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Legionella pneumophila and Rickettsia prowazekii
are human pathogenic bacteria that cause Legionnaire’s disease
and Rocky Mountain spotted fever, respectively. Each of these bacteria
reproduce within organelles they establish inside the macrophages
that engulf them. These replicative vacuoles result from pathogenic
subversion of the normal membrane traffic machinery. One protein
isolated from L. pneumophila, termed RalF, was recently shown able
to activate Arf proteins, ubiquitous and essential regulators of
eukaryotic membrane traffic. All Arf activators known, including
RalF, share a region of sequence homology termed the Sec7 domain.
Despite this sequence homology, mutagenesis data suggest important
differences in the mechanism of Arf activation. We hypothesize that
RalF activates Arfs by a mechanism different from eukaryotic Sec7
proteins. The critical test of this hypothesis is the solution of
the structure of RalF bound to Arf and comparison to previously
determined eukaryotic Sec7-Arf complexes. We began efforts to express
large amounts of RalF as well as the RalF Sec7 domain for structural
studies. RalF and the RalF Sec7 domain were expressed in bacteria
as His6 fusion proteins. Purification yielded 30 and 80 mgs of protein
per liter of bacterial culture, respectively, that were suitable
for protein crystallization trials. Three conditions were found
to give protein crystals in initial screens. One of these was optimized
for the Sec7 domain of RalF. Preliminary diffraction data were collected
locally and indicated that high quality data can be obtained. High
resolution data collection at the Brookhaven National Laboratory
synchotron is scheduled for early August. These data are expected
to be suitable for structure determination to better than 3D resolution.
The structure of the RalF Sec7 domain will be compared to eukaryotic
Sec7 domain structures. An effort to crystallize the Ralf-Arf complex
is underway and comparison of this structure to eukaryotic Sec7-Arf
complex structures will be the critical test of our hypothesis.
ARF activating proteins contain the Sec7 domain, with
sequence homology and the ability to promote the exchange of GDP
for GTP. The currently accepted mechanism for this exchange is based
on both crystal structures and mutagenesis data from eukaryotic
GEF proteins. Crystal structures of a Sec7-ARF complex show a glutamate
filling the space normally occupied by GDP. Mutation of the glutamate
caused eukaryotic Sec7 proteins to lose activity. Researchers suggested
that the glutamate physically knocks the GDP molecule off of ARF,
leaving the binding site open for GTP. RalF is a prokaryotic Sec7
protein secreted by the pathogenic bacteria L. pneumophila, which
causes Legionnaire’s disease. Although the glutamate is conserved
in RalF, mutation does not result in a loss of ARF exchange factor
activity. This critical difference suggests that RalF acts through
a mechanism different from eukaryotic ARF activating proteins. A
mechanistic difference of this type may provide the basis for the
development of inhibitors and potential clinical treatments for
Legionnaire’s disease.
L. pneumophila injects RalF into host cell cytoplasm.
RalF activates ARF, contributing to the development of reproductive
vacuoles for infectious particles and Legionnaire’s Disease.
ARF is activated when bound to GTP. The active ARF-GTP complex gains
affinity for membranes. RalF facilitates the exchange of GDP for
GTP, thereby recruiting ARF to the early endosome surface. The presence
of active ARF on endosomal membranes blocks their fusion with lysosomes
and promotes replication of the pathogen.
RalF and RalF-Sec7 open reading frames were amplified,
subcloned into the pHis-1 plasmid and expressed in BL21 DE3* cells
as His6 fusion proteins.
RalF and RalF-Sec7 open reading frames were PCR amplified.
PCR was performed using a plasmid template and Pfu polymerase with
initial annealing temperatures of 30ºC or 40ºC for two cycles and
then continued for 25 cycles with a 62ºC annealing temperature.
Products shown match predicted sizes (RalF: 1.2kb, RalF-Sec7: 0.6kb).
RalF and RalF-Sec7 proteins were purified by FPLC.
