L1 Loop Research Articles

Why do the outer membrane proteins OmpF from E. coli and OprP from P. aeruginosa prefer trimers? Simulation studies

Porins are water-filled protein channels across the outer membrane of gram-negative bacteria. They facilitate the uptake of nutrients and essential ions. Solutes are filtered by a constriction loop L3 at the mid of a pore. Porins are heat-stable and resistant to toxic agents and detergents. Most porins are trimer, but no clear explanation why trimeric form is preferable. In this work, we thus studied effects of oligomerization on porin structure and function in microscopic detail. A well-studied OmpF (general porin from Escherichia coli) and well-characterised OprP (phosphate-specific pore from Pseudomonas aeruginosa) are used as samples from 2 types of porins found in gram-negative bacteria. MD simulations of trimeric and monomeric pores in pure water and 1M NaCl solution were performed. With a salt solution, the external electric field was applied to mimic a transmembrane potential. Expectedly, OprP is more stable than OmpF. Interestingly, being a monomer turns OmpF into an anion-selective pore. The dislocation of D113’s side chain on L3 in OmpF causes the disruption of cation pathway resulting in the reduction of cation influx. In contrast, OprP's structure and function are less dependent on oligomeric states. Both monomeric and trimeric OprP can maintain their anion selectivity. Our findings suggest that trimerization is crucial for both structure and function of general porin OmpF, whereas being trimer in substrate-specific channel OprP supports a pore function.

Conformational Effects of the C-terminal Tail on the Human Neuronal Calcium Sensor-1 Protein: An Atomistic Simulation Study

Functional neuronal calcium sensor-1 protein plays multiple neuronal functions and binds partners mostly through a largely exposed hydrophobic crevice (HC). The C-terminal tail (loop L3, spanning residues D176∼V190) binds directly to the HC pocket and regulates its conformational stability in the absence of a ligand. A NMR experimental study demonstrated that L3 deletion resulted in global structure destabilization. However, the influence of C-terminal deletion on the conformations of NCS-1 protein is unclear at atomic level. In order to delineate the role of L3 in human NCS-1 protein, we investigated the structural properties and the conformational dynamics of wild type (WT) NCS-1 and the L3 truncation variant by extensive all-atom molecular dynamics simulations. Our multiple simulations starting from two different initial structures demonstrated that the secondary structure content of WT NCS-1 was closely similar to that of the L3 truncation variant, consistent with a recent NMR experimental study. The structural flexibilities of the C-domain and the N-domain distal to C-terminal tail were increased, indicating local and global perturbations of C-terminal tail on the conformation of NCS-1 protein. The global structure and HC were simultaneously expanded, inducing the HC to be fully exposed to the solvent. The salt-bridges, especially in the C-domain, were significantly disrupted. The community network analysis illustrated that the L3 truncation weakened the inter-domain correlation and altered the residue-residue connections. These results reveal that L3 plays a critical role in the conformation dynamics and these structural changes caused by the variant may affect the regulation of target interactions. Our study provides atomic details for the conformational dynamics effects of the C-terminal tail on human wild type NCS-1.

The Structural Kinetics of Switch-1 and the Neck Linker Explain the Functions of Kinesin-1 and Eg5

Kinesins perform mechanical work in order to power a variety of cellular functions. Distinct functions shape distinct enzymologies, and this is illustrated by comparing kinesin-1, a highly processive transport motor that can work alone, to Eg5, a minimally processive mitotic motor that works in large ensembles. While crystallographic models for both motors reveal similar structures for the domains involved in mechanochemical transduction—including switch-1 and the neck linker—how movement of these two domains is coordinated through the ATPase cycle remains unknown. We have addressed this issue by utilizing a novel method—transient, time resolved fluorescence resonance energy transfer (TR2-FRET)—in order to measure the mole fractions of docked and undocked neck linker and closed and open switch 1 in the absence of nucleotide and to monitor how these equilibria change after mixing with nucleotide. This allows us to conclude that: 1) in kinesin-1, the conformational equilibria of the neck linker and switch 1 are closely coordinated, while in Eg5, they are not; 2) in kinesin 1, ATP binding is rate limited by the closed→open conformational change in switch 1, while in Eg5, it is rate limited by a conformational change in loop L5. We find that differences between the structural kinetics of Eg5 and kinesin-1 yield insights into how these two motors adapt their enzymologies for their distinct functions.

