Binding of Protein Kinase Inhibitors to Synapsin I Inferred from Pair-Wise Binding Site Similarity Measurements

Predicting off-targets by computational methods is getting increasing importance in early drug discovery stages. We herewith present a computational method based on binding site three-dimensional comparisons, which prompted us to investigate the cross-reaction of protein kinase inhibitors with synapsin I, an ATP-binding protein regulating neurotransmitter release in the synapse. Systematic pair-wise comparison of the staurosporine-binding site of the proto-oncogene Pim-1 kinase with 6,412 druggable protein-ligand binding sites suggested that the ATP-binding site of synapsin I may recognize the pan-kinase inhibitor staurosporine. Biochemical validation of this hypothesis was realized by competition experiments of staurosporine with ATP-γ³⁵S for binding to synapsin I. Staurosporine, as well as three other inhibitors of protein kinases (cdk2, Pim-1 and casein kinase type 2), effectively bound to synapsin I with nanomolar affinities and promoted synapsin-induced F-actin bundling. The selective Pim-1 kinase inhibitor quercetagetin was shown to be the most potent synapsin I binder (IC50  = 0.15 µM), in agreement with the predicted binding site similarities between synapsin I and various protein kinases. Other protein kinase inhibitors (protein kinase A and chk1 inhibitor), kinase inhibitors (diacylglycerolkinase inhibitor) and various other ATP-competitors (DNA topoisomerase II and HSP-90α inhibitors) did not bind to synapsin I, as predicted from a lower similarity of their respective ATP-binding sites to that of synapsin I. The present data suggest that the observed downregulation of neurotransmitter release by some but not all protein kinase inhibitors may also be contributed by a direct binding to synapsin I and phosphorylation-independent perturbation of synapsin I function. More generally, the data also demonstrate that cross-reactivity with various targets may be detected by systematic pair-wise similarity measurement of ligand-annotated binding sites.     

Ligand Binding Site Similarity Identification Based on Chemical and Geometric Similarity

The similarity comparison of binding sites based on amino acid between different proteins can facilitate protein function identification. However, Binding site usually consists of several crucial amino acids which are frequently dispersed among different regions of a protein and consequently make the comparison of binding sites difficult. In this study, we introduce a new method, named as chemical and geometric similarity of binding site (CGS-BSite), to compute the ligand binding site similarity based on discrete amino acids with maximum-weight bipartite matching algorithm. The principle of computing the similarity is to find a Euclidean Transformation which makes the similar amino acids approximate to each other in a geometry space, and vice versa. CGS-BSite permits site and ligand flexibilities, provides a stable prediction performance on the flexible ligand binding sites. Binding site prediction on three test datasets with CGS-BSite method has similar performance to Patch-Surfer method but outperforms other five tested methods, reaching to 0.80, 0.71 and 0.85 based on the area under the receiver operating characteristic curve, respectively. It performs a marginally better than Patch-Surfer on the binding sites with small volume and higher hydrophobicity, and presents good robustness to the variance of the volume and hydrophobicity of ligand binding sites. Overall, our method provides an alternative approach to compute the ligand binding site similarity and predict potential special ligand binding sites from the existing ligand targets based on the target similarity.     

Myelin Basic Protein Is an Endogenous Inhibitor of the High-Affinity Cannabinoid Binding Site in Brain

Radioligand binding studies with the water-soluble cannabinoid [3H]5'-trimethylammonium delta 8-tetrahydrocannabinol ([3H]TMA) have revealed a saturable high-affinity site in brain that is specific for cannabinoids. To determine whether endogenous compounds of brain might act upon the site physiologically, we sought inhibitors in extracts of brain. An endogenous inhibitor has been purified to homogeneity by acid extraction of rat brain followed by adsorption to a reverse-phase matrix and gel filtration chromatography. The purified inhibitor has a subunit molecular mass of 14,500 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Inhibition of [3H]TMA binding by the purified inhibitor occurs with a Ki of about 4 nM in a noncompetitive manner. The molecular weight, abundance, and extraction properties are the same as a species of myelin basic protein (MBP). The MBPs of rat, rabbit, pig, and cow also inhibit [3H]TMA binding noncompetitively with similar potencies. The purified inhibitor comigrates with rat MBP-small form on SDS-PAGE, has a similar amino acid composition, and is recognized by antibody directed against MBP. Studies of fragments of rabbit MBP suggest that the determinants of affinity for the [3H]TMA site are contained primarily within the C-terminal half of the rabbit MBP. Synthetic polycationic peptides such as polylysine and polyarginine mimic the effects of MBP, suggesting that the high-affinity cannabinoid binding site recognizes large polycations. The identification of the endogenous inhibitor of [3H]TMA binding as MBP suggests that MBP interacts physiologically with the high-affinity cannabinoid site.     

