Substrate Recognition Pattern Research Articles

Temperature-Dependent Affinity Changes in Substrate Binding Affect the Cleavage Activity of BthC2c1.

The CRISPR-Cas system is an adaptive immune mechanism for bacteria and archaea to resist foreign invasion. Currently, Cas9 and Cpf1 have been widely studied and applied in gene editing. C2c1 is a newly discovered CRISPR-Cas system endonuclease. It has broad application prospects due to its small molecular weight and high substrate recognition specificity. Bacillus thermoamylovorans C2c1(BthC2c1) was expressed in E. coli C43 (DE3) competent cells, purified, and the BthC2c1-sgRNA-dsDNA complex was assembled. The effect of temperature on the cleavage ability of the BthC2c1 system was investigated. The cDNA of BthC2c1 was cloned into the vector pGEX-6P-1. BthC2c1 was expressed in E. coli C43(DE3) cells and purified using a GST affinity column and FPLC. The sgRNAs were transcribed and purified in vitro, and the complexes were assembled by gel filtration chromatography. The enzyme cleavage activity of BthC2c1 at different temperatures was investigated using an in vitro cleavage assay. Microscale Thermophoresis detected the affinity of the BthC2c1-sgRNA complexes to substrate DNA. BthC2c1 proteins were prokaryotically expressed and purified. The complex of BthC2c1 with sgRNA and dsDNA was assembled. In vitro cleavage assay results showed that BthC2c1 cleaved the target DNA at temperatures ranging from 37°C to 67°C. The cleavage ability of BthC2c1 at 42oC was stronger than that at 37oC. The results of affinity detection showed that the affinity between the BthC2c1-sgRNA complex and ds36/36 at 42oC was stronger than that at 37oC. In this study, BthC2c1 was expressed, purified, and assembled into a complex with sgRNA and dsDNA. BthC2c1 cleaved DNA within the temperature range of 37oC to 67oC. The affinity of BthC2c1-sgRNA to DNA at 42°C was significantly enhanced than that at 37°C. It may be related to its stringent substrate recognition pattern, which differs from Cas9 and Cpf1. The temperature-dependent affinity changes of substrate binding may be part of the reason for the stronger cleavage activity of BthC2c1 at 42oC. This study may provide an experimental basis for optimizing and modifying the C2c1 gene editing system.

Knockout of ABCC1 in NCI-H441 cells reveals CF to be a suboptimal substrate to study MRP1 activity in organotypic in vitro models

Multidrug resistance-associated protein 1 (MRP1/ABCC1) is an efflux transporter responsible for the extrusion of endogenous substances as well as xenobiotics and their respective metabolites. Its high expression levels in lung tissue imply a key role in pulmonary drug disposition. Moreover, its association with inflammatory lung diseases underline MRP1’s relevance in drug development and precision-medicine. With the aim to develop a tool to better understand MRP1’s role in drug disposition and lung disease, we generated an ABCC1−/− clone based on the human distal lung epithelial cell line NCI-H441 via a targeted CRISPR/Cas9 approach. Successful knockout (KO) of MRP1 was confirmed by qPCR, immunoblot and Sanger sequencing. To assess potential compensatory upregulation of transporters with a similar substrate recognition pattern as MRP1, expression levels of MRP2–9 as well as OAT1–4, 6, 7 and 10 were measured. Functional transporter activity was determined via release studies with two prodrug/substrate pairs, i.e. 5(6)-carboxyfluorescein (CF; formed from its diacetate prodrug) and S-(6-(7-methylpurinyl))glutathione (MPG; formed from its prodrug 6-bromo-7-methylpurine, BMP), respectively. Lastly, transepithelial electrical resistance (TEER) of monolayers of the KO clone were compared with wildtype (WT) NCI-H441 cells. Of eight initially generated clones, the M2 titled clone showed complete absence of mRNA and protein in accordance with the designed genome edit. In transport studies using the substrate CF, however, no differences between the KO clone and WT NCI-H441 cells were observed, whilst no differences in expression of potential compensatory transporters was noted. On the other hand, when using BMP/MPG, the release of MPG was reduced to 11.5% in the KO clone. Based on these results, CF appears to be a suboptimal probe for the study of MRP1 function, particularly in organotypic in vitro and ex vivo models. Our ABCC1−/− NCI-H441 clone further retained the ability to form electrically tight barriers, making it a useful model to study MRP1 function in vitro.

