Current Science - Biochemistry Publications

Targeting a prokaryotic protein in a eukaryotic pathogen:  identification of lead compounds against Cryptosporidiosis

Nwakaso N. Umejiego, Deviprasad Gollapalli, Lisa Sharling, Anna Volftsun, Jennifer Lu,
Nicole N. Benjamin, Adam H. Stroupe, Thomas V. Riera, Boris Striepen and Lizbeth Hedstrom
Chem Biol. 2008 Jan;15:70-7. [more information]

Cryptosporidium parvum is an important human pathogen and potential bioterrorism agent. No vaccines exist against C. parvum, the drugs currently approved to treat cryptosporidiosis are ineffective, and drug discovery is challenging because the parasite cannot be maintained continuously in cell culture. Mining the sequence of the C. parvum genome has revealed that the only route to guanine nucleotides is via inosine-5'-monophosphate dehydrogenase (IMPDH). Moreover, phylogenetic analysis suggests that the IMPDH gene was obtained from bacteria by lateral gene transfer. Here we exploit the unexpected evolutionary divergence of parasite and host enzymes by designing a high-throughput screen to target the most diverged portion of the IMPDH active site. We have identified four parasite-selective IMPDH inhibitors that display antiparasitic activity with greater potency than paromomycin, the current gold standard for anticryptosporidial activity.

Transducin activation by nanoscale lipid bilayers containing one and two rhodopsins

Bayburt TH, Leitz AJ, Xie G, Oprian DD, Sligar SG.
The Journal of Biological Chemistry, 2007 [Epub]

Nanodiscs are nanometer scale planar membranes of controlled size that are rendered soluble in aqueous solution via an encircling amphipathic membrane scaffold protein "belt" (Bayburt, T. H., Grinkova, Y. V., and Sligar, S. G. (2002) Nano Letters 2, 853-856.). Integral membrane proteins can be self-assembled into the Nanodisc bilayer with defined stoichiometry which allows an unprecedented opportunity to investigate the nature of the oligomerization state of a G-protein coupled receptor and its coupling to heterotrimeric G-proteins. We generated Nanodiscs having one and two rhodopsins present in the 10 nm diameter lipid bilayer domain. Efficient transducin activation and isolation of a high-affinity transducin-Metarhodopsin II complex was demonstrated for a monodisperse and monomeric receptor. A population of Nanodiscs containing two rhodopsins was generated using an increased ratio of receptor to membrane scaffold protein in the self-assembly mixture. The two rhodopsin population was isolated and purified by density gradient centrifugation. Interestingly, in this case only one of the two receptors present in the Nanodisc were able to form a stable metarhodopsin II - G-protein complex. Thus there is clear evidence that a monomeric rhodopsin is capable of full coupling to transducin. Importantly, presumably due to steric interactions, it appears that only a single receptor in the Nanodiscs containing two rhodopsins can interact with G-protein. These results have important implications for the stoichiometry of receptor-G-protein coupling and cross talk in signaling pathways.

Structure and dynamics of pin1 during catalysis by NMR

Labeikovsky W, Eisenmesser EZ, Bosco DA, Kern D
Journal of Molecular Biology, 2007; 367:1370-81

The link between internal enzyme motions and catalysis is poorly understood. Correlated motions in the microsecond-to-millisecond timescale may be critical for enzyme function. We have characterized the backbone dynamics of the peptidylprolyl isomerase (Pin1) catalytic domain in the free state and during catalysis. Pin1 is a prolyl isomerase of the parvulin family and specifically catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds. Pin1 has been shown to be essential for cell-cycle progression and to interact with the neuronal tau protein inhibiting its aggregation into fibrillar tangles as found in Alzheimer's disease. (15)N relaxation dispersion measurements performed on Pin1 during catalysis reveal conformational exchange processes in the microsecond timescale. A subset of active site residues undergo kinetically similar exchange processes even in the absence of a substrate, suggesting that this area is already "primed" for catalysis. Furthermore, structural data of the turning-over enzyme were obtained through inter- and intramolecular nuclear Overhauser enhancements. This analysis together with a characterization of the substrate concentration dependence of the conformational exchange allowed the distinguishing of regions of the enzyme active site that are affected primarily by substrate binding versus substrate isomerization. Together these data suggest a model for the reaction trajectory of Pin1 catalysis.