Effects of H2A Histone Variants on DNA Sequence and Nucleosome Structure using Coarse Grain Simulations

Coarse grain molecular dynamics (CG-MD) simulations are seeing a rising tendency in a wide range of applications because their potential to enhance the sampling of all-atomistic simulations (AA-MD) and the capacity to make near-atomistic millisecond-timescale simulations practical, putting the second threshold on the horizon. In this field, the coarse-grained Martini force field has a prominent position and has recently extended its already range of applications to DNA-containing biomolecules [1].Eukaryotic genomic DNA exists as highly compacted nucleosome arrays called chromatin. Each nucleosome contains a 147-base-pair (bp) stretch of DNA, which is sharply bent and tightly wrapped around a histone protein octamer [2]. Several mechanisms regulate DNA accessibility, including replacement of canonical histones with specialized histone variants. Among the core histone variants, the H2A family is the biggest one -to date 5 histones variants are documented- showing also the highest sequence divergence among histone families. H2A variants show striking differences in three sites that are critical in intra- and inter-nucleosome interactions: the docking domain, which is close to the DNA entry/exit region, the L1 loop and the acidic patch, which is involved in nucleosome-nucleosome interactions. We have studied the structural and dynamic differences in those regions among H2A histone variants-containing nucleosome structures.Furthermore, it has been shown that DNA sequence effects vary between nucleosomes resulting in a significant factor in their stability. For this reason, we have also analyzed the differences in the associated DNA flexibility along the wrapped DNA sequence derived from MD simulations.

Allosteric Networks in the Nucleosome Core Particle

Eukaryotic organisms package their genetic material using the nucleosome core particle (NCP), a histone octamer that stably wraps approximately 146 basepairs of DNA. Accessibility of DNA is modulated by a variety of biochemical processes, including post-translational modifications (PTMs) and histone variant substitution. The significant global effects resulting from such highly localized changes suggest the presence of allosteric mechanisms. In this molecular dynamics study, we present a comprehensive analysis of allosteric networks within the canonical NCP and nucleosomes containing transcription-related H2A variants: H2A.Z (modulator), macroH2A (repressor), and one in which the canonical H2A sequence was mutated to possess macroH2A-like L1 loops. While these variants are structurally similar to the canonical form, the altered sequence at key locations - such as the dimer-tetramer and the dimer-dimer (L1-L1) interfaces - provides significant dynamic differences. We observe that macroH2A systems rely most heavily on the L1 loops for propagating dynamic correlations, while the major type and H2A.Z nucleosomes display a preference for utilizing DNA base-pairs and protein-DNA contacts. The increased intra-protein correlation of macroH2A systems creates stronger dynamic couplings throughout the entire NCP. Lastly, we quantify the importance of individual residues to networks within each molecule via the betweenness centrality metric and identify several known PTM sites in the canonical system. These data suggest that strategic changes caused by PTMs may alter stability locally as well as globally via these networks. Furthermore, variant inclusion alters the structure of allosteric pathways and may act to modulate chromatin stability.

Non-Canonical Microtubule Interaction by Yeast Kinesin-5, Cin8

Kinesin-5's were originally described as slow, plus-end-directed motors that are required for proper establishment and maintenance of the mitotic spindle. They share a bipolar homotetrameric structure with four heavy chains organized such that two motor domains are positioned at each end of a coiled-coil stalk, allowing the motor to cross-link and slide two antiparallel microtubules. Interestingly, recent studies have demonstrated bidirectional motility for yeast kinesin-5 motors, which represents a significant biophysical conundrum given our previous understanding of directed kinesin motility. We aim to understand the structural and biochemical features of yeast kinesin-5 motors that promote directional switching of motility. Saccharomyces cerevisiae Cin8 has been shown to switch directionality based on ionic strength, motor coupling and binding between antiparallel microtubules. Using ionic strength to promote switching of directionality, we have determined the microtubule binding behavior, steady-state kinetics, and cryo-EM structure of the Cin8 motor domain. Cosedimentation assays revealed that Cin8 binds super-stoichiometrically along the microtubule lattice with 3±1 motor domains binding per tubulin dimer. Competition assays with human kinesin-5 (Eg5) indicate that Cin8 binds at the canonical kinesin binding site as well as at a yet unidentified non-canonical site. Interestingly, Cin8 has microtubule-stimulated ATPase activity on both the canonical and non-canonical sites. We have probed the structural explanation for these biochemical observations by obtaining a 7Å cryo-EM reconstruction of the microtubule-Cin8 complex in the ADP+AlFx state. Deletion of the large insert in Cin8's loop L8 retains similar steady-state ATPase activity yet abolishes super-stoichiometric microtubule binding, providing a powerful tool to dissect its ATPase mechanism. Cin8's microtubule interaction offers a mechanism for bidirectional movement, such that the motor utilizes a non-canonical microtubule site for directional switching.

