Determinants Of Binding Specificity Research Articles

Methods of Cryptococcal Polysaccharide Analysis Using ELISA.

One of the standard assays for the fungal pathogen Cryptococcus neoformans is the glucuronoxylomannan (GXM) ELISA. This assay utilizes monoclonal antibodies targeted against the critical virulence factor, the polysaccharide (PS) capsule. GXM ELISA is one of the most used assays in the field used for diagnosis of cryptococcal infection, quantification of PS content, and determination of binding specificity for antibodies. Here we present three variations of the GXM ELISA used by our group-indirect, capture, and competition ELISAs. We have also provided some history, perspective, and notes on these methods, which we hope will help the reader choose, and implement, the best assay for their research.While it has long been referred to as the GXM ELISA, we also suggest a name update to better reflect our updated understanding of the polysaccharide antigens targeted by this assay. The Cryptococcal PS ELISA is a more accurate description of this set of methodologies and the antigens they measure. Finally, we discuss the limitations of this assay and put forth future plans for expanding the antigens assayed by ELISA.

The c-di-AMP-binding protein CbpB modulates the level of ppGpp alarmone in Streptococcus agalactiae.

Cyclic di-AMP is an essential signalling molecule in Gram-positive bacteria. This second messenger regulates the osmotic pressure of the cell by interacting directly with the regulatory domains, either RCK_C or CBS domains, of several potassium and osmolyte uptake membrane protein systems. Cyclic di-AMP also targets stand-alone CBS domain proteins such as DarB in Bacillus subtilis and CbpB in Listeria monocytogenes. We show here that the CbpB protein of Group B Streptococcus binds c-di-AMP with a very high affinity. Crystal structures of CbpB reveal the determinants of binding specificity and significant conformational changes occurring upon c-di-AMP binding. Deletion of the cbpB gene alters bacterial growth in low potassium conditions most likely due to a decrease in the amount of ppGpp caused by a loss of interaction between CbpB and Rel, the GTP/GDP pyrophosphokinase.

Molecular Determinants of Tissue Specificity of Flavivirus Nonstructural Protein 1 Interaction with Endothelial Cells.

Members of the mosquito-borne flavivirus genus such as dengue (DENV), West Nile (WNV), and Zika (ZIKV) viruses cause distinct diseases and affect different tissues. We previously found that the secreted flaviviral nonstructural protein 1 (NS1) interacts with endothelial cells and disrupts endothelial barrier function in a tissue-specific manner consistent with the disease tropism of the respective viruses. However, the underlying molecular mechanism of this tissue-specific NS1-endothelial cell interaction is not well understood. To elucidate the distinct role(s) that the wing and β-ladder domains of NS1 play in NS1 interactions with endothelial cells, we constructed flavivirus NS1 chimeras that exchanged the wing and β-ladder domains in a pairwise manner between DENV, WNV, and ZIKV NS1. We found that both the NS1 wing and β-ladder domains conferred NS1 tissue-specific endothelial dysfunction, with the wing conferring cell binding and the β-ladder involved in inducing endothelial hyperpermeability as measured by transendothelial electrical resistance. To narrow down the amino acids dictating cell binding specificity, we utilized the DENV-WNV NS1 chimera and identified residues 91 to 93 (GDI) of DENV NS1 as a molecular motif determining binding specificity. Further, using an in vivo mouse model of localized leak, we found that the GDI motif of the wing domain was essential for triggering DENV NS1-induced vascular leak in mouse dermis. Taken together, we identify molecular determinants of flavivirus NS1 that confer NS1 binding and vascular leak and highlight the importance of the NS1 wing domain for flavivirus pathogenesis. IMPORTANCE Flavivirus NS1 is secreted into the bloodstream from infected cells during a viral infection. Dengue virus NS1 contributes to severe dengue pathology such as endothelial dysfunction and vascular leak independently of the virus. We have shown that multiple flavivirus NS1 proteins result in endothelial dysfunction in a tissue-specific manner consistent with their respective viral tropism. Here, we aimed to identify the molecular determinants that make some, but not other, flavivirus NS1 proteins bind to select endothelial cells in vitro and cause vascular leak in a mouse model. We identified the wing domain of NS1 as a primary determinant conferring differential endothelial dysfunction and vascular leak and narrowed the contributing amino acid residues to a three-residue motif within the wing domain. The insights from this study pave the way for future studies on the effects of flavivirus NS1 on viral dissemination and pathogenesis and offer potential new avenues for antiviral therapies.