Mutant Ribosomal Protein with Defective RNA Binding Site

THE 30S ribosomal subunits of Escherichia coli contain twenty-one different proteins^1–4, which together with 16S RNA can reassemble in vitro to form functional 30S particles⁵. Five proteins can individually bind to specific sites on the 16S RNA^6–8 and these are S4, S7, S8, S15 and S20 (in the nomenclature recently adopted by several laboratories to report results with the E. coli system⁹). We report here the first identification of a mutation that affects a ribosomal protein-nucleic acid interaction.     

Characterization of EPPIN's Semenogelin I Binding Site: A Contraceptive Drug Target

Epididymal protease inhibitor (EPPIN) is found on the surface of spermatozoa and works as a central hub for a sperm surface protein complex (EPPIN protein complex [EPC]) that inhibits sperm motility on the binding of semenogelin I (SEMG1) during ejaculation. Here, we identify EPPIN's amino acids involved in the interactions within the EPC and demonstrate that EPPIN's sequence C102-P133 contains the major binding site for SEMG1. Within the same region, the sequence F117-P133 binds the EPC-associated protein lactotransferrin (LTF). We show that residues Cys102, Tyr107, and Phe117 in the EPPIN C-terminus are required for SEMG1 binding. Additionally, residues Tyr107 and Phe117 are critically involved in the interaction between EPPIN and LTF. Our findings demonstrate that EPPIN is a key player in the protein-protein interactions within the EPC. Target identification is an important step toward the development of a novel male contraceptive, and the functionality of EPPIN's residues Cys102, Tyr107, and Phe117 offers novel opportunities for contraceptive compounds that inhibit sperm motility by targeting this region of the molecule.     

BIPSPI+: Mining Type-Specific Datasets of Protein Complexes to Improve Protein Binding Site Prediction

Computational approaches for predicting protein-protein interfaces are extremely useful for understanding and modelling the quaternary structure of protein assemblies. In particular, partner-specific binding site prediction methods allow delineating the specific residues that compose the interface of protein complexes. In recent years, new machine learning and other algorithmic approaches have been proposed to solve this problem. However, little effort has been made in finding better training datasets to improve the performance of these methods. With the aim of vindicating the importance of the training set compilation procedure, in this work we present BIPSPI+, a new version of our original server trained on carefully curated datasets that outperforms our original predictor. We show how prediction performance can be improved by selecting specific datasets that better describe particular types of protein interactions and interfaces (e.g. homo/hetero). In addition, our upgraded web server offers a new set of functionalities such as the sequence-structure prediction mode, hetero- or homo-complex specialization and the guided docking tool that allows to compute 3D quaternary structure poses using the predicted interfaces. BIPSPI+ is freely available at https://bipspi.cnb.csic.es.     

Ranking Enzyme Structures in the PDB by Bound Ligand Similarity to Biological Substrates

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Heart-Type Fatty Acid Binding Protein Is Associated with Proteinuria in Obesity

Rationale

Lipid metabolism contributes to the formation of obesity-related glomerulopathy (ORG). Heart-type fatty acid binding protein (H-FABP or FABP3) is involved in lipid metabolism and was predicted to relate to renal lesions in obesity.

Methods

A total of 28 patients with ORG were investigated, and renal tissue from 7 kidney donors served as controls. Db/db mice with albuminuria were treated with Simvastatin for 12 weeks.