Synthesis of Fluorescent-NTA and its Application to the Labeling of Photo-Controlled Kinesin Eg5

All members of the kinesin superfamily contain a structurally conserved loop L5 near the ATP-binding site. The length and the amino acids composition of L5 vary among the kinesin superfamily members. It is believed that L5 may be related to the enzymatic and motor function of kinesin. Interestingly, L5 of Eg5 is uniquely longer than that of other kinesins. Moreover, it was demonstrated that this loop undergoes conformational changes that are related to the nucleotide binding and neck linker docking. Previously we attempted to control Eg5 function photo-reversibly by incorporating photochromic molecules into the L5. We prepared Eg5 mutants, E116C, E118C, T125C, W127C, D130C, which have a single cysteine residue in L5 in order to incorporate photochromic molecules specifically. The Eg5 mutants modified with azobenzene and spiropyran derivatives showed photo-reversible alteration of microtubules dependent ATPase activities.In this study, we synthesized a novel thiol reactive photochromic molecule, monoiodoacetyl-flugide (IAFG). Fulgimide performs photo-reversible isomerization between non-polar opened-ring form and polar closed-ring form upon visible light and ultraviolet light, respectively. IAFG was incorporated into Eg5 mutant W127C stoichiometrically. Although the modified Eg5 mutant W127C-IAFG showed slightly decreased ATPase activity, the ATPase activity showed photo-reversible alteration upon UV and visible light irradiations. We utilized fluorescent probe bound NTA-Ni to label the His-tagged Eg5 modified with photochromic molecules. According to the methods of Soh et al., Dansyl-NTA and other fluorescent-NTA were synthesized and conjugated with Ni2+. The Dansyl-NTA-Ni2+ bound to His tagged Eg5. Using the fluorescent NTA-Ni2+, interaction of photo-regulated Eg5 with microtubules was monitored.

Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus

Psychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic β-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (Tm). Mutation of His299 to Tyr increased the optimal temperature, the Tm, and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures.

Effects of MacroH2A and H2A.Z on Nucleosome Dynamics as Elucidated by Molecular Dynamics Simulations

Eukaryotes tune the transcriptional activity of their genome by altering the nucleosome core particle through multiple chemical processes. In particular, replacement of the canonical H2A histone with the variants macroH2A and H2A.Z has been shown to affect DNA accessibility and nucleosome stability; however, the processes by which this occurs remain poorly understood. In this study, we elucidate the molecular mechanisms of these variants with an extensive molecular dynamics study of the canonical nucleosome along with three variant-containing structures: H2A.Z, macroH2A, and an H2A mutant with macroH2A-like L1 loops. Simulation results show that variant L1 loops play a pivotal role in stabilizing DNA binding to the octamer through direct interactions, core structural rearrangements, and altered allosteric networks in the nucleosome. All variants influence dynamics; however, macroH2A-like systems have the largest effect on energetics. In addition, we provide a comprehensive analysis of allosteric networks in the nucleosome and demonstrate that variants take advantage of stronger interactions between L1 loops to propagate dynamics throughout the complex. Furthermore, we show that posttranslational modifications are enriched at key locations in these networks. Taken together, these results provide, to our knowledge, new insights into the relationship between the structure, dynamics, and function of the nucleosome core particle and chromatin fibers, and how they are influenced by chromatin remodeling factors.

Cluster Analysis of p53 Binding Site Sequences Reveals Subsets with Different Functions.

p53 is an important regulator of cell cycle arrest, senescence, apoptosis and metabolism, and is frequently mutated in tumors. It functions as a tetramer, where each component dimer binds to a decameric DNA region known as a response element. We identify p53 binding site subtypes and examine the functional and evolutionary properties of these subtypes. We start with over 1700 known binding sites and, with no prior labeling, identify two sets of response elements by unsupervised clustering. When combined, they give rise to three types of p53 binding sites. We find that probabilistic and alignment-based assessments of cross-species conservation show no strong evidence of differential conservation between types of binding sites. In contrast, functional analysis of the genes most proximal to the binding sites provides strong bioinformatic evidence of functional differentiation between the three types of binding sites. Our results are consistent with recent structural data identifying two conformations of the L1 loop in the DNA binding domain, suggesting that they reflect biologically meaningful groups imposed by the p53 protein structure.