Molecular mechanism of methyl-dependent and spatial-specific DNA recognition of c-Jun homodimer.

DNA methylation is important in regulation of gene expression and normal development because it alters the interplay between protein and DNA. Experiments have shown that a single 5-methylcytosine at different CpG sites (mCpG) might have different effects on specific recognition, but the atomistic origin and dynamic details are largely unclear. In this work, we investigated the mechanism of monomethylation at different CpG sites in the cognate motif and the cooperativity of full methylation. By constructing four models of c-Jun/Jun protein binding to the 5[Formula: see text]-XGAGTCA-3[Formula: see text] (X represents C or methylated C) motif, we characterized the dynamics of the contact interface using the all-atom molecular dynamics method. Free energy analysis of MM/GBSA suggests that regardless of whether the C12pG13 site of the bottom strand is methylated, the effects from mC25 of the top strand are dominant and can moderately enhance the binding by [Formula: see text] 31 kcal/mol, whereas mC12 showed a relatively small contribution, in agreement with the experimental data. Remarkably, we found that this spatial-specific influence was induced by different regulatory rules. The influence of the mC25 site is mainly mediated by steric hindrance. The additional methyl group leads to the conformational changes in nearby residues and triggers an obvious structural bending in the protein, which results in the formation of a new T-Asn-C triad that enhances the specific recognition of TCA half-sites. The substitution of the methyl group at the mC12 site of the bottom strand breaks the original H-bonds directly. Such changes in electrostatic interactions also lead to the remote allosteric effects of protein by multifaceted interactions but have negligible contributions to binding. Although these two influence modes are different, they can both fine-tune the local environment, which might produce remote allosteric effects through protein-protein interactions. Further analysis reveals that the discrepancies in these two modes are primarily due to their location. Moreover, when both sites are methylated, the major determinant of binding specificity depends on the context and the location of the methylation site, which is the result of crosstalk and cooperativity.

Imprinted Particles for Direct Fluorescence Detection of Sialic Acid in Polar Media and on Cancer Cells with Enhanced Control of Nonspecific Binding

Glycoproteins are abundant on the cell surface of mammals, providing structural support, modulating cell membrane properties, and acting as signaling agents. Variation of glycosylation patterns has been found to indicate various disease states, including cell malignancy. Sialic acid (SA) is present as a terminating group on cell-surface glycans, and its overexpression has been linked to several types of cancer. Detection of SA on the cell surface is therefore critical for detection of cancer in its early stages. In this work, a fluorescent molecularly imprinted polymer layer targeting SA was synthesized on the surface of silica-coated polystyrene (PS) particles. Compared to previous works, a PS core supplies a lighter, lower-density support for improved suspension stability and scattering properties. Moreover, their smaller size provides a higher surface-area-to-volume ratio for binding. The incorporation of a fluorescent monomer in the MIP shell allowed for simple and rapid determination of binding specificity in polar media due to a deprotonation–reprotonation interaction mechanism between the fluorescent monomer and SA, which led to spectral changes. Upon titration of the MIP particles with SA in suspension, an increase in fluorescence emission of the particles was observed, with the MIP particles binding SA more selectively compared to the nonimprinted polymer (NIP) control particles. In cell staining experiments performed by flow cytometry, the binding behavior of the MIP particles compared favorably with that of SA-binding lectins. NIPs prepared with a “dummy” template served as a better negative control in cell binding assays due to the favorable inward orientation of template-binding functional groups in the polymer shell, which reduced nonspecific binding. The results show that fluorescent MIPs targeting SA are a promising tool for in vitro fluorescence staining of cancerous cells and for future diagnosis of cancer at early stages.