Results

Immunohistochemistry demonstrated the H-FABP staining in glomerular and tubular areas of patients with ORG, and the percentage of H-FABP in the glomerular area was significantly higher than in controls (15.8±1.62 versus 4.51±0.56%, P<0.001). Moreover, H-FABP expression correlated with proteinuria, high-density lipoprotein (HDL) cholesterol, waist circumference and the homeostatic model assessment – insulin resistance (HOMA-IR) among patients with ORG. Enhanced expression of H-FABP was also detected in the db/db mice, and expression increased from 8 to 20 weeks of age and was weakly related to increased albuminuria (r = 0.433; P = 0.020). Furthermore, H-FABP was co-localized with synaptopodin and demonstrated a podocyte pattern distribution. After Simvastation treatment, the urine albumin levels decreased with lipid levels and H-FABP expression in the glomeruli. The expression of H-FABP was related to Simvastatin treatment, albuminuria and triglycerides, while it was only linked with triglycerides and albuminuria (r = 0.643, P = 0.036).

Conclusions

This study confirmed an association of H-FABP with the pathogenesis of clinical and experimental ORG, and suggests that such a process might be related to podocytes and lipid dysmetabolism.     

Novel Reticular Calcium Binding Protein Is Purified on Taipoxin Columns

Abstract: We identified, by affinity chromatography, two putative binding proteins for the presynaptic snake venom toxin taipoxin. We have previously characterized one of these proteins [neuronal pentraxin (NP)] as a neuronally secreted protein with homology to acute-phase proteins. Here we report the identification of the second protein as a 49-kDa lumenal calcium binding protein that we have named taipoxin-associated calcium binding protein 49 (TCBP-49). This protein contains six EF-hand putative calcium binding domains and the carboxyl-terminal sequence His-Asp-Glu-Leu (HDEL), identical to the yeast endoplasmic reticulum retention signal. Message for this protein is present in brain, liver, muscle, heart, kidney, and testis. Antibodies to this protein label reticular organelles of neurons and glia. This localization and the specific enrichment of native and recombinant TCBP-49 on columns of immobilized taipoxin raise the possibility that this protein interacts with internalized taipoxin, perhaps mediating its activation. The availability of pure TCBP-49 will allow direct tests of whether TCBP-49 alters the integrity of the oligomeric structure, phospholipase activity, or toxicity of taipoxin.     

The Receptor Binding Site of Feline Leukemia Virus Surface Glycoprotein Is Distinct from the Site Involved in Virus Neutralization

The external surface glycoprotein (SU) of feline leukemia virus (FeLV) contains sites which define the viral subgroup and induce virus-neutralizing antibodies. The subgroup phenotypic determinants have been located to a small variable region, VR1, towards the amino terminus of SU. The sites which function as neutralizing epitopes in vivo are unknown. Recombinant SU proteins were produced by using baculoviruses that contained sequences encoding the SUs of FeLV subgroup A (FeLV-A), FeLV-C, and two chimeric FeLVs (FeLV-215 and FeLV-VC) in which the VR1 domain of FeLV-A had been replaced by the corresponding regions of FeLV-C isolates. The recombinant glycoproteins, designated Bgp70-A, -C, -215, and -VC, respectively, were similar to their wild-type counterparts in several immunoblots and inhibited infection of susceptible cell lines in a subgroup-specific manner. Thus, Bgp70-A interfered with infection by FeLV-A, whereas Bgp70-C, -VC, and -215 did not. Conversely, Bgp70-C, -VC, and -215 blocked infection with FeLV-C, while Bgp70-A had no effect. These results indicate that the site on SU which binds to the FeLV cell surface receptor was preserved in the recombinant glycoproteins. It was also found that the recombinant proteins were able to bind naturally occurring neutralizing antibodies. Bgp70-A, -VC, and -215 interfered with the action of anti-FeLV-A neutralizing antibodies, whereas Bgp70-C did not. Furthermore, Bgp70-C interfered with the action of anti-FeLV-C neutralizing antibodies, while the other proteins did not. These results indicate that the neutralizing epitope(s) of FeLV SU lies outside the subgroup-determining VR1 domain.     