Uncoupling and turnover in a Cl-/H+ exchange transporter

Walden M, Accardi A, Wu F, Xu C, Williams C, Miller C.
The Journal of General Physiology, 2007;129:317-29

The CLC-family protein CLC-ec1, a bacterial homologue of known structure, stoichiometrically exchanges two Cl(-) for one H(+) via an unknown membrane transport mechanism. This study examines mutations at a conserved tyrosine residue, Y445, that directly coordinates a Cl(-) ion located near the center of the membrane. Mutations at this position lead to "uncoupling," such that the H(+)/Cl(-) transport ratio decreases roughly with the volume of the substituted side chain. The uncoupled proteins are still able to pump protons uphill when driven by a Cl(-) gradient, but the extent and rate of this H(+) pumping is weaker in the more uncoupled variants. Uncoupling is accompanied by conductive Cl(-) transport that is not linked to counter-movement of H(+), i.e., a "leak." The unitary Cl(-) transport rate, measured in reconstituted liposomes by both a conventional initial-velocity method and a novel Poisson dilution approach, is approximately 4,000 s(-1) for wild-type protein, and the uncoupled mutants transport Cl(-) at similar rates.

Structure of acid beta-glucosidase with pharmacological chaperone provides insight into Gaucher disease

Lieberman RL, Wustman BA, Huertas P, Powe AC Jr, Pine CW, Khanna R, Schlossmacher MG, Ringe D, Petsko GA
Nature Chemical Biology, 2007; 3:101-7

Gaucher disease results from mutations in the lysosomal enzyme acid beta-glucosidase (GCase). Although enzyme replacement therapy has improved the health of some affected individuals, such as those with the prevalent N370S mutation, oral treatment with pharmacological chaperones may be therapeutic in a wider range of tissue compartments by restoring sufficient activity of endogenous mutant GCase. Here we demonstrate that isofagomine (IFG, 1) binds to the GCase active site, and both increases GCase activity in cell lysates and restores lysosomal trafficking in cells containing N370S mutant GCase. We also compare the crystal structures of IFG-bound GCase at low pH with those of glycerol-bound GCase at low pH and apo-GCase at neutral pH. Our data indicate that IFG induces active GCase, which is secured by interactions with Asn370. The design of small molecules that stabilize substrate-bound conformations of mutant proteins may be a general therapeutic strategy for diseases caused by protein misfolding and mistrafficking.

Identification of functional subclasses in the DJ-1 superfamily proteins

Wei Y, Ringe D, Wilson MA, Ondrechen MJ
PLoS Computational Biology, 2007;3:e10

Genomics has posed the challenge of determination of protein function from sequence and/or 3-D structure. Functional assignment from sequence relationships can be misleading, and structural similarity does not necessarily imply functional similarity. Proteins in the DJ-1 family, many of which are of unknown function, are examples of proteins with both sequence and fold similarity that span multiple functional classes. THEMATICS (theoretical microscopic titration curves), an electrostatics-based computational approach to functional site prediction, is used to sort proteins in the DJ-1 family into different functional classes. Active site residues are predicted for the eight distinct DJ-1 proteins with available 3-D structures. Placement of the predicted residues onto a structural alignment for six of these proteins reveals three distinct types of active sites. Each type overlaps only partially with the others, with only one residue in common across all six sets of predicted residues. Human DJ-1 and YajL from Escherichia coli have very similar predicted active sites and belong to the same probable functional group. Protease I, a known cysteine protease from Pyrococcus horikoshii, and PfpI/YhbO from E. coli, a hypothetical protein of unknown function, belong to a separate class. THEMATICS predicts a set of residues that is typical of a cysteine protease for Protease I; the prediction for PfpI/YhbO bears some similarity. YDR533Cp from Saccharomyces cerevisiae, of unknown function, and the known chaperone Hsp31 from E. coli constitute a third group with nearly identical predicted active sites. While the first four proteins have predicted active sites at dimer interfaces, YDR533Cp and Hsp31 both have predicted sites contained within each subunit. Although YDR533Cp and Hsp31 form different dimers with different orientations between the subunits, the predicted active sites are superimposable within the monomer structures. Thus, the three predicted functional classes form four different types of quaternary structures. The computational prediction of the functional sites for protein structures of unknown function provides valuable clues for functional classification.