Characterize the Interaction of the DNA Helicase PriA with the Stalled DNA Replication Fork Using Atomic Force Microscopy.

In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. ssDNA-binding protein (SSB) is typically present at the abandoned forks, protecting the ssDNA from nucleases. Research that is based on the assays for junction dissociation, surface plasmon resonance, single-molecule FRET, and x-ray crystal structure has revealed the helicase activity of PriA, the SSB-PriA interaction, and structural information of PriA helicase. Here, we used Atomic Force Microscopy (AFM) to visualize the interaction between PriA and DNA substrates with or without SSB in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. The protocol describes the steps to obtain high-resolution AFM images and the details of data analysis and presentation.

Atomic force microscopy–based characterization of the interaction of PriA helicase with stalled DNA replication forks

In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. Single-stranded DNA-binding protein (SSB) is typically present at the abandoned forks, but it is unclear how SSB and PriA interact, although it has been shown that the two proteins interact both physically and functionally. Here, we used atomic force microscopy to visualize the interaction of PriA with DNA substrates with or without SSB. These experiments were done in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. These analyses revealed that in the absence of SSB, PriA binds preferentially to a fork substrate with a gap in the leading strand. Such a preference has not been observed for 5'- and 3'-tailed duplexes, suggesting that it is the fork structure that plays an essential role in PriA's selection of DNA substrates. Furthermore, we found that in the absence of SSB, PriA binds exclusively to the fork regions of the DNA substrates. In contrast, fork-bound SSB loads PriA onto the duplex DNA arms of forks, suggesting a remodeling of PriA by SSB. We also demonstrate that the remodeling of PriA requires a functional C-terminal domain of SSB. In summary, our atomic force microscopy analyses reveal key details in the interactions between PriA and stalled DNA replication forks with or without SSB.

Elucidation of degradation pattern and immobilization of a novel alginate lyase for preparation of alginate oligosaccharides

Alginate lyases are important enzymes to prepare alginate oligosaccharides for industrial applications and elucidating the degradation pattern of alginate lyases is essential for expanding their applications. Herein, we cloned and expressed AlyPL6, a novel member of polysaccharide lyase family-6 (PL6) with high activity from Pedobacter hainanensis NJ-02. It was found that AlyPL6 could recognize tetrasaccharide as the minimal substrate to release oligosaccharides with low degrees of polymerization (DPs). As a result, it could be speculated that the cleavage site of substrate is located between −1 and +1 subsites. For further application, AlyPL6 was then immobilized onto mesoporous titanium oxide particles (MTOPs) with >55.4% of maximal activity retained at 45 °C after it was reused for 10 times. The research provided extended insights into the substrate recognition and degradation pattern of PL 6 alginate lyases, which may benefit the further application of alginate lyases a lot.

Biochemical Characterization and Elucidation of Action Pattern of a Novel Polysaccharide Lyase 6 Family Alginate Lyase from Marine Bacterium Flammeovirga sp. NJ-04.

Alginate lyases have been widely used to prepare alginate oligosaccharides in food, agricultural, and medical industries. Therefore, discovering and characterizing novel alginate lyases with excellent properties has drawn increasing attention. Herein, a novel alginate lyase FsAlyPL6 of Polysaccharide Lyase (PL) 6 family is identified and biochemically characterized from Flammeovirga sp. NJ-04. It shows highest activity at 45 °C and could retain 50% of activity after being incubated at 45 °C for 1 h. The Thin-Layer Chromatography (TLC) and Electrospray Ionization Mass Spectrometry (ESI-MS) analysis indicates that FsAlyPL6 endolytically degrades alginate polysaccharide into oligosaccharides ranging from monosaccharides to pentasaccharides. In addition, the action pattern of the enzyme is also elucidated and the result suggests that FsAlyPL6 could recognize tetrasaccharide as the minimal substrate and cleave the glycosidic bonds between the subsites of −1 and +3. The research provides extended insights into the substrate recognition and degradation pattern of PL6 alginate lyases, which may further expand the application of alginate lyases.