水稻RNA结合蛋白C3H12与RNA结合位点的鉴定

CCH型锌指蛋白质C3H12是进化上保守的RNA结合蛋白质,它含有5个串联的CCCH锌指结构域ZnF1-5,形成2个紧密的锌指簇ZnF1-3和ZnF4-5。早期的研究发现,C3H12可能通过与mRNA结合的方式在转录后水平调控基因的表达。然而,与C3H12结合的mRNA类型和他们的结合模式,并未通过实验得到证明。本文表达纯化了一系列C3H12截短及全长蛋白质,并合成了一些潜在RNA底物ARE9、ARE19及对照Random21。通过等温滴定量热法 (isothermal titration calorimetry, ITC) 确定了C3H12与富含腺嘌呤尿嘧啶单元 (AU-rich element, ARE) mRNA底物的结合,并揭示了互作核心区域和热力学性质。通过荧光光谱分析和微型热泳动 (microscale thermophoresis, MST)技术对ITC的结果进一步佐证。结果表明：(1) C3H12与ARE底物的相互作用是焓驱动的能量有利的 (ΔG<0) 特异性结合,结合比为1:1。(2) C3H12与ARE19的亲和力较ARE9更高（约2倍）。(3) C3H12中ZnF1-3在与ARE类底物的结合活性中发挥主导作用。(4) C3H12结构中的141个氨基酸残基的接头不直接参与和ARE底物的相互作用。本研究揭示的CCCH型锌指蛋白质C3H12与ARE底物结合模式,将为进一步在分子结构水平阐明C3H12与ARE底物结合的机制奠定基础。     

水稻RNA结合蛋白C3H12与RNA结合位点的鉴定

CCH型锌指蛋白质C3H12是进化上保守的RNA结合蛋白质,它含有5个串联的CCCH锌指结构域ZnF1-5,形成2个紧密的锌指簇ZnF1-3和ZnF4-5。早期的研究发现,C3H12可能通过与mRNA结合的方式在转录后水平调控基因的表达。然而,与C3H12结合的mRNA类型和他们的结合模式,并未通过实验得到证明。本文表达纯化了一系列C3H12截短及全长蛋白质,并合成了一些潜在RNA底物ARE9、ARE19及对照Random21。通过等温滴定量热法 (isothermal titration calorimetry, ITC) 确定了C3H12与富含腺嘌呤尿嘧啶单元 (AU-rich element, ARE) mRNA底物的结合,并揭示了互作核心区域和热力学性质。通过荧光光谱分析和微型热泳动 (microscale thermophoresis, MST)技术对ITC的结果进一步佐证。结果表明：(1) C3H12与ARE底物的相互作用是焓驱动的能量有利的 (ΔG<0) 特异性结合,结合比为1:1。(2) C3H12与ARE19的亲和力较ARE9更高（约2倍）。(3) C3H12中ZnF1-3在与ARE类底物的结合活性中发挥主导作用。(4) C3H12结构中的141个氨基酸残基的接头不直接参与和ARE底物的相互作用。本研究揭示的CCCH型锌指蛋白质C3H12与ARE底物结合模式,将为进一步在分子结构水平阐明C3H12与ARE底物结合的机制奠定基础。     

NNvPDB: Neural Network based Protein Secondary Structure Prediction with PDB Validation

Availabilityhttp://bit.srmuniv.ac.in/cgi-bin/bit/cfpdb/nnsecstruct.pl     

Achieving Peptide Binding Specificity and Promiscuity by Loops: Case of the Forkhead-Associated Domain

The regulation of a series of cellular events requires specific protein–protein interactions, which are usually mediated by modular domains to precisely select a particular sequence from diverse partners. However, most signaling domains can bind to more than one peptide sequence. How do proteins create promiscuity from precision? Moreover, these complex interactions typically occur at the interface of a well-defined secondary structure, α helix and β sheet. However, the molecular recognition primarily controlled by loop architecture is not fully understood. To gain a deep understanding of binding selectivity and promiscuity by the conformation of loops, we chose the forkhead-associated (FHA) domain as our model system. The domain can bind to diverse peptides via various loops but only interact with sequences containing phosphothreonine (pThr). We applied molecular dynamics (MD) simulations for multiple free and bound FHA domains to study the changes in conformations and dynamics. Generally, FHA domains share a similar folding structure whereby the backbone holds the overall geometry and the variety of sidechain atoms of multiple loops creates a binding surface to target a specific partner. FHA domains determine the specificity of pThr by well-organized binding loops, which are rigid to define a phospho recognition site. The broad range of peptide recognition can be attributed to different arrangements of the loop interaction network. The moderate flexibility of the loop conformation can help access or exclude binding partners. Our work provides insights into molecular recognition in terms of binding specificity and promiscuity and helpful clues for further peptide design.