24 research outputs found
Changing Cellular Location of CheZ Predicted by Molecular Simulations
In the chemotaxis pathway of the bacterium Escherichia coli, signals are carried from a cluster of receptors to the flagellar motors by the diffusion of the protein CheY-phosphate (CheYp) through the cytoplasm. A second protein, CheZ, which promotes dephosphorylation of CheYp, partially colocalizes with receptors in the plasma membrane. CheZ is normally dimeric in solution but has been suggested to associate into highly active oligomers in the presence of CheYp. A model is presented here and supported by Brownian dynamics simulations, which accounts for these and other experimental data: A minority component of the receptor cluster (dimers of CheA(short)) nucleates CheZ oligomerization and CheZ molecules move from the cytoplasm to a bound state at the receptor cluster depending on the current level of cellular stimulation. The corresponding simulations suggest that dynamic CheZ localization will sharpen cellular responses to chemoeffectors, increase the range of detectable ligand concentrations, and make adaptation more precise and robust. The localization and activation of CheZ constitute a negative feedback loop that provides a second tier of adaptation to the system. Subtle adjustments of this kind are likely to be found in many other signaling pathways
Molecular analysis of 'hobo' and 'Himar1' transposition
EThOS - Electronic Theses Online ServiceGBUnited Kingdo
Molecular analysis of hobo and Himar 1 transposition
The aim of this study is to find out more about how eukaryotic transposons work. Transposons are widespread genetic elements that can autonomously change their position within a genome. Their translocation is mediated by the transposase enzyme, encoded by the transposon itself. Transposition is often associated with mutations and recombination in the host and may promote evolutionary change. Most knowledge of the principles and molecular mechanisms has so far come from prokaryotic transposons. Eukaryotic transposons have mostly been studied on the level of population dynamics. In this thesis, I studied two eukaryotic transposons on a molecular level, hobo and Himarl are members of very widespread transposon families, hAT and mariner-like elements, respectively. Their interest lies mainly in the preposition that the order of catalytic steps is the opposite of all prokaryotic transposons studied so far. I developed several protocols for the purification of hobo transposase. These preparations were assayed by several methods in vitro. DNA hairpin structures, which had been postulated to arise in the flanking DNA upon hobo excision, were assayed for with a newly developed very sensitive method, frayed duplex PCR. Here, and in reconstructed in vivo transposition systems, no specific transpositional activity, apart from a high toxicity for the host cells, was detected. This suggests a requirement for as yet unidentified host factors. For Himarl, I improved the published transposase purification protocol and optimised the in vitro reaction conditions, achieving a c. 10-fold increase in specific activity. Specific transposase-DNA binding is shown, representing the earliest intermediates in transposition. Later complexes studied are target capture and strand transfer complexes, which are the final stage of the transposition reaction. Hydroxyradical footprinting and band shift assays suggest that Himarl transposase binds a single DNA end as multimers while occupying only a single DNA binding site. Himarl can bind and commit to target DNA before as well as after excision from the donor. Target commitment is only clearly visible under conditions of macromolecular crowding, and insertions were found to be more efficient if the target is supercoiled. These are both conditions closely resembling the situation in the eukaryotic nucleus.</p
Schematic of the Dynamic CheZ Hypothesis
<div><p>(A) A layer of CheA dimers is positioned at the cytoplasmic face of the
polar chemoreceptor cluster. Interspersed with the catalytically active
CheA dimers are CheA<sub>short</sub> homodimers, which act as anchoring
points for CheZ dimers. In the absence of CheYp, a condition produced by
saturating concentration of attractants, the remaining CheZ dimers
diffuse freely in the cytoplasm.</p><p>(B) Upon increased phosphorylation of CheY, which occurs after exposure
to repellent, CheZ dimers bind CheYp and oligomerize by assembly at the
CheA<sub>short</sub>-CheZ nuclei. These clustered oligomers have a
greatly increased CheYp dephosphorylation activity, providing negative
feedback to the system.</p></div
Graphical Output of the <i>Smoldyn</i> Simulations, Showing Differential Localization of CheZ and Its Oligomeric Forms
<div><p>(A) In the absence of CheA<sub>long</sub> kinase activity and
phosphorylated CheY, all CheZ dimers are unbound and freely diffusing in
the cytoplasm.</p><p>(B, C) Upon sudden increase of kinase activity, the level of CheYp rises
initially in the anterior part (B), and then, (C), in the entire cell.</p><p>(D) After 1 min at constant kinase activity, the increase of CheYp has
led to the formation of oligomeric CheZ<i><sub>2</sub></i>Yp clusters at the inner face of the polar receptor cluster.</p><p>Snapshots of animations in OpenGL. Reactions 1, 3, 8, 9 (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020039#pcbi-0020039-t001" target="_blank">Table 1</a>).</p></div
Proposed Structure of the CheZ<sub>2</sub>Yp-Oligomeric Clusters
<div><p>(A) In the
(CheY-BeF<sub>3</sub><sup>−</sup>-Mg<sup>2+</sup>)<sub>2</sub>CheZ<sub>2</sub>
co-crystal structure (PDB entry 1KM1, [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020039#pcbi-0020039-b011" target="_blank">11</a>]), CheZ (green and gray)
exists as a stable dimer. On each side of its four-helix bundle is an
active site with loose affinity for a CheYp monomer (orange). The main
binding affinity for CheYp is in a short C-terminal helix, which is
connected to the main body of CheZ by a flexible peptide tether (dashed
lines). Instead of bending back on itself, the unstructured domain,
which is invisible to the crystallographer, could connect a CheYp
molecule bound to the C-terminus of one CheZ dimer to the catalytic site
of a neighboring one. This allows for the formation of extended
oligomers. Anchorage to the polar cluster could occur via the
CheZ-apical helices to CheA<sub>S</sub> homodimers (salmon-colored
ovals), as suggested by mutagenesis [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020039#pcbi-0020039-b005" target="_blank">5</a>].</p><p>(B) In oligomeric networks, each CheZ dimer can be connected to a maximum
of four neighboring CheZ dimers, via flexible tethers and CheYp. A
looser network will exist if not all CheYp binding sites are occupied.