Pathogenicity and Genotyping of Fowl Adenoviruses Associated with Gizzard Erosion in Commerciallayer Grower Chicken in India

During the year 2013–2014, commercial layer birds showed heavy economic losses due to gizzard erosion (GE) and mortality (10 to 30%). The affected birds revealed dullness, uneven growth, decreased feed and water intake. GE with detachment of the koilin layer and blackish discoloration of gizzard contents were observed during necropsy examination. Fowl adenoviruses (FAdVs) were isolated using passage in chicken embryo liver cells and genotyped by DNA sequencing. Phylogenetic analysis of the hexon loop L1 gene FAdVs isolates was grouped into FAdV-A (serotype 1), FAdV-C (serotype 4), D (serotypes 2, 3 and 11) and E (serotype 8) species. The pathogenicity of FAdVs was investigated in 4-week-old specific pathogen-free chickens; FAdV serotypes 4, 8 and 11 were found pathogenic and cause GE. Histologically, basophilic intranuclear inclusion bodies were seen in the gizzard mucosal layer from day 7 to 11-day post infection. This is the first report of the GE associated with FAdV serotypes 4, 8 and 11 in layer birds. These findings suggest that GE outbreaks associated with FAdVs lead to economic losses in commercial layer grower birds in India.

Loop dynamics of thymidine diphosphate-rhamnose 3'-O-methyltransferase (CalS11), an enzyme in calicheamicin biosynthesis.

Structure analysis and ensemble refinement of the apo-structure of thymidine diphosphate (TDP)-rhamnose 3′-O-methyltransferase reveal a gate for substrate entry and product release. TDP-rhamnose 3′-O-methyltransferase (CalS11) catalyses a 3′-O-methylation of TDP-rhamnose, an intermediate in the biosynthesis of enediyne antitumor antibiotic calicheamicin. CalS11 operates at the sugar nucleotide stage prior to glycosylation step. Here, we present the crystal structure of the apo form of CalS11 at 1.89 Å resolution. We propose that the L2 loop functions as a gate facilitating and/or providing specificity for substrate entry or promoting product release. Ensemble refinement analysis slightly improves the crystallographic refinement statistics and furthermore provides a compelling way to visualize the dynamic model of loop L2, supporting the understanding of its proposed role in catalysis.

Molecular dynamics simulations elucidate the mode of protein recognition by Skp1 and the F-box domain in the SCF complex.

Polyubiquitination of the target protein by a ubiquitin transferring machinery is key to various cellular processes. E3 ligase Skp1-Cul1-F-box (SCF) is one such complex which plays crucial role in substrate recognition and transfer of the ubiquitin molecule. Previous computational studies have focused on S-phase kinase-associated protein 2 (Skp2), cullin, and RING-finger proteins of this complex, but the roles of the adapter protein Skp1 and F-box domain of Skp2 have not been determined. Using sub-microsecond molecular dynamics simulations of full-length Skp1, unbound Skp2, Skp2-Cks1 (Cks1: Cyclin-dependent kinases regulatory subunit 1), Skp1-Skp2, and Skp1-Skp2-Cks1 complexes, we have elucidated the function of Skp1 and the F-box domain of Skp2. We found that the L16 loop of Skp1, which was deleted in previous X-ray crystallography studies, can offer additional stability to the ternary complex via its interactions with the C-terminal tail of Skp2. Moreover, Skp1 helices H6, H7, and H8 display vivid conformational flexibility when not bound to Skp2, suggesting that these helices can recognize and lock the F-box proteins. Furthermore, we observed that the F-box domain could rotate (5°-129°), and that the binding partner determined the degree of conformational flexibility. Finally, Skp1 and Skp2 were found to execute a domain motion in Skp1-Skp2 and Skp1-Skp2-Cks1 complexes that could decrease the distance between ubiquitination site of the substrate and the ubiquitin molecule by 3 nm. Thus, we propose that both the F-box domain of Skp2 and Skp1-Skp2 domain motions displaying preferential conformational control can together facilitate polyubiquitination of a wide variety of substrates.

Role of Residues W228 and Y233 in the Structure and Activity of Metallo-β-Lactamase GIM-1.