Learning peptide recognition rules for a low-specificity protein.

Many proteins interact with short linear regions of target proteins. For some proteins, however, it is difficult to identify a well-defined sequence motif that defines its target peptides. To overcome this difficulty, we used supervised machine learning to train a model that treats each peptide as a collection of easily-calculated biochemical features rather than as an amino acid sequence. As a test case, we dissected the peptide-recognition rules for human S100A5 (hA5), a low-specificity calcium binding protein. We trained a Random Forest model against a recently released, high-throughput phage display dataset collected for hA5. The model identifies hydrophobicity and shape complementarity, rather than polar contacts, as the primary determinants of peptide binding specificity in hA5. We tested this hypothesis by solving a crystal structure of hA5 and through computational docking studies of diverse peptides onto hA5. These structural studies revealed that peptides exhibit multiple binding modes at the hA5 peptide interface-all of which have few polar contacts with hA5. Finally, we used our trained model to predict new, plausible binding targets in the human proteome. This revealed a fragment of the protein α-1-syntrophin that binds to hA5. Our work helps better understand the biochemistry and biology of hA5, as well as demonstrating how high-throughput experiments coupled with machine learning of biochemical features can reveal the determinants of binding specificity in low-specificity proteins.

The ancillary N-terminal region of the yeast AP-1 transcription factor Yap8 contributes to its DNA binding specificity.

Activator protein 1 (AP-1) is one of the largest families of basic leucine zipper (bZIP) transcription factors in eukaryotic cells. How AP-1 proteins achieve target DNA binding specificity remains elusive. In Saccharomyces cerevisiae, the AP-1-like protein (Yap) family comprises eight members (Yap1 to Yap8) that display distinct genomic target sites despite high sequence homology of their DNA binding bZIP domains. In contrast to the other members of the Yap family, which preferentially bind to short (7–8 bp) DNA motifs, Yap8 binds to an unusually long DNA motif (13 bp). It has been unclear what determines this unique specificity of Yap8. In this work, we use molecular and biochemical analyses combined with computer-based structural design and molecular dynamics simulations of Yap8–DNA interactions to better understand the structural basis of DNA binding specificity determinants. We identify specific residues in the N-terminal tail preceding the basic region, which define stable association of Yap8 with its target promoter. We propose that the N-terminal tail directly interacts with DNA and stabilizes Yap8 binding to the 13 bp motif. Thus, beside the core basic region, the adjacent N-terminal region contributes to alternative DNA binding selectivity within the AP-1 family.

Control of Ligand-Binding Specificity Using Photocleavable Linkers in AFM Force Spectroscopy.

In recent decades, atomic force microscopy (AFM), in particular the force spectroscopy mode, has become a method of choice to study biomolecular interactions at the single-molecule level. However, grafting procedures as well as determining binding specificity remain challenging. We report here an innovative approach based on a photocleavable group that enables in situ release of the ligands bound to the AFM tip and thus allows direct assessment of the binding specificity. Applicable to a wide variety of molecules, the strategy presented here provides new opportunities to study specific interactions and deliver single molecules with high spatiotemporal resolution in a wide range of applications, including AFM-based cell biology.

Cul5-type Ubiquitin Ligase KLHDC1 Contributes to the Elimination of Truncated SELENOS Produced by Failed UGA/Sec Decoding.

SummaryThe UGA codon signals protein translation termination, but it can also be translated into selenocysteine (Sec, U) to produce selenocysteine-containing proteins (selenoproteins) by dedicated machinery. As Sec incorporation can fail, Sec-containing longer and Sec-lacking shorter proteins co-exist. Cul2-type ubiquitin ligases were recently shown to destabilize such truncated proteins; however, which ubiquitin ligase targets truncated proteins for degradation remained unclear. We report that the Cul5-type ubiquitin ligase KLHDC1 targets truncated SELENOS, a selenoprotein, for proteasomal degradation. SELENOS is involved in endoplasmic reticulum (ER)-associated degradation, which is linked to reactive oxygen species (ROS) production, and the knockdown of KLHDC1 in U2OS cells decreased ER stress-induced cell death. Knockdown of SELENOS increased the cell population with lower ROS levels. Our findings reveal that, in addition to Cul2-type ubiquitin ligases, KLHDC1 is involved in the elimination of truncated oxidoreductase-inactive SELENOS, which would be crucial for maintaining ROS levels and preventing cancer development.