Prediction of prkC-mediated protein serine/threonine phosphorylation sites for bacteria

As an abundant post-translational modification, reversible phosphorylation is critical for the dynamic regulation of various biological processes. prkC, a critical serine/threonine-protein kinase in bacteria, plays important roles in regulation of signaling transduction. Identification of prkC-specific phosphorylation sites is fundamental for understanding the molecular mechanism of phosphorylation-mediated signaling. However, experimental identification of substrates for prkC is time-consuming and labor-intensive, and computational methods for kinase-specific phosphorylation prediction in bacteria have yet to be developed. In this study, we manually curated the experimentally identified substrates and phosphorylation sites of prkC from the published literature. The analyses of the sequence preferences showed that the substrate recognition pattern for prkC might be miscellaneous, and a complex strategy should be employed to predict potential prkC-specific phosphorylation sites. To develop the predictor, the amino acid location feature extraction method and the support vector machine algorithm were employed, and the methods achieved promising performance. Through 10-fold cross validation, the predictor reached a sensitivity of 91.67% at the specificity of 95.12%. Then, we developed freely accessible software, which is provided at http://free.cancerbio.info/prkc/. Based on the predictor, hundreds of potential prkC-specific phosphorylation sites were annotated based on the known bacterial phosphorylation sites. prkC-PSP was the first predictor for prkC-specific phosphorylation sites, and its prediction performance was promising. We anticipated that these analyses and the predictor could be helpful for further studies of prkC-mediated phosphorylation.

Molecular modeling and docking of protease from Bacillus sp. for the keratin degradation

Abstract In this present study, the 3D structure of protease from Bacillus sp. was modeled by using modeler 9v9. The generated structure was accessed for geometrical errors and energy stability using RAMPAGE, VERFIY 3D, ERRAT and PROSA. The proteolytic nature of this modeled protease was determined by docking studies using FlexX. The kertinolytic activity was revealed by using polysacchadire sub units such as arabinose, rhamnose, galactose, and glucose; aminopeptidase activity was revealed by using amino acids like methionine, leucine, rivastigmine and also by protease inhibitor like N-carbobenzyloxy-L-alanine and 4-amidino phenyl methanesulfonyl flurode. The docking studies showed that Asp160 and Try246 residues were mainly involved in binding of all the compounds and other important amino acid residues such as His85, Asp97, Glu131, Asp160, Glu131, Glu132, Asp160, Tyr246, and His247 were involved in hydrogen bonding with the substrates. These studies also determined the substrate recognition pattern and the development of suitable protease can potentially enhance its applications in keratin degradation.

Profiling the substitution pattern of xyloglucan derivatives by integrated enzymatic hydrolysis, hydrophilic-interaction liquid chromatography and mass spectrometry

Plant polysaccharides constitute arguably the most complex family of biomacromolecules in terms of the stereochemistry and regiochemistry of their intramolecular linkages. The chemical modification of such polysaccharides introduces an additional level of complexity for structural determinations. We have developed an integrated analytical procedure combining selective enzymatic hydrolysis, hydrophilic interaction liquid chromatography (HILIC), and mass spectrometry (MS) to describe the substitution pattern of xyloglucan (XyG) and its chemo-enzymatic derivatives (cationic, anionic, and benzyl aminated). Enzymatic hydrolysis of XyG derivatives by a xyloglucan-specific endoglucanase (XEG) generates oligosaccharides amenable for mass spectrometric identification with distinct structures, based on enzymatic substrate recognition and hydrolytic pattern. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF-MS) and electrospray ionisation mass spectrometry (ESI–MS) offer qualitative mass profiling of the chemical derivatives. Separation and identification of the complex oligosaccharide profiles released by enzymatic hydrolysis is achieved by hyphenation of hydrophilic interaction liquid chromatography with mass spectrometry (HILIC-ESI–MS). Further fragmentation by tandem mass spectrometry (ESI–MS/MS) in positive mode enables the structural sequencing of modified XyG oligosaccharides and the identification of the substituent position without further derivatisation. This integrated approach can be used to obtain semi-quantitative information of the substitution pattern of hemicellulose derivatives, with fundamental implications for their modification mechanisms and performance.