Metallo-β-lactamases (MBLs) hydrolyze virtually all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. The worldwide emergence of antibiotic-resistant bacteria harboring MBLs poses an increasing clinical threat. The MBL German imipenemase-1 (GIM-1) possesses an active site that is narrower and more hydrophobic than the active sites of other MBLs. The GIM-1 active-site groove is shaped by the presence of the aromatic side chains of tryptophan at residue 228 and tyrosine at residue 233, positions where other MBLs harbor hydrophilic residues. To investigate the importance of these two residues, eight site-directed mutants of GIM-1, W228R/A/Y/S and Y233N/A/I/S, were generated and characterized using enzyme kinetics, thermostability assays, and determination of the MICs of representative β-lactams. The structures of selected mutants were obtained by X-ray crystallography, and their interactions with β-lactam substrates were modeled in silico. Steady-state kinetics revealed that both positions are important to GIM-1 activity but that the effects of individual mutations vary depending on the β-lactam substrate. Activity against type 1 substrates bearing electron-donating C-3/C-4 substituents (cefoxitin, meropenem) could be enhanced by mutations at position 228, whereas hydrolysis of type 2 substrates (benzylpenicillin, ampicillin, ceftazidime, imipenem) with methyl or positively charged substituents was favored by mutations at position 233. The crystal structures showed that mutations at position 228 or the Y233A variant alters the conformation of GIM-1 loop L1 rather than that of loop L3, on which the mutations are located. Taken together, these data show that point mutations at both positions 228 and 233 can influence the catalytic properties and the structure of GIM-1.

Structural and Dynamic Basis for Low-Affinity, High-Selectivity Binding of L-Glutamine by the Glutamine Riboswitch.

Naturally occurring L-glutamine riboswitches occur in cyanobacteria and marine metagenomes, where they reside upstream of genes involved in nitrogen metabolism. By combining X-ray, NMR, and MD, we characterized an L-glutamine-dependent conformational transition in the Synechococcus elongatus glutamine riboswitch from tuning fork to L-shaped alignment of stem segments. This transition generates an open ligand-binding pocket with L-glutamine selectivity enforced by Mg(2+)-mediated intermolecular interactions. The transition also stabilizes the P1 helix through a long-range "linchpin" Watson-Crick G-C pair-capping interaction, while melting a short helix below P1 potentially capable of modulating downstream readout. NMR data establish that the ligand-free glutamine riboswitch in Mg(2+) solution exists in a slow equilibrium between flexible tuning fork and a minor conformation, similar, but not identical, to the L-shaped bound conformation. We propose that an open ligand-binding pocket combined with a high conformational penalty for forming the ligand-bound state provide mechanisms for reducing binding affinity while retaining high selectivity.

The p53 tetramer shows an induced-fit interaction of the C-terminal domain with the DNA-binding domain.

The Trp53 gene is the most frequently mutated gene in all human cancers. Its protein product p53 is a very powerful transcription factor that can activate different biochemical pathways and affect the regulation of metabolism, senescence, DNA damage response, cell cycle and cell death. The understanding of its function at the molecular level could be of pivotal relevance for therapy. Investigation of long-range intra- and interdomain communications in the p53 tetramer–DNA complex was performed by means of an atomistic model that included the tetramerization helices in the C-terminal domain, the DNA-binding domains and a consensus DNA-binding site of 18 base pairs. Nonsymmetric dynamics are illustrated in the four DNA-binding domains, with loop L1 switching from inward to outward conformations with respect to the DNA major groove. Direct intra- and intermonomeric long-range communications between the tetramerization and DNA-binding domains are noted. These long-distance conformational changes link the C terminus with the DNA-binding domain and provide a biophysical rationale for the reported functional regulation of the p53 C-terminal region. A fine characterization of the DNA deformation caused by p53 binding is obtained, with ‘static' deformations always present and measured by the slide parameter in the central thymine–adenine base pairs; we also detect ‘dynamic' deformations switched on and off by particular p53 tetrameric conformations and measured by the roll and twist parameters in the same base pairs. These different conformations can indeed modulate the electrostatic potential isosurfaces of the whole p53–DNA complex. These results provide a molecular/biophysical understanding of the evident role of the C terminus in post-translational modification that regulates the transcriptional function of p53. Furthermore, the unstructured C terminus is able to facilitate contacts between the core DNA-binding domains of the tetramer.