Understanding Protein-DNA Recognition in the Context of Multi-Scale Genome Organization

Many structures of protein-DNA complexes have been solved and high-throughput binding assays were developed because structural biology and genomics researchers were equally puzzled by the question of how proteins bind DNA with high specificity. The understanding of protein-DNA binding has advanced through data generated by structural biology and genomics, and the combination of insights gained by both fields of research. Genomic data has become available for protein-DNA binding under cellular conditions. However, a mechanistic understanding of in vivo binding of transcription factors to DNA is still lacking. Different determinants for in vivo binding have been revealed but it is not clear to which extent each of those layers contributes to specificity [Slattery, et al. Absence of a simple code: how transcription factors read the genome. Trends Biochem. Sci. 39, 381-399 (2014)]. Moreover, these determinants play various roles for DNA binding of proteins from different families. Various layers of specificity relate to the genome organization in the nucleus at multiple scales. The factors discussed in this talk will include DNA methylation at the nucleotide scale [Rao, et al. Systematic prediction of DNA shape changes due to CpG methylation explains epigenetic effects on protein-DNA binding. Epigenetics Chromatin 11, 6 (2018)] and histone modifications at the nucleosome scale [Xin and Rohs. Relationship between histone modifications and transcription factor binding is protein family specific. Genome Res. 28, 321-333 (2018)]. The talk will also show how new computational such as deep learning can be used to study the impact of binding specificity determinants at multiple scales.

Abstract 4712: Selective polo-like kinase 1 PBD inhibitor

Abstract Polo Like Kinase 1 (PLK1) is only expressed in dividing cells and plays a critical role in several stages of mitosis. PLK1 is highly expressed in tumors of various origins while its expression is largely absent in normal tissues. PLK1 inhibition selectively kills cancer cells because cancer cells utterly depend on the mitotic functions of PLK1 overexpression. PLK1 consists of a highly conserved N-terminal catalytic kinase domain and a unique, functionally essential C-terminal Polo Box Domain (PBD). The PBD of each PLK is a phospho-peptide binding motif that determines substrate recognition and sub-cellular localization. PLK1 catalytic inhibitorshave advanced to clinical trials but they inhibit other kinases including family members PLK2 and PLK3. This is problematic because PLK3 is a tumor suppressor and partial inhibition of PLK3 could lead to a long-term malignant progression. Moreover, a single point mutation (C67V) in the PLK1 kinase domain confers substantial cancer cell resistance against PLK1 catalytic inhibitors. An alternative approach to developing potent and selective PLK1 inhibitors is to allosterically target the PBD. We generated fragment ligated inhibitory peptides (FLIPs) through a strategy called REPLACE. This is a computational and synthetic approach that uses structure activity relationships of peptide inhibitors to generate pharmaceutically acceptable lead molecules. Fragments are docked into the crystal structure of a truncated peptide/receptor complex, then prioritized for synthesis with the peptide and subsequently tested in an in vitro binding assay. PBD-interacting protein (PBIP) and Cdc25c are natural substrates of PLK1. We replaced individual amino acid residues in the PBIP (PLHSpTAI) and Cdc25C PBD substrate peptides (LLCSpTPNGL) with benzoic acid fragments. Here we show PLK1 specificity for PBD inhibitors in vitro. A fluorescent polarization (FP) competitive binding assay to the PBD of PLK1 was used to determine binding specificity. A PLK3 counter screen was used to determine the binding selectivity of our PBD inhibitors. To measure the PLK1 inhibitory activity of the FLIPs in cells, phosphorylation of mitotic proteins was measured. Candidate FLIPs induce mitotic arrest, block PLK1 mediated phosphorylation, and inhibit growth of human cancer cells. To make more drug-like molecules, we modified FLIPs to Small Molecule Inhibitors (SMIs). Further, we are optimizing SMIs to improve biochemical selectivity. SMIs bind to the PLK1 PBD as determined by FP assay. Also, SMIs inhibit cancer cell growth as determined by viability measurements and live cell imaging. Overall these results highlight that targeting the PBD of PLK1 has promise as an antitumor strategy. Citation Format: Danda P. Chapagai, Merissa Baxter, Gurusankar Ramamoorthy, Campbell McInnes, Michael Wyatt. Selective polo-like kinase 1 PBD inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4712.

