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Laboratory of Molecular Biophysics
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Variations in environmental factors, such as reduced aeration, extremes of
temperature and the availability of essential nutrients, restrict microbial
growth. As a result, bacteria spend much of their life cycle in stationary
phase [1]. The requirement for acclimatisation to the environment has forced
bacteria to develop a series of complex adaptive responses to stress [2]. The
initial and rapid response to stress of some Gram+ bacteria is to synthesise
two classes of proteins. One class is specific to the particular type of stress
that has been imposed; the second is a large and diverse family of
B-dependent general stress proteins that are expressed in response to a wide
variety of environmental pressures. The expression of the
B regulon provides
bacteria with a protective multiple stress resistance.
B regulates the expression of a wide and varied group of genes which are
strongly induced either by starvation of nutrients, leading to a fall in the
concentration of ATP, or by physical or chemical insult.
B is kept under
strict control by the gene products of seven
regulators of sigma
B (rsb) found
in the sigB operon, rsbR, rsbS,
rsbT, rsbX, rsbV, rsbW and rsbU. These Rsb
proteins may be functionally divided into an "upstream"
and a "downstream"
module [3]. Each module comprises a protein kinase, a protein phosphatase, and
a "switch" protein substrate for the kinase and phosphatase. The
phosphorylation state of the switch molecule indirectly controls the activity
of
B and regulation of the state of phosphorylation is brought about by the
antagonistic activities of the two classes of enzymes.
In the downstream module, RsbW is the key, bifunctional, protein. It forms a
complex with
B, thus acting as an anti-sigma factor, and also acts as an
ATP-dependent serine/threonine kinase, the substrate for which is the switch
protein, RsbV. RsbV has been termed an anti-anti-sigma factor. RsbU is a
protein phosphatase whose substrate is the phosphorylated form of RsbV,
RsbV-P. During normal growth, RsbW utilises the high ATP concentration to
specifically phosphorylate RsbV. RsbW has a lower affinity for RsbV-P than
RsbV: thus in the presence of RsbV-P, RsbW forms a protein:protein complex
with
B and inactivates it. Under conditions that diminish the ATP
concentration, the kinase activity of RsbW is decreased in comparison to the
phosphatase activity of RsbU. The ratio of RsbV:RsbV-P rises, RsbW binds
preferentially to RsbV and relinquishes
B, with the result that
B is
activated and general stress proteins are synthesised. The activity of
B is
thus directly controlled by the phosphorylation state of RsbV [4].
The components of the upstream module are highly homologous in amino acid
sequence to their downstream counterparts. In the upstream module, RsbT is the
kinase, RsbX the phosphatase and RsbS the substrate for these two mutually
antagonistic enzymes. In many respects, the RsbX-RsbS-RsbT components of the
upstream module function similarly to their downstream RsbU-RsbV-RsbW
homologues. In the presence of RsbS-P, the RsbT kinase from the upstream
module forms a protein:protein complex with the RsbU phosphatase of the
downstream module, stimulating its phosphatase activity against RsbV-P some
20-fold. The product of this reaction, RsbV, liberates
B from its inactive
complex with RsbW and induces expression of the general stress proteins. Thus,
RsbT links the upstream and downstream modules.
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RsbW and RsbT are believed to be members of a growing family of
kinases/ATPases, called the GHKL family, members of which includes proteins
such as DNA gyrase, Hsp90, MutL and CheA [5]. Little is known about why some of
these proteins are ATPases, and why some are kinases, nor why RsbW can also
sequester sigma factors, whereas RsbT (and others) cannot. Accordingly, RsbW
has been overexpressed in E. coli and purified for structural studies. This
protein - a dimer - has crystallized from solutions of MPD in a hexagonal
crystal form that diffracts using CuK
radiation to
about 7Å
(Figure 1).
A second crystal form has also been obtained in the presence of a
non-hydrolysable ATP analogue, AMP-PNP and the co-factor magnesium. These
crystals are smaller and have a different morphology to those of the apo form
(Figure 2),
but do not yet show appreciable diffraction using the in-house
source of X-rays.