Anionic Phospholipids Stabilize RecA Filament Bundles in Escherichia coli

We characterize the interaction of RecA with membranes in vivo and in vitro and demonstrate that RecA binds tightly to the anionic phospholipids cardiolipin (CL) and phosphatidylglycerol (PG). Using computational models, we identify two regions of RecA that interact with PG and CL: (1) the N-terminal helix and (2) loop L2. Mutating these regions decreased the affinity of RecA to PG and CL in vitro. Using 3D super-resolution microscopy, we demonstrate that depleting Escherichia coli PG and CL altered the localization of RecA foci and hindered the formation of RecA filament bundles. Consequently, E. coli cells lacking aPLs fail to initiate a robust SOS response after DNA damage, indicating that the membrane acts as a scaffold for nucleating the formation of RecA filament bundles and plays an important role in the SOS response.

Effects of the C-Terminal Tail on the Conformational Dynamics of Human Neuronal Calcium Sensor-1 Protein.

Neuronal calcium sensor-1 (NCS-1) protein has been implicated in multiple neuronal functions by binding partners mostly through a largely exposed hydrophobic crevice (HC). In the absence of a ligand, the C-terminal tail (loop L3, residues D176 to V190) binds directly to the HC pocket as a ligand mimetic, occupying the HC and regulating its conformational stability. A recent experimental study reported that L3 deletion resulted in global structure destabilization. However, the influence of C-terminal tail on the conformations of NCS-1 protein is unclear at the atomic level. In this study, we investigated the structural properties and the conformational dynamics of wild type NCS-1 and L3 truncation variant by extensive all-atom molecular dynamics (MD) simulations. Our cumulative 2 μs MD simulations demonstrated that L3 deletion increased the structural flexibility of the C-domain and the distant N-domain. The community network analysis illustrated that C-terminal tail truncation weakened the interdomain correlation. Moreover, our data showed that the variant significantly disrupted the salt bridges network and expanded simultaneously the global structure and HC. These conformational changes caused by C-terminal tail truncation may affect the regulation of target interactions. Our study provides atomic details of the conformational dynamics effects of the C-terminal tail on human wild type NCS-1.

Phylogenetic and geographic analysis of fowl adenovirus field strains isolated from poultry in Poland.

Fowl adenoviruses (FAdVs) are widely distributed in chickens in Poland and throughout the world. FAdV infections have been reported in the United States, Australia, Europe, and the Mediterranean basin. Detection of FAdVs strains is very important from the epidemiological point of view and for monitoring disease outbreaks and developing strategies for vaccine development. Several molecular epidemiology and phylogenetic studies have been performed, but the results obtained are still limited, because FAdV strains, even of the same serotype, have very diverse characteristics. Some strains are pathogenic and some are nonpathogenic. This report describes the successful isolation of 96 FAdV field strains from chickens in Poland. A PCR assay specific for the L1 loop region of the hexon gene was conducted, and the products were subjected to sequence analysis. The sequences were analysed using BLAST and Geneious 6.0 software and compared to adenovirus field and reference strain sequences from different parts of the world that are accessible in the NCBI GenBank database. The sequences of the adenovirus strains indicated that they belonged to five species, Fowl aviadenovirus A-E, represented by eight serotypes FAdV-1, FAdV-4, FAdV-5, FAdV-7, FAdV-8a, FAdV-8b, and FAdV-2/11 (FAdV-D). The relationships between FAdVs isolated in Poland and isolates from other regions of the world were determined.

Synthetic Studies on Centromere-Associated Protein-E (CENP-E) Inhibitors: 2. Application of Electrostatic Potential Map (EPM) and Structure-Based Modeling to Imidazo[1,2-a]pyridine Derivatives as Anti-Tumor Agents.

To develop centromere-associated protein-E (CENP-E) inhibitors for use as anticancer therapeutics, we designed novel imidazo[1,2-a]pyridines, utilizing previously discovered 5-bromo derivative 1a. By site-directed mutagenesis analysis, we confirmed the ligand binding site. A docking model revealed the structurally important molecular features for effective interaction with CENP-E and could explain the superiority of the inhibitor (S)-isomer in CENP-E inhibition vs the (R)-isomer based on the ligand conformation in the L5 loop region. Additionally, electrostatic potential map (EPM) analysis was employed as a ligand-based approach to optimize functional groups on the imidazo[1,2-a]pyridine scaffold. These efforts led to the identification of the 5-methoxy imidazo[1,2-a]pyridine derivative (+)-(S)-12, which showed potent CENP-E inhibition (IC50: 3.6 nM), cellular phosphorylated histone H3 (p-HH3) elevation (EC50: 180 nM), and growth inhibition (GI50: 130 nM) in HeLa cells. Furthermore, (+)-(S)-12 demonstrated antitumor activity (T/C: 40%, at 75 mg/kg) in a human colorectal cancer Colo205 xenograft model in mice.