high network centrality with a propensity to be energetic hot spots for drug binding. We have found that selective (Nilotinib) and promiscuous (Bosutinib, Dasatinib) kinase inhibitors can use their energetic hot spots to differentially modulate stability of the residue interaction networks, thus inhibiting or promoting conformational equilibrium between inactive and active states. According to our results, Nilotinib binding may induce a significant network-bridging effect and enhance centrality of the hot spot residues that stabilize structural environment favored by the specific kinase form. In contrast, Bosutinib and Dasatinib can incur modest changes in the residue interaction network in which ligand binding is primarily coupled only with the identity of the gate-keeper residue. These factors may promote structural adaptability of the active kinase states in binding with these promiscuous inhibitors. Our results have related ligand-induced changes in the residue interaction networks with drug resistance effects, showing that network robustness may be compromised by targeted mutations of key mediating residues. This study has outlined mechanisms by which inhibitor binding could modulate resilience and efficiency of allosteric interactions in the kinase structures, while preserving structural topology required for catalytic activity and regulation.     

Identification and Characterization of the Fourth Single-Stranded-DNA Binding Domain of Replication Protein A

Replication protein A (RPA), the heterotrimeric single-stranded-DNA (ssDNA) binding protein (SSB) of eukaryotes, contains two homologous ssDNA binding domains (A and B) in its largest subunit, RPA1, and a third domain in its second-largest subunit, RPA2. Here we report that Saccharomyces cerevisiae RPA1 contains a previously undetected ssDNA binding domain (domain C) lying in tandem with domains A and B. The carboxy-terminal portion of domain C shows sequence similarity to domains A and B and to the region of RPA2 that binds ssDNA (domain D). The aromatic residues in domains A and B that are known to stack with the ssDNA bases are conserved in domain C, and as in domain A, one of these is required for viability in yeast. Interestingly, the amino-terminal portion of domain C contains a putative Cys₄-type zinc-binding motif similar to that of another prokaryotic SSB, T4 gp32. We demonstrate that the ssDNA binding activity of domain C is uniquely sensitive to cysteine modification but that, as with gp32, ssDNA binding is not strictly dependent on zinc. The RPA heterotrimer is thus composed of at least four ssDNA binding domains and exhibits features of both bacterial and phage SSBs.     

Crystallization and Characterization of Pumilio: A Novel RNA Binding Protein

Axis determination in early Drosophila embryos is controlled, in part, by regulation of translation of mRNAs transcribed in maternal cells during oogenesis. The Pumilio protein is essential in posterior determination, binding to hunchback mRNA in complex with Nanos to suppress hunchback translation. In order to understand the structural basis of RNA binding, Nanos recruitment, and translational control, we have crystallized a domain of the Drosophila Pumilio protein that binds RNA. The crystals belong to the space group P6₃ with unit cell dimensions of a = b = 94.5 Å, c = 228.9 Å, α = β = 90°, γ = 120° and diffract to 2.6 Å with synchrotron radiation. We show that the purified protein actively binds RNA and is likely to have a novel RNA binding fold due to a very high content of α-helical secondary structure.     

Presaging Critical Residues in Protein interfaces-Web Server (PCRPi-W): A Web Server to Chart Hot Spots in Protein Interfaces

Background

It is well established that only a portion of residues that mediate protein-protein interactions (PPIs), the so-called hot spot, contributes the most to the total binding energy, and thus its identification is an important and relevant question that has clear applications in drug discovery and protein design. The experimental identification of hot spots is however a lengthy and costly process, and thus there is an interest in computational tools that can complement and guide experimental efforts.

Principal Findings

Here, we present Presaging Critical Residues in Protein interfaces-Web server (http://www.bioinsilico.org/PCRPi), a web server that implements a recently described and highly accurate computational tool designed to predict critical residues in protein interfaces: PCRPi. PRCPi depends on the integration of structural, energetic, and evolutionary-based measures by using Bayesian Networks (BNs).

Conclusions

PCRPi-W has been designed to provide an easy and convenient access to the broad scientific community. Predictions are readily available for download or presented in a web page that includes among other information links to relevant files, sequence information, and a Jmol applet to visualize and analyze the predictions in the context of the protein structure.     