A bacterial arginine-agmatine exchange transporter involved in extreme acid resistance

Fang Y, Kolmakova-Partensky L, Miller C.
The Journal of Biological Chemistry, 2007;282:176-82

The arginine-dependent extreme acid resistance response of Escherichia coli operates by decarboxylating arginine. AdiC, a membrane antiporter, catalyzes arginine influx coupled to efflux of the decarboxylation product agmatine, effectively exporting a proton in each turnover. Using the adiC coding sequence under control of a tetracycline promoter in an E. coli vector, we expressed and purified the transport-protein with a yield of approximately 10 mg/liter bacterial culture. Glutaraldehyde cross-linking experiments indicate that the protein is a homodimer in detergent micelles and lipid membranes. Purified AdiC reconstituted into liposomes exchanges arginine and agmatine in a strictly coupled, electrogenic fashion. Kinetic analysis yields K(m) approximately 80 microm for Arg, in the same range as its dissociation constant determined by isothermal titration calorimetry.

FREALIGN: high-resolution refinement of single particle structures

Grigorieff N
Journal of Structural Biology, 2007;157:117-25

The refinement of three-dimensional reconstructions and correction for the contrast transfer function of the microscope are important steps in the determination of macromolecular structures by single particle electron microscopy. The algorithms implemented in the computer program FREALIGN are optimized to perform these tasks efficiently. A general overview and details on how to use FREALIGN are provided. The program is free and available for download on the author's web page.

Ab initio resolution measurement for single particle structures

Sousa D, Grigorieff N
Journal of Structural Biology, 2007;157:201-10

A computational method is described that allows the measurement of the signal-to-noise ratio and resolution of a three-dimensional structure obtained by single particle electron microscopy and reconstruction. The method does not rely on the availability of the original image data or the calculation of several structures from different parts of the data that are needed for the commonly used Fourier Shell Correlation criterion. Instead, the correlation between neighboring Fourier pixels is calculated and used to distinguish signal from noise. The new method has been conveniently implemented in a computer program called RMEASURE and is available to the microscopy community.

 

SIGNATURE: a single-particle selection system for molecular electron microscopy.

Chen JZ, Grigorieff N.
Journal of Structural Biology, 2007;157:168-73

SIGNATURE is a particle selection system for molecular electron microscopy. It applies a hierarchical screening procedure to identify molecular particles in EM micrographs. The user interface of the program provides versatile functions to facilitate image data visualization, particle annotation and particle quality inspection. The system design emphasizes both functionality and usability. This software has been released to the EM community and has been successfully applied to macromolecular structural analyses.

Current Science - Biophysics & Structural Biology Publications

Biological consequences of tightly bent DNA: the other life of a macromolecular celebrity

Garcia HG, Grayson P, Han L, Inamdar M, Kondev J, Nelson PC, Phillips R, Widom J, Wiggins PA.
Biopolymers. 2007;85:115-30.

The mechanical properties of DNA play a critical role in many biological functions. For example, DNA packing in viruses involves confining the viral genome in a volume (the viral capsid) with dimensions that are comparable to the DNA persistence length. Similarly, eukaryotic DNA is packed in DNA-protein complexes (nucleosomes), in which DNA is tightly bent around protein spools. DNA is also tightly bent by many proteins that regulate transcription, resulting in a variation in gene expression that is amenable to quantitative analysis. In these cases, DNA loops are formed with lengths that are comparable to or smaller than the DNA persistence length. The aim of this review is to describe the physical forces associated with tightly bent DNA in all of these settings and to explore the biological consequences of such bending, as increasingly accessible by single-molecule techniques.

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Current Science - Molecular & Cell Biology Publications

Tryptophan 32 potentiates aggregation and cytotoxicity of an sod-1 mutant associated with familial amyotrophic lateral sclerosis

Taylor DM, Gibbs BF, Kabashi E, Minotti S, Durham HD, Agar JN
The Journal of Biological Chemistry, 2007

One familial form of the neurodegenerative disease, amyotrophic lateral sclerosis, is caused by gain-of-function mutations in the gene encoding. This study provides in vivo evidence that normally occurring oxidative modification to SOD-1 promotes aggregation and toxicity of mutant proteins. The oxidation of Trp32 was identified as a normal modification of SOD-1, being present in both wild-type enzyme and SOD-1 with the disease-causing mutation, G93A, isolated from erythrocytes. Mutating Trp32 to a residue with a slower rate of oxidative modification, phenylalanine, decreased both the cytotoxicity of mutant SOD-1 and its propensity to form cytoplasmic inclusions in motor neurons of dissociated mouse spinal cord cultures.