Structural and Functional Study of d-Glucuronyl C5-epimerase

Heparan sulfate (HS) is a glycosaminoglycan present on the cell surface and in the extracellular matrix, which interacts with diverse signal molecules and is essential for many physiological processes including embryonic development, cell growth, inflammation, and blood coagulation. D-glucuronyl C5-epimerase (Glce) is a crucial enzyme in HS synthesis, converting D-glucuronic acid to L-iduronic acid to increase HS flexibility. This modification of HS is important for protein ligand recognition. We have determined the crystal structures of Glce in apo-form (unliganded) and in complex with heparin hexasaccharide (product of Glce following O-sulfation), both in a stable dimer conformation. A Glce dimer contains two catalytic sites, each at a positively charged cleft in C-terminal α-helical domains binding one negatively charged hexasaccharide. Based on the structural and mutagenesis studies, three tyrosine residues, Tyr(468), Tyr(528), and Tyr(546), in the active site were found to be crucial for the enzymatic activity. The complex structure also reveals the mechanism of product inhibition (i.e. 2-O- and 6-O-sulfation of HS keeps the C5 carbon of L-iduronic acid away from the active-site tyrosine residues). Our structural and functional data advance understanding of the key modification in HS biosynthesis.

Determination of substrate specificities against β-glucosidase A (BglA) from Thermotoga maritime: a molecular docking approach.

Thermostable enzymes derived from Thermotoga maritima have attracted worldwide interest for their potential industrial applications. Structural analysis and docking studies were preformed on T. maritima β-glucosidase enzyme with cellobiose and pNP-linked substrates. The 3D structure of the thermostable β-glucosidase was downloaded from the Protein Data Bank database. Substrates were downloaded from the PubCehm database and were minimized using MOE software. Docking of BglA and substrates was carried out using MOE software. After analyzing docked enzyme/substrate complexes, it was found that Glu residues were mainly involved in the reaction, and other important residues such as Asn, Ser, Tyr, Trp, and His were involved in hydrogen bonding with pNP-linked substrates. By determining the substrate recognition pattern, a more suitable β-glucosidase enzyme could be developed, enhancing its industrial potential.

Human ABC Transporters at blood-CNS Interfaces as Determinants of CNS Drug Penetration

Since the discovery of P-glycoprotein (P-gp) in brain microvessels composing the human blood-brain barrier (BBB), ATP-binding cassette (ABC) transporters have been recognized as bottlenecks in the development and delivery of neuropharmaceuticals. ABC transporters are expressed predominately at the plasma luminal membrane of brain capillary endothelial cells. These ABC transporters are responsible for the efflux of their substrates from the endothelial cells to the bloodstream against the concentration gradient and thus limit the entry of some drugs within the central nervous system (CNS). Advanced quantitative molecular biology tools allowed gene and protein quantification of the components of microvessels isolated from different species including human. Recently, positron emission tomography using radiolabelled probes that are substrates of ABC transporters allowed the determination of their functional activity at the human BBB. Here, we summarized new information regarding the relative expression, substrate recognition pattern for CNS drugs and functional activity of ABC transporters that are quantitatively expressed at the human BBB.

The β-Glucanase ZgLamA from Zobellia galactanivorans Evolved a Bent Active Site Adapted for Efficient Degradation of Algal Laminarin

Laminarinase is commonly used to describe β-1,3-glucanases widespread throughout Archaea, bacteria, and several eukaryotic lineages. Some β-1,3-glucanases have already been structurally and biochemically characterized, but very few from organisms that are in contact with genuine laminarin, the storage polysaccharide of brown algae. Here we report the heterologous expression and subsequent biochemical and structural characterization of ZgLamAGH16 from Zobellia galactanivorans, the first GH16 laminarinase from a marine bacterium associated with seaweeds. ZgLamAGH16 contains a unique additional loop, compared with other GH16 laminarinases, which is composed of 17 amino acids and gives a bent shape to the active site cleft of the enzyme. This particular topology is perfectly adapted to the U-shaped conformation of laminarin chains in solution and thus explains the predominant specificity of ZgLamAGH16 for this substrate. The three-dimensional structure of the enzyme and two enzyme-substrate complexes, one with laminaritetraose and the other with a trisaccharide of 1,3-1,4-β-d-glucan, have been determined at 1.5, 1.35, and 1.13 Å resolution, respectively. The structural comparison of substrate recognition pattern between these complexes allows the proposition that ZgLamAGH16 likely diverged from an ancestral broad specificity GH16 β-glucanase and evolved toward a bent active site topology adapted to efficient degradation of algal laminarin.