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The residues of RsbW, and the wider anti-sigma factor family, which are
involved in molecular recognition events are not known. Similarly, those
residues of RsbV, and the anti-anti-sigma factor family, which are involved in
protein:protein interactions have not been indentified. In order to study
this, the complexes formed between
B, RsbW and RsbV are being investigated.
These complexes can simply be monitored by non-denaturing polyacrylamide gel
electrophoresis. By using a variety of techniques, e.g. electrophoresis
coupled with titration and/or densitometry, sedimentation equilibrium and
nano-spray mass spectrometry the stoichiometries of the RsbW:RsbV and
B:RsbW
complexes have been determined. For RsbW:RsbV, the apparent molecular mass is
60.4 kDa, consistent with one dimer of RsbW binding to two monomers of RsbV.
The complex formed between RsbW and
B has an apparent molecular mass of 66.5
kDa, in good agreement to that predicted from the mass of the gene products
assuming that the RsbW dimer binds a single
B monomer. Sufficient quantities
of the RsbW:RsbV and
B:RsbW complexes have been purified for crystallization
trials, and micro-crystals of the RsbW:RsbV complex have obtained
(Figure 3),
but they are as yet too small for diffraction studies.
Tenuous sequence homology places RsbX and RsbU in the PP2C-type of M2+-dependent phosphatase family, which is poorly characterised from a structural perspective [6]. Unlike other PP2C-type phosphatases, such as SpoIIE, RsbU requires magnesium ions, and is inhibited by the presence of manganese. No crystals have yet been grown of the apo-form of RsbU and at the high protein concentrations used for crystallization, addition of catalytic magnesium ions induces almost complete protein precipitation. This may indicate that a significant conformational change takes place on binding metal ions. RsbX has also been purified in large amounts, but is insoluble in low ionic strength buffers. This insolubility has precluded crystallization trials, and will continue to do so until a more suitable buffering system can be identified.
RsbU encodes an N-terminal domain of approximately 100 amino acids, with no obvious clue to its function from its sequence. It has been predicted that this N-terminal extension binds to RsbT, which is required to realise the full catalytic potential of RsbU towards its substrate, RsbV-P [7]. Certainly, RsbT and the regulatory domain of RsbU form long-lived protein:protein complexes, which can also be visualised by native gel electrophoresis. By mild trypsinolysis, the N-terminal domain can be cleaved and separated from the C-terminal, catalytic domain of ~200 amino acids. The observation that the N-terminal domain is not completely destroyed by tryptic digest implies that it has a fully folded form, and thus presumably also a function. The purified catalytic domain of RsbU shows much higher phosphatase activity towards RsbV-P than intact RsbU alone, and is not stimulated by the presence of RsbT. Therefore, the function of the N-terminal domain of RsbU is to negatively regulate the inherent phosphatase activity of the intact protein, in the absence of RsbT. The molecular mechanism behind the activation process remains obscure. The effect on the stress-activation pathway of an in-frame deletion of the 5' end of the chromosomal copy of rsbU is the focus of continuing experiments, in collaboration with the laboratories of Profs. Michael Yudkin and Chet Price.
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The effects of phosphorylation on protein structure vary extensively, and cover the spectrum of movements of >50Å to <0.5Å [8]. The related switch molecule, SpoIIAA, falls into this latter category, since comparison of the recently determined structures in both phosphorylated and non-phosphorylated forms [9] reveals few significant differences (Figure 4). Is the same true of RsbV and RsbS? His-tagged RsbV has been purified to homogeneity, but as yet, no crystals have been grown. This may reflect that RsbV has a tendency to aggregate. Better progress has been made, however, with RsbS, where small crystals have been obtained from solutions containing polyethyleneimine as the precipitant, but unfortunately these are not yet suitable for diffraction studies (Figure 5).
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Funding: Wellcome Trust
Collaborators: O. Delumeau, C.W. Price, D.J. Scott, M.D. Yudkin
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