Regulated binding of adenomatous polyposis coli protein to actin

Moseley JB, Bartolini F, Okada K, Wen Y, Gundersen GG, Goode BL
The Journal of Biological Chemistry, 2007, 282:12661-8

Adenomatous polyposis coli (APC) protein is a large tumor suppressor that is truncated in most colorectal cancers. The carboxyl-terminal third of APC protein mediates direct interactions with microtubules and the microtubule plus-end tracking protein EB1. In addition, APC has been localized to actin-rich regions of cells, but the mechanism and functional significance of this localization have remained unclear. Here we show that purified carboxyl-terminal basic domain of human APC protein (APC-basic) bound directly to and bundled actin filaments and associated with actin stress fibers in microinjected cells. Actin filaments and microtubules competed for binding to APC-basic, but APC-basic also could cross-link actin filaments and microtubules at specific concentrations, suggesting a possible role in cytoskeletal cross-talk. APC interactions with actin in vitro were inhibited by its ligand EB1, and co-microinjection of EB1 prevented APC association with stress fibers. Point mutations in EB1 that disrupted APC binding relieved the inhibition in vitro and restored APC localization to stress fibers in vivo, demonstrating that EB1-APC regulation is direct. Because tumor formation and metastasis involve coordinated changes in the actin and microtubule cytoskeletons, this novel function for APC and its regulation by EB1 may have direct implications for understanding the molecular basis of tumor suppression.

Anaphase onset before complete DNA replication with intact checkpoint responses

Torres-Rosell J, De Piccoli G, Cordon-Preciado V, Farmer S, Jarmuz A, Machin F, Pasero P, Lisby M, Haber JE, Aragon L.
Science, 2007; 315:1411-5

Cellular checkpoints prevent mitosis in the presence of stalled replication forks. Whether checkpoints also ensure the completion of DNA replication before mitosis is unknown. Here, we show that in yeast smc5-smc6 mutants, which are related to cohesin and condensin, replication is delayed, most significantly at natural replication-impeding loci like the ribosomal DNA gene cluster. In the absence of Smc5-Smc6, chromosome nondisjunction occurs as a consequence of mitotic entry with unfinished replication despite intact checkpoint responses. Eliminating processes that obstruct replication fork progression restores the temporal uncoupling between replication and segregation in smc5-smc6 mutants. We propose that the completion of replication is not under the surveillance of known checkpoints.

RecA-independent recombination is efficient but limited by exonucleases

Dutra BE, Sutera VA Jr, Lovett ST.
Proceedings of the National Academy of Sciences of the USA, 2007;104:216-21

Genetic recombination in bacteria is facilitated by the RecA strand transfer protein and strongly depends on the homology between interacting sequences. RecA-independent recombination is detectable but occurs at extremely low frequencies and is less responsive to the extent of homology. In this article, we show that RecA-independent recombination in Escherichia coli is depressed by the redundant action of single-strand exonucleases. In the absence of multiple single-strand exonucleases, the efficiency of RecA-independent recombination events, involving either gene conversion or crossing-over, is markedly increased to levels rivaling RecA-dependent events. This finding suggests that RecA-independent recombination is not intrinsically inefficient but is limited by single-strand DNA substrate availability. Crossing-over is inhibited by exonucleases ExoI, ExoVII, ExoX, and RecJ, whereas only ExoI and RecJ abort gene-conversion events. In ExoI(-) RecJ(-) strains, gene conversion can be accomplished by transformation of short single-strand DNA oligonucleotides and is more efficient when the oligonucleotide is complementary to the lagging-strand replication template. We propose that E. coli encodes an unknown mechanism for RecA-independent recombination (independent of prophage recombination systems) that is targeted to replication forks. The potential of RecA-independent recombination to mediate exchange at short homologies suggests that it may contribute significantly to genomic change in bacteria, especially in species with reduced cellular exonuclease activity or those that encode DNA protection factors.