Binding Specificity of Native Odorant-Binding Protein Isoforms Is Driven by Phosphorylation and O-N-Acetylglucosaminylation in the Pig Sus scrofa.

Odorant-binding proteins (OBP) are secreted in the nasal mucus at the vicinity of olfactory receptors (ORs). They act, at least, as an interface between hydrophobic and volatile odorant molecules and the hydrophilic medium bathing the ORs. They have also been hypothesized to be part of the molecular coding of odors and pheromones, by forming specific complexes with odorant molecules that could ultimately stimulate ORs to trigger the olfactory transduction cascade. In a previous study, we have evidenced that pig olfactory secretome was composed of numerous olfactory binding protein isoforms, generated by O-GlcNAcylation and phosphorylation. In addition, we have shown that recombinant OBP (stricto sensu) produced in yeast is made up of a mixture of isoforms that differ in their phosphorylation pattern, which in turn determines binding specificity. Taking advantage of the high amount of OBP secreted by a single animal, we performed a similar study, under exactly the same experimental conditions, on native isoforms isolated from pig, Sus scrofa, nasal tissue. Four fractions were obtained by using strong anion exchange HPLC. Mapping of phosphorylation and O-GlcNAcylation sites by CID-nanoLC-MS/MS allowed unambiguous localization of phosphosites at S13 and T122 and HexNAc sites at S13 and S19. T112 or T115 could also be phosphorylated. BEMAD analysis suggested extra phosphosites located at S23, S24, S41, S49, S57, S67, and T71. Due to the very low stoichiometry of GlcNAc-peptides and phosphopeptides, these sites were identified on total mixture of OBP isoforms instead of HPLC-purified OBP isoforms. Nevertheless, binding properties of native OBP isoforms to specific ligands in S. scrofa were monitored by fluorescence spectroscopy. Recombinant phosphorylated OBP-Pichia isoforms bind steroids and fatty acids with slight differences. Native isoforms, that are phosphorylated but also O-GlcNAcylated show radically different binding affinities for the same compounds, which strongly suggests that O-GlcNAcylation increases the binding specificity of OBP isoforms. These findings extend the role of O-GlcNAc in regulating the function of proteins involved in many mechanisms of metabolic homeostasis, including extracellular signaling in olfaction. Data is available via ProteomeXChange with identifier PXD011371.