Bacterial cell lysate was run through a nickel column followed by
a mono-Q column, yielding 30 mgs of RalF protein and 80 mgs of RalF-Sec7
protein per liter of bacterial culture.
The hanging drop method was used to screen for optimal
RalF and RalF-Sec7 crystallization conditions. The concentration
of the mother liquor is diluted in the crystallization droplet,
resulting in a net movement of water out of the droplet and into
the mother liquor at the bottom of the well. The loss of water causes
an increase in protein concentration that promotes crystallization.
Four protein concentrations were tested during the trials: 10 mg/ml,
20 mg/ml, 30 mg/ml and 40 mg/ml.
RalF forms small crystals under one of fifty conditions
tested. In a solution of 2.0 M sodium formate and 0.1 M sodium acetate
at pH 4.6, RalF showed an excess of crystalline nucleation with
little or no growth in size. Finer screening around this condition
will be designed to promote growth of crystals suitable for x-ray
analysis.
RalF-Sec7 crystallized under several conditions and
crystal quality was optimized in the presence of cryoprotectants.
Initial screens showed two conditions yielding crystals. These conditions
were varied with respect to salt, PEG, and pH to produce smooth,
monomeric crystals. Cryoprotectants were added to facilitate x-ray
data collection.
RalF-Sec7 crystal diffracts to high resolution. A
crystal from the condition yielding the highest quality crystals
was put on the local x-ray beam to collect diffraction data. Initial
data indicate that the RalF-Sec7 protein structure can be solved
to a resolution of at least 2.6Å. Crystals are in the primitive
orthorhombic space group. Analysis of data collected from the high
energy synchotron beam should allow solution of the first structure
of a bacterial ARF activating protein.
RalF and RalF-Sec7 were subcloned into the pHis-1 plasmid and expressed
in BL21 DE3* cells as His6 fusion proteins. · Each protein
was purified using sequential Ni2+ and Mono-Q columns. ·
Crystallization trials revealed two conditions in which RalF-Sec7
crystallized and one condition in which RalF formed microcrystals.
· RalF-Sec7 crystallization conditions were optimized to
yield high quality crystals capable of x-ray diffraction. ·
The RalF-Sec7 structure is expected to be solved following data
collection at the Brookhaven National Laboratory synchotron source.
· Further efforts to co-crystallize RalF and ARF will yield
greater insight into the mechanism by which RalF activates ARF.
We would like to thank Matt Bennett, Brad Bowzard, Karen Hill,
Yawei Li, Melissa McKay, Jack Shern, Corey Snelson, and Ian Stevenson
for advice and support throughout this project. We would also like
to thank John Horton for help with the x-ray machine and data interpretation.
This work is supported by the Howard Hughes Medical Institute under
Grant No. 52003071.
Legionella pneumophila is a bacteria that causes a
severe type of pneumonia called Legionnaire's disease. When engulfed
by a victim's immune cells, L. pneumophila takes over the cell signalling
mechanism. Instead of being destroyed, the bacteria forms an organelle
in which it multiplies, eventually killing the cell. RalF is a protein
that is injected by the bacteria into the host. It activates an
important membrane traffic protein(ARF), causing it to stick to
the membrane containing the bacteria. RalF is one of only two bacterial
proteins able to activate ARF. It seems to cause activation differently
from ARF activating proteins produced by non-bacterial organisms.
The proteins produced by more advanced species no longer activate
ARF when a certain piece of the protein is mutated, but RalF maintains
activity even after mutation. We are trying to determine the structure
of RalF in order to discover how it activates ARF. To do this, we
put the RalF gene into bacteria and caused them to produce large
quantities of protein. We then purified the protein from other cell
proteins by running it over several affinity columns. The protein
was set up for crystallization in a variety of conditions and crystal
quality was optimized by testing around the most promising ones.
The best crystals were analyzed with an x-ray beam and were found
to diffract with high resolution. In order for the structure to
be completely solved, data will be collected by a high energy synchotron
x-ray beam in the Brookhaven National Laboratory. The next project
is to crystallize RalF bound with ARF to see exactly how the two
proteins interact.
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