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Current Science - Neuroscience Publications

Ptpmeg is required for the proper establishment and maintenance of axon projections in the central brain of Drosophila

Whited JL, Robichaux MB, Yang JC, Garrity PA.
Development. 2007;134(1):43-53

Ptpmeg is a cytoplasmic tyrosine phosphatase containing FERM and PDZ domains. Drosophila Ptpmeg and its vertebrate homologs PTPN3 and PTPN4 are expressed in the nervous system, but their developmental functions have been unknown. We found that ptpmeg is involved in neuronal circuit formation in the Drosophila central brain, regulating both the establishment and the stabilization of axonal projection patterns. In ptpmeg mutants, mushroom body (MB) axon branches are elaborated normally, but the projection patterns in many hemispheres become progressively abnormal as the animals reach adulthood. The two branches of MB alpha/beta neurons are affected by ptpmeg in different ways; ptpmeg activity inhibits alpha lobe branch retraction while preventing beta lobe branch overextension. The phosphatase activity of Ptpmeg is essential for both alpha and beta lobe formation, but the FERM domain is required only for preventing alpha lobe retraction, suggesting that Ptpmeg has distinct roles in regulating the formation of alpha and beta lobes. ptpmeg is also important for the formation of the ellipsoid body (EB), where it influences the pathfinding of EB axons. ptpmeg function in neurons is sufficient to support normal wiring of both the EB and MB. However, ptpmeg does not act in either MB or EB neurons, implicating ptpmeg in the regulation of cell-cell signaling events that control the behavior of these axons.

Neuronal morphology and neuropil structure in the stomatogastric ganglion of the lobster, Homarus americanus

Bucher D, Johnson CD, Marder E.
The Journal of Comparative Neurology, 2007; 501(2):185-205

The stomatogastric nervous system (STNS) has long been used as a model system for the study of central pattern generation, neuromodulation, and network dynamics. Anatomical studies of the crustacean stomatogastric ganglion (STG) in different species have mostly been restricted to subsets of neurons and/or general structural features. For the first time, we describe the morphology of all STG neurons belonging to the two circuits that produce the well-described pyloric and gastric rhythms in the lobster, Homarus americanus. Somata sit on the dorsal and lateral surface of the STG and send a single primary neurite into the core of the neuropil, which is mostly made up of larger lower order branches. The perimeter of the neuropil consists mostly of finer higher order branches. Immunohistochemical labeling for synaptic proteins is associated with the small diameter branches. Somata positions are not constant but show preferred locations across individuals. The number of copies is constant for all neuron types except the PY and GM neurons (PY neuron number ranges from 3 to 7, and GM neuron number ranges from 6 to 9). Branch structure is largely nondichotomous, and branches can deviate substantially from cylindrical shape. Diameter changes at branch points can be as large as 20-fold. Clearly, the morphology of a specific neuron type can be quite variable from animal to animal.

Understanding circuit dynamics using the stomatogastric nervous system of lobsters and crabs

Marder E, Bucher D.
Annual Review of Physiology, 2007;69:291-316

Studies of the stomatogastric nervous systems of lobsters and crabs have led to numerous insights into the cellular and circuit mechanisms that generate rhythmic motor patterns. The small number of easily identifiable neurons allowed the establishment of connectivity diagrams among the neurons of the stomatogastric ganglion. We now know that (a) neuromodulatory substances reconfigure circuit dynamics by altering synaptic strength and voltage-dependent conductances and (b) individual neurons can switch among different functional circuits. Computational and experimental studies of single-neuron and network homeostatic regulation have provided insight into compensatory mechanisms that can underlie stable network performance. Many of the observations first made using the stomatogastric nervous system can be generalized to other invertebrate and vertebrate circuits.

Auditory Short-Term Memory Behaves Like Visual Short-Term Memory

Visscher KM, Kaplan E, Kahana MJ, Sekuler R.
PLoS Biology, 2007; 5(3):e56

Are the information processing steps that support short-term sensory memory common to all the senses? Systematic, psychophysical comparison requires identical experimental paradigms and comparable stimuli, which can be challenging to obtain across modalities. Participants performed a recognition memory task with auditory and visual stimuli that were comparable in complexity and in their neural representations at early stages of cortical processing. The visual stimuli were static and moving Gaussian-windowed, oriented, sinusoidal gratings (Gabor patches); the auditory stimuli were broadband sounds whose frequency content varied sinusoidally over time (moving ripples). Parallel effects on recognition memory were seen for number of items to be remembered, retention interval, and serial position. Further, regardless of modality, predicting an item's recognizability requires taking account of (1) the probe's similarity to the remembered list items (summed similarity), and (2) the similarity between the items in memory (inter-item homogeneity). A model incorporating both these factors gives a good fit to recognition memory data for auditory as well as visual stimuli. In addition, we present the first demonstration of the orthogonality of summed similarity and inter-item homogeneity effects. These data imply that auditory and visual representations undergo very similar transformations while they are encoded and retrieved from memory.