Insights into the substrate binding specificity of quorum-quenching acylase PvdQ

Quorum sensing is a cell to cell signaling mechanism that enables them to coordinate their behaviors in a density-dependent manner mediated by small diffusible signaling molecules, which can control the virulence and biofilm gene expression in many Gram-negative and positive bacteria. N-acyl homoserine lactone acylase PvdQ from human opportunistic pathogen Pseudomonas aeruginosa is a quorum-quenching enzyme that can hydrolyze the amide bond of the quorum signaling N-acyl homoserine lactones (AHLs) thereby degrading the signaling molecules, turning off the biofilm phenotype and resulting in a reduction of bacterial virulence. Previous studies demonstrated that PvdQ has different preferences for N-acyl substrates with different acyl chain lengths and substituents. However, the substrate binding specificity determinants of the quorum-quenching enzyme PvdQ with the different bacterial ligands are unknown and unintuitive. Further, elucidation of these determinants can lead to mutants with efficiency and broader substrate promiscuity. To investigate this question, a computational study was carried out combining multiple molecular docking methods, molecular dynamics simulations, residue interaction network analysis, and binding free energy calculations. The main findings are: firstly, the results from pKa predictions support that the pKa of the N-terminus of Serβ1 was depressed due to the surrounding residues. Multiple molecular docking studies provide useful information about the detailed binding modes and binding affinities. Secondly, 300 ns molecular dynamics simulations were carried out to analyze the overall molecular motions of substrate-bound and substrate-free PvdQ. The specific interactions between the active site of PvdQ and different ligands revealed the determinants for the preference among the ligands. A systematic comparison and analysis of the protein dynamic fingerprint of each complex demonstrated that binding of the most favorable ligand, C12-homoserine lactone (C12-HSL), reduced the global motions of the complex and maintained the correct arrangement of the catalytic site. Further, the residue interaction network analysis of each system illustrated that there are more communication contacts and pathways between the residues in the C12-HSL complex as compared to complexes with the other ligands. The binding of the C12-HSL ligand facilitates structural communication between the two knobs and the active site. While the binding of the other ligands tend to impair specific communication pathways between the two knobs and the active site, and lead to a catalytically inefficient state. Finally, simulation results from free energy landscape and binding free energy analysis revealed that the C12-HSL ligand has the lowest binding free energy and greater stability than the less favored ligands. Each of the following residues: Serβ1, Hisβ23, Pheβ24, Metβ30, Pheβ32, Leuβ50, Asnβ57, Thrβ69, Valβ70, Trpβ162, Trpβ186, Asnβ269, Argβ297 and Leuα146, play different roles in substrate binding specificity. This is the first computational study that provides molecular information for structure-dynamic-function relationships of PvdQ with different ligands and demonstrates determinants of bacterial substrate binding specificity.

Engineered SH2 domains with tailored specificities and enhanced affinities for phosphoproteome analysis.

Protein phosphorylation is the most abundant post-translational modification in cells. Src homology 2 (SH2) domains specifically recognize phosphorylated tyrosine (pTyr) residues to mediate signaling cascades. A conserved pocket in the SH2 domain binds the pTyr side chain and the EF and BG loops determine binding specificity. By using large phage-displayed libraries, we engineered the EF and BG loops of the Fyn SH2 domain to alter specificity. Engineered SH2 variants exhibited distinct specificity profiles and were able to bind pTyr sites on the epidermal growth factor receptor, which were not recognized by the wild-type Fyn SH2 domain. Furthermore, mass spectrometry showed that SH2 variants with additional mutations in the pTyr-binding pocket that enhanced affinity were highly effective for enrichment of diverse pTyr peptides within the human proteome. These results showed that engineering of the EF and BG loops could be used to tailor SH2 domain specificity, and SH2 variants with diverse specificities and high affinities for pTyr residues enabled more comprehensive analysis of the human phosphoproteome. STATEMENT: Src Homology 2 (SH2) domains are modular domains that recognize phosphorylated tyrosine embedded in proteins, transducing these post-translational modifications into cellular responses. Here we used phage display to engineer hundreds of SH2 domain variants with altered binding specificities and enhanced affinities, which enabled efficient and differential enrichment of the human phosphoproteome for analysis by mass spectrometry. These engineered SH2 domain variants will be useful tools for elucidating the molecular determinants governing SH2 domains binding specificity and for enhancing analysis and understanding of the human phosphoproteome.

Changes to the core and flanking sequences of G-box elements lead to increases and decreases in gene expression in both native and synthetic soybean promoters.