Substrate Recognition by the Multifunctional Cytochrome P450 MycG in Mycinamicin Hydroxylation and Epoxidation Reactions

The majority of characterized cytochrome P450 enzymes in actinomycete secondary metabolic pathways are strictly substrate-, regio-, and stereo-specific. Examples of multifunctional biosynthetic cytochromes P450 with broader substrate and regio-specificity are growing in number and are of particular interest for biosynthetic and chemoenzymatic applications. MycG is among the first P450 monooxygenases characterized that catalyzes both hydroxylation and epoxidation reactions in the final biosynthetic steps, leading to oxidative tailoring of the 16-membered ring macrolide antibiotic mycinamicin II in the actinomycete Micromonospora griseorubida. The ordering of steps to complete the biosynthetic process involves a complex substrate recognition pattern by the enzyme and interplay between three tailoring modifications as follows: glycosylation, methylation, and oxidation. To understand the catalytic properties of MycG, we structurally characterized the ligand-free enzyme and its complexes with three native metabolites. These include substrates mycinamicin IV and V and their biosynthetic precursor mycinamicin III, which carries the monomethoxy sugar javose instead of the dimethoxylated sugar mycinose. The two methoxy groups of mycinose serve as sensors that mediate initial recognition to discriminate between closely related substrates in the post-polyketide oxidative tailoring of mycinamicin metabolites. Because x-ray structures alone did not explain the mechanisms of macrolide hydroxylation and epoxidation, paramagnetic NMR relaxation measurements were conducted. Molecular modeling based on these data indicates that in solution substrate may penetrate the active site sufficiently to place the abstracted hydrogen atom of mycinamicin IV within 6 Å of the heme iron and ~4 Å of the oxygen of iron-ligated water.

HIV-1 Protease and Substrate Coevolution Validates the Substrate Envelope As the Substrate Recognition Pattern

Drug resistance of HIV-1 protease alters the balance in the molecular recognition events in favor of substrate processing versus inhibitor binding. To develop robust inhibitors targeting ensembles of drug-resistant variants, the code of this balance needs to be cracked. For this purpose, the principles governing the substrate recognition are required to be revealed. Previous crystallographic studies on the WT protease-substrate complexes showed that the substrates have a conserved consensus volume in the protease active site despite their low sequence homology. This consensus volume is termed as the substrate envelope. The substrate envelope was recently reevaluated by taking the substrate dynamics into account, and the dynamic substrate envelope was reported to better define the substrate specificity for HIV-1 protease. Drug resistance occurs mostly through mutations in the protease, occasionally accompanied by cleavage site mutations. In this study, three coevolved protease-substrate complexes (AP2VNC-p1V82A, LP1'Fp1-p6D30N/N88D, and SP3'Np1-p6D30N/N88D) were investigated for structural and dynamic properties by molecular modeling and dynamics simulations. The results show the substrate envelope is preserved by these cleavage site mutations in the presence of drug-resistance mutations in the protease, if not enhanced. This study on the conformational and mutational ensembles of protease-substrate complexes validates the substrate envelope as the substrate recognition motif for HIV-1 protease. The substrate envelope hypothesis allows for the elucidation of possible drug resistance mutation patterns in the polyprotein cleavage sites.

Understanding the structural and energetic basis of inhibitor and substrate bound to the full-length NS3/4A: insights from molecular dynamics simulation, binding free energy calculation and network analysis

Hepatitis C virus (HCV) bifunctional NS3/4A is an attractive anti-HCV drug target, as both the protease and helicase functions are required for viral infection and replication. Although the first generation of NS3/4A protease inhibitors (PIs) has focused almost exclusively on the interaction with the protease domain alone, recent studies have shown that PIs also inhibit the full-length NS3/4A protein. However, the detailed molecular mechanism of the interaction between protease inhibitors, as well as the peptide substance with the full-length NS3/4A protein, remains poorly understood. Herein, starting from the recently determined crystal structure of an inhibitor (inhibitor ) bound to the full-length NS3/4A protein, the structures of the full-length NS3/4A complexed with inhibitor ITMN-191 (by InterMune/Roche; Phase II) and substrate 4B5A (the viral cleavage product peptide) were built. Then, residue interaction network (RIN) analysis, molecular dynamics (MD) simulation, binding free energy calculation, decomposition of free energies on per-residue and dynamic substrate recognition pattern analysis were employed to uncover the structural and energetic basis of inhibitor and substrate binding mode in the binding cleft located at the interface of the protease and helicase domains of the full-length NS3/4A. The results from our study reveal that both the protease and helicase residues of the NS3/4A participate in the interactions with the inhibitor , ITMN-191 and 4B5A. Additional analysis of the NS3/4A substrate and inhibitor envelopes reveals the areas where the consensus inhibitor volume extended beyond the substrate envelope. These areas correspond to drug resistance mutations including Arg155, Ala156 and Asp168 at the protease active site as well as the two conserved helicase residues Gln526 and His528 that strongly interact with the inhibitors. Thus, the findings of this study will be very useful for understanding the interaction mechanism between the inhibitor (substrate) and NS3/4A and also for the rational design and development of new potent molecules targeting the full-length NS3/4A.