Combinatorics of Contacts in Protein Contact Maps

Contacts play a fundamental role in the study of protein structure and folding problems. The contact map of a protein can be represented by arranging its amino acids on a horizontal line and drawing an arc between two residues if they form a contact. In this paper, we are mainly concerned with the combinatorial enumeration of the arcs in m-regular linear stack, an elementary structure of the protein contact map, which was introduced by Chen et al. (J Comput Biol 21(12):915–935, 2014). We modify the generating function for m-regular linear stacks by introducing a new variable y regarding to the number of arcs and obtain an equation satisfied by the generating function for m-regular linear stacks with n vertices and k arcs. Consequently, we also derive an equation satisfied by the generating function of the overall number of arcs in m-regular linear stacks with n vertices.     

Characterization of the Single-Stranded DNA Binding Protein Encoded by the Vaccinia Virus I3 Gene

The 34-kDa protein encoded by the I3 gene of vaccinia virus is expressed at early and intermediate times postinfection and is phosphorylated on serine residues. Recombinant I3 has been expressed in Escherichia coli and purified to near homogeneity, as has the protein from infected cells. Both recombinant and endogenous I3 protein demonstrate a striking affinity for single-stranded, but not for double-stranded, DNA. The interaction with DNA is resistant to salt, exhibits low cooperativity, and appears to involve a binding site of approximately 10 nucleotides. Electrophoretic mobility shift assays indicate that numerous I3 molecules can bind to a template, reflecting the stoichiometric interaction of I3 with DNA. Sequence analysis reveals that a pattern of aromatic and charged amino acids common to many replicative single-stranded DNA binding proteins (SSBs) is conserved in I3. The inability to isolate viable virus containing an interrupted I3 allele provides strong evidence that the I3 protein plays an essential role in the viral life cycle. A likely role for I3 as an SSB involved in DNA replication and/or repair is discussed.     

Ligand Binding Mechanics of Maltose Binding Protein

In the past decade, single-molecule force spectroscopy has provided new insights into the key interactions stabilizing folded proteins. A few recent studies probing the effects of ligand binding on mechanical protein stability have come to quite different conclusions. While some proteins seem to be stabilized considerably by a bound ligand, others appear to be unaffected. Since force acts as a vector in space, it is conceivable that mechanical stabilization by ligand binding is dependent on the direction of force application. In this study, we vary the direction of the force to investigate the effect of ligand binding on the stability of maltose binding protein (MBP). MBP consists of two lobes connected by a hinge region that move from an open to a closed conformation when the ligand maltose binds. Previous mechanical experiments, where load was applied to the N and C termini, have demonstrated that MBP is built up of four building blocks (unfoldons) that sequentially detach from the folded structure. In this study, we design the pulling direction so that force application moves the two MBP lobes apart along the hinge axis. Mechanical unfolding in this geometry proceeds via an intermediate state whose boundaries coincide with previously reported MBP unfoldons. We find that in contrast to N-C-terminal pulling experiments, the mechanical stability of MBP is increased by ligand binding when load is applied to the two lobes and force breaks the protein-ligand interactions directly. Contour length measurements indicate that MBP is forced into an open conformation before unfolding even if ligand is bound. Using mutagenesis experiments, we demonstrate that the mechanical stabilization effect is due to only a few key interactions of the protein with its ligand. This work illustrates how varying the direction of the applied force allows revealing important details about the ligand binding mechanics of a large protein.     

Architecture of Protein and DNA Contacts within the TFIIIB-DNA Complex

The RNA polymerase III factor TFIIIB forms a stable complex with DNA and can promote multiple rounds of initiation by polymerase. TFIIIB is composed of three subunits, the TATA binding protein (TBP), TFIIB-related factor (BRF), and B". Chemical footprinting, as well as mutagenesis of TBP, BRF, and promoter DNA, was used to probe the architecture of TFIIIB subunits bound to DNA. BRF bound to TBP-DNA through the nonconserved C-terminal region and required 15 bp downstream of the TATA box and as little as 1 bp upstream of the TATA box for stable complex formation. In contrast, formation of complete TFIIIB complexes required 15 bp both upstream and downstream of the TATA box. Hydroxyl radical footprinting of TFIIIB complexes and modeling the results to the TBP-DNA structure suggest that BRF and B" surround TBP on both faces of the TBP-DNA complex and provide an explanation for the exceptional stability of this complex. Competition for binding to TBP by BRF and either TFIIB or TFIIA suggests that BRF binds on the opposite face of the TBP-DNA complex from TFIIB and that the binding sites for TFIIA and BRF overlap. The positions of TBP mutations which are defective in binding BRF suggest that BRF binds to the top and N-terminal leg of TBP. One mutation on the N-terminal leg of TBP specifically affects the binding of the B" subunit.