Summary Cis‐regulatory elements in promoters are major determinants of binding specificity of transcription factors (TFs) for transcriptional regulation. To improve our understanding of how these short DNA sequences regulate gene expression, synthetic promoters consisting of both classical (CACGTG) and variant G‐box core sequences along with different flanking sequences derived from the promoters of three different highly expressing soybean genes, were constructed and used to regulate a green fluorescent protein (gfp) gene. Use of the classical 6‐bp G‐box provided information on the base level of GFP expression while modifications to the 2–4 flanking bases on either side of the G‐box influenced the intensity of gene expression in both transiently transformed lima bean cotyledons and stably transformed soybean hairy roots. The proximal 2‐bp sequences on either flank of the G‐box significantly affected G‐box activity, while the distal 2‐bp flanking nucleotides also influenced gene expression albeit with a decreasing effect. Manipulation of the upstream 2‐ to 4‐bp flanking sequence of a G‐box variant (GACGTG), found in the proximal region of a relatively weak soybean glycinin promoter, significantly enhanced promoter activity using both transient and stable expression assays, if the G‐box variant was first converted into a classical G‐box (CACGTG). In addition to increasing our understanding of regulatory element composition and structure, this study shows that minimal targeted changes in native promoter sequences can lead to enhanced gene expression, and suggests that genome editing of the promoter region can result in useful and predictable changes in native gene expression.

Identification and Immunological Characterization of the Ligand Domain of Plasmodium vivax Reticulocyte Binding Protein 1a.

Erythrocyte invasion by malaria parasites is essential for blood-stage development. Consequently, parasite proteins critically involved in erythrocyte invasion, such as the Plasmodium vivax reticulocyte binding proteins (RBPs) that mediate preferential invasion of reticulocytes, are considered potential vaccine targets. Thus, targeting the RBPs could prevent blood-stage infection and disease. The RBPs are large, and little is known about their functional domains and whether individuals naturally exposed to P. vivax acquire binding-inhibitory antibodies to these critical binding regions. This study aims to functionally and immunologically characterize Plasmodium vivax RBP1a. Recombinant proteins of overlapping fragments of RBP1a were used to determine binding specificity to erythrocytes and immunogenicity in laboratory animals. The naturally acquired antibody response to these proteins was evaluated using serum samples from individuals in regions of endemicity. The N-terminal extracellular region, RBP1157-650 (RBP1:F8), was determined to bind both reticulocytes and normocytes, with a preference for immature reticulocytes. Antibodies elicited against rRBP1:F8 blocked binding between RBP1:F8 and erythrocytes. Naturally acquired anti-RBP1 binding-inhibitory antibodies were detected in serum specimens from P. vivax-exposed individuals from Papua New Guinea and Brazil. Recombinant RBP1:F8 binds human erythrocytes, elicits artificially induced functional blocking antibodies, and is a target of naturally acquired binding-inhibitory antibodies.

Uncovering the mechanistic basis for specific recognition of monomethylated H3K4 by the CW domain of Arabidopsis histone methyltransferase SDG8

Chromatin consists of DNA and histones, and specific histone modifications that determine chromatin structure and activity are regulated by three types of proteins, called writer, reader, and eraser. Histone reader proteins from vertebrates, vertebrate-infecting parasites, and higher plants possess a CW domain, which has been reported to read histone H3 lysine 4 (H3K4). The CW domain of Arabidopsis SDG8 (also called ASHH2), a histone H3 lysine 36 methyltransferase, preferentially binds monomethylated H3K4 (H3K4me1), unlike the mammalian CW domain protein, which binds trimethylated H3K4 (H3K4me3). However, the molecular basis of the selective binding by the CW domain of SDG8 (SDG8-CW) remains unclear. Here, we solved the 1.6-Å-resolution structure of SDG8-CW in complex with H3K4me1, which revealed that residues in the C-terminal α-helix of SDG8-CW determine binding specificity for low methylation levels at H3K4. Moreover, substitutions of key residues, specifically Ile-915 and Asn-916, converted SDG8-CW binding preference from H3K4me1 to H3K4me3. Sequence alignment and mutagenesis studies revealed that the CW domain of SDG725, the homolog of SDG8 in rice, shares the same binding preference with SDG8-CW, indicating that preference for low methylated H3K4 by the CW domain of ASHH2 homologs is conserved among higher-order plants. Our findings provide first structural insights into the molecular basis for specific recognition of monomethylated H3K4 by the H3K4me1 reader protein SDG8 from Arabidopsis.

Relationship between histone modifications and transcription factor binding is protein family specific.