View from below, as compared to (A). Created with MacPyMOL (DeLano
Scientific LLC, San Carlos, California, United States).</p></div
CheYp Levels, CheZ Clustering Dynamics, and Motor Occupancy in Response to an Extreme Activity Profile
<div><p>Simulations were carried out in cells of the architectures in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020039#pcbi-0020039-g003" target="_blank">Figure 3</a> but with four
copies of each motor #1 (0.2 μm from the anterior end)
and motor #2 (1.8 μm from the anterior end), one on each
lateral face. As input, the phosphorylation state of CheA kinase was
changed sharply from 0% to 100% and back (yellow
line); this activity profile, shown in full in column 1 (A–D)
was repeated 25 times. Red (free CheYp/total CheY), mean of 25 runs;
black (CheZ localized in oligomers/total CheZ), mean of 25 runs. Columns
2 and 3 (E–L) are expanded sections of these simulations.
Blue, occupancy (FliMYp/total FliM) of motor #1, mean of (25
runs × 4 motors =) 100 traces; cyan, occupancy of
motor #2, mean of 100 traces; thin gray, corresponding curves
from the top two panels in the same column. Note the differences in time
scales.</p><p>Row 1 (A, E, I) All CheZ dimers are in an immobile lattice 40 nm from the
anterior end. Reactions 2, 3, 4 (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020039#pcbi-0020039-t001" target="_blank">Table 1</a>).</p><p>Row 2 (B, F, J), All CheZ are dimers freely diffusing in the entire cell
volume. Reactions 2, 3, 5 (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020039#pcbi-0020039-t001" target="_blank">Table 1</a>).</p><p>Row 3 (C, G, K), Formation of freely diffusing CheZ oligomers in the
entire cell volume. Reactions 2, 3, 6, 7 (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020039#pcbi-0020039-t001" target="_blank">Table 1</a>).</p><p>Row 4 (D, H, L), Dynamic CheZ localization with oligomerization at
CheA<sub>short</sub> according to our hypothesis. Reactions 2, 3, 8,
9 (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020039#pcbi-0020039-t001" target="_blank">Table 1</a>).</p></div
Dose-Response Curves
<div><p>CheA activity (orange) was increased in ten equal steps from steady-state
to maximum level to mimic an increasing repellent concentration
(A–D) or decreased to mimic an increase in attractant
(E–H). Orange, ratio of active CheA; red, free CheYp; black,
oligomeric CheZ.</p><p>(A, E) CheZ all dimeric and fixed at the cluster. Reactions 1, 3, 4
(<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020039#pcbi-0020039-t001" target="_blank">Table 1</a>).</p><p>(B, F) CheZ all dimeric and cytoplasmic. Reactions 1, 3, 5 (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020039#pcbi-0020039-t001" target="_blank">Table 1</a>).</p><p>(C, G) Cytoplasmic CheZ oligomerization. Reactions 1, 3, 6, 7 (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020039#pcbi-0020039-t001" target="_blank">Table 1</a>).</p><p>(D, H) Dynamic CheZ clustering. Reactions 1, 3, 8, 9 (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020039#pcbi-0020039-t001" target="_blank">Table 1</a>).</p></div
Molecular Movements: Results of a Typical Simulation
<div><p>(A) The input to the simulations is the proportion of active CheA kinase
dimers (orange; A<sub>2</sub>* in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020039#pcbi-0020039-t001" target="_blank">Table 1</a>), which undergoes three
cycles of halving and doubling. It does not include any
methylation-dependent adaptation and is thus equivalent to a
Δ<i>cheR cheB</i> strain. CheAp (yellow) is
generated by autophosphorylation of active CheA. (Shown is the sum of
species A<sub>2</sub>*p and A<sub>2</sub>p.) Black arrows indicate the
addition and removal of attractant in an equivalent experimental system.</p><p>(B) Fraction of CheYp (red), generated by phosphotransfer from CheAp and
autophosphorylation. Only unbound CheYp monomers are shown (CheYp free).</p><p>(C) CheZ dimers bound in oligomers of increasing size (light to dark
green) and total CheZ in these polar oligomers (black).</p><p>(D) CheYp concentration, calculated from experimental measurements of CCW
bias of Δ<i>cheR cheB</i> strain ST447, stimulated
with 1 μM l-serine (modified from [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020039#pcbi-0020039-b026" target="_blank">26</a>]). Curves
were calculated with two different degrees of motor cooperativity
(<i>H</i> = 5 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020039#pcbi-0020039-b051" target="_blank">51</a>,<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020039#pcbi-0020039-b052" target="_blank">52</a>] (magenta) or
<i>H</i> = 10.3 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020039#pcbi-0020039-b025" target="_blank">25</a>] (purple), see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020039#s4" target="_blank">Materials and Methods</a>). Open circles
are values where the real CCW bias was estimated to be 1%
instead of the published 0%; this accounts for inaccuracies
of the Hill equation at low numbers.</p><p>Reactions 1, 3, 8, 9 (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020039#pcbi-0020039-t001" target="_blank">Table 1</a>).</p></div