PET and SPECT Radiotracers to Assess Function and Expression of ABC Transporters In Vivo

Adenosine triphosphate-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp, ABCB1), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance-associated proteins (MRPs) are expressed in high concentrations at various physiological barriers (e.g. blood-brain barrier, blood-testis barrier, blood-tumor barrier), where they impede the tissue accumulation of various drugs by active efflux transport. Changes in ABC transporter expression and function are thought to be implicated in various diseases, such as cancer, epilepsy, Alzheimer's and Parkinson's disease. The availability of a non-invasive imaging method which allows for measuring ABC transporter function or expression in vivo would be of great clinical use in that it could facilitate the identification of those patients that would benefit from treatment with ABC transporter modulating drugs. To date three different kinds of imaging probes have been described to measure ABC transporters in vivo: i) radiolabelled transporter substrates ii) radiolabelled transporter inhibitors and iii) radiolabelled prodrugs which are enzymatically converted into transporter substrates in the organ of interest (e.g. brain). The design of new imaging probes to visualize efflux transporters is inter alia complicated by the overlapping substrate recognition pattern of different ABC transporter types. The present article will describe currently available ABC transporter radiotracers for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) and critically discuss strengths and limitations of individual probes and their potential clinical applications.

Comparison of Contemporary Feature Selection Algorithms: Application to the Classification of ABC‐Transporter Substrates

AbstractMultidrug ABC‐transporters are highly polyspecific in their substrate recognition pattern and influence the pharmacokinetics of a broad variety of structurally diverse compounds. Thus, prediction of ABC‐transporter substrate properties of compound libraries is of major interest. In this study, we use k‐nearest neighbor (kNN) classification in combination with five different feature subset selection (FS) algorithms to create predictive models for classification of ABCB1, ABCC1, and ABCG2 substrates. Our results show that FS methods that incorporate the classification algorithm give the best results and contain only a small subset of descriptors. For ABCB1 and ABCG2 cross validated accuracies of higher than 80% were achieved. The interpretation of the best performing feature subsets showed that descriptors consisting of simple counts as well as of projections of physicochemical properties on subdivided surface areas have highest contribution to the models.

Functional and Structural Characterization of a Prokaryotic Peptide Transporter with Features Similar to Mammalian PEPT1

The ydgR gene of Escherichia coli encodes a protein of the proton-dependent oligopeptide transporter (POT) family. We cloned YdgR and overexpressed the His-tagged fusion protein in E. coli BL21 cells. Bacterial growth inhibition in the presence of the toxic phosphonopeptide alafosfalin established YgdR functionality. Transport was abolished in the presence of the proton ionophore carbonyl cyanide p-chlorophenylhydrazone, suggesting a proton-coupled transport mechanism. YdgR transports selectively only di- and tripeptides and structurally related peptidomimetics (such as aminocephalosporins) with a substrate recognition pattern almost identical to the mammalian peptide transporter PEPT1. The YdgR protein was purified to homogeneity from E. coli membranes. Blue native-polyacrylamide gel electrophoresis and transmission electron microscopy of detergent-solubilized YdgR suggest that it exists in monomeric form. Transmission electron microscopy revealed a crown-like structure with a diameter of approximately 8 nm and a central density. These are the first structural data obtained from a proton-dependent peptide transporter, and the YgdR protein seems an excellent model for studies on substrate and inhibitor interactions as well as on the molecular architecture of cell membrane peptide transporters.

Cleavage preference distinguishes the two-component NS2B–NS3 serine proteinases of Dengue and West Nile viruses

Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1'-P2'-P3'-P4'-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1' or the P2' site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.