The very small fraction of putative binding sites (BSs) that are occupied by transcription factors (TFs) in vivo can be highly variable across different cell types. This observation has been partly attributed to changes in chromatin accessibility and histone modification (HM) patterns surrounding BSs. Previous studies focusing on BSs within DNA regulatory regions found correlations between HM patterns and TF binding specificities. However, a mechanistic understanding of TF–DNA binding specificity determinants is still not available. The ability to predict in vivo TF binding on a genome-wide scale requires the identification of features that determine TF binding based on evolutionary relationships of DNA binding proteins. To reveal protein family–dependent mechanisms of TF binding, we conducted comprehensive comparisons of HM patterns surrounding BSs and non-BSs with exactly matched core motifs for TFs in three cell lines: 33 TFs in GM12878, 37 TFs in K562, and 18 TFs in H1-hESC. These TFs displayed protein family–specific preferences for HM patterns surrounding BSs, with high agreement among cell lines. Moreover, compared to models based on DNA sequence and shape at flanking regions of BSs, HM-augmented quantitative machine-learning methods resulted in increased performance in a TF family–specific manner. Analysis of the relative importance of features in these models indicated that TFs, displaying larger HM pattern differences between BSs and non-BSs, bound DNA in an HM-specific manner on a protein family–specific basis. We propose that TF family–specific HM preferences reveal distinct mechanisms that assist in guiding TFs to their cognate BSs by altering chromatin structure and accessibility.

Interaction of Fluorescently Labeled Cadherin G Protein-coupled Receptor with the Cry1Ab Toxin of Bacillus thuringiensis.

The Cry1Ab toxin produced by Bacillus thuringiensis binds to a conserved structural motif in the 12th ectodomain module (EC12) of BT-R1, a cadherin G protein-coupled receptor (GPCR) contained in the membrane of midgut epithelial cells of the tobacco hornworm Manduca sexta. Toxin binding transmits a signal into the cells and turns on a multi-step signal transduction pathway, culminating in cell death. Using chromatographically purified Cry1Ab and EC12 proteins, we demonstrated the direct formation of a stable complex between these two proteins in solution and visualized it on a native polyacrylamide gel. Moreover, we generated a fluorescent EC12 probe by converting the 36th residue to cysteine to enable maleimide-mediated conjugation of Alexa-488 fluorescent dye to EC12 by site-directed mutagenesis. In addition, we changed the 44th residue of EC12 to tryptophan, which greatly improved accuracy of protein quantification and traceability. Using the fluorescently labeled EC12 probe for direct and competitive binding assays, we were able to determine binding specificity in solution. These accomplishments will facilitate identification and characterization of the interface sequences for both the Cry1Ab toxin and BT-R1.

Characterization of LL25X, a Newly Isolated Anti-SIV Monoclonal Antibody, to Determine Binding Specificity

A phage display library was previously constructed from an SIV-infected rhesus macaque. Several single chain Fv (scFv), including SU24, SU343 and LL25X, were selected using phage display technology. Sequences corresponding to SU24, SU343 and LL25X were optimized for expression in a mammalian system and commercially synthesized. SU24 and SU343 had previously been cloned into a mammalian expression vector. In this study, we aimed to characterize the specificity of SU24, SU343, and LL25X.. The codon-optimized version of the scFv LL25X gene sequence was cloned into a mammalian expression vector (pCEP4). LL25X DNA was amplified by PCR, and the PCR product and mammalian expression vector were both digested with KpnI/SapI restriction enzymes. Digested fragments were purified, and the fragments were ligated using T4DNA ligase. E. coli cells were transformed with the ligation reaction. Single colonies were selected on LB agar plates containing the selective antibiotic (ampicillin). Positive colonies were identified after DNA mini-preparation and test-digestion with KpnI and SapI. Sanger sequencing confirmed cloning results and DNA sequence accuracy. Following transfection of mammalian cells (293T), LL25X-Fc cells, and purifying our protein, the binding of LL25X-Fc to the SIV gp140 envelope protein was confirmed via ELISA and Western Blotting.