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Structural and Functional Studies of theFusidic Acid Resistance
Protein FusB
Xiaohu Guo
Degree project in applied biotechnology, Master of Science (2
years), 2010Examensarbete i tillämpad bioteknik 45 hp till
masterexamen, 2010Biology Education Centre and Structural biology,
Dept. of Cellular and Molecular Biology,
UppsalaUniversitySupervisor: Maria Selmer
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Abstract
............................................................................................................................................2
Introduction
.....................................................................................................................................3
Fusidic acid
..............................................................................................................................3
Staphylococcus aureus, a major antibiotic resistant
pathogen........................................3 Staphylococcus
aureus antibiotic resistance
mechanism..................................................3 The
identification of FusB, another fusidic acid resistant determinant
..........................4 FusB and its
homologues.......................................................................................................4
Aim of the study and
result...................................................................................................5
Material and Method
......................................................................................................................5
Plasmid pUB101
extraction....................................................................................................5
TA cloning
................................................................................................................................5
Cleavable His-tagged FusB (FusB_LN) TA cloning
.....................................................5
Non-cleavable His-tagged FusB (FusB_SN) TA cloning
.............................................6 Non His-tagged EF-G
(EF-G_NoT) TA cloning
............................................................6
Transformation and large-scale Expression
........................................................................6
His-tagged FusB transformation and large scale
expression....................................6 None His-tagged
EF-G (EF-G_NoT) transformation and large-scale expression ...7
His-tagged EF-G transformation and large-scale expression
...................................7
Protein purification
.................................................................................................................7
His-tagged FusB purification
.........................................................................................7
His-tagged EF-G purification
.........................................................................................7
TEV protease purification
..............................................................................................8
FusB – EF-G complex purification
................................................................................8
Digested S.a EF-G and FusB binding
test............................................................................8
Crystallization of FusB and complex
....................................................................................9
Data
collection.........................................................................................................................9
Results
..............................................................................................................................................9
FusB and EF-G plasmid construction
...................................................................................9
Tev protease His-tag cutting test
.......................................................................................10
FusB binds to S. aureus EF-G
.............................................................................................11
FusB does not bind to E. coli
EF-G.....................................................................................11
Complex purification
............................................................................................................12
FusB and S. aureus EF-G affinity test by fluorescence spectroscopy
...........................13 Digested S. aureus EF-G and FusB
binding
test...............................................................13
Crystallization and
crystallography.....................................................................................15
Discussion
......................................................................................................................................18
Structure determination of FusB and FusB - EF-G complex
...................................................18
The binding target of
FusB..................................................................................................19
Hypothetical resistance mechanisms
.................................................................................21
Acknowledgement
........................................................................................................................23
Reference
.......................................................................................................................................24
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Abstract Fusidic acid, first derived from the Fusidium
coccineum, is an antibiotic used against Staphylococcus aureus. It
functions by blocking the release of elongation factor G (EF-G)
from the ribosome, thus preventing the binding of a new aminoacyl
tRNA to the ribosome and blocking the translation process. One
resistance mechanism for S. aureus to fusidic acid involves a
single gene fusB, carried by plasmid pUB101. The FusB protein has
previously been shown to interact with S. aureus EF-G but not with
E. coli EF-G. Further, FusB confers fusidic-acid resistance to an
S. aureus in vitro translation system, but fails from protecting an
E. coli in vitro translation system from fusidic-acid inhibition.
With the aim of structure determination and biochemical studies of
FusB and the FusB-EF-G complex, we have cloned, expressed and
purified FusB and S. aureus EF-G. FusB has been crystallized. The
crystals diffract to 3.9 Å resolution and belong to space group
P21212. Crystals are being further optimized. We also study the in
vitro interaction of S. aureus EF-G and FusB. Our results show that
the FusB can not bind to S. aureus EF-G domainⅠtogether with domain
Ⅱ, and probably functions by its interaction with domainⅤ.
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Introduction
Fusidic acid
Fusidic acid, first derived from the fungus Fusidium coccineum,
is an antibiotic used to against gram-positive bacteria such as
Staphylococcus and Corynebaterium [1]. Shortly after this finding
in the 1960s, sodium salt of fusidic acid was induced into clinical
use, especially against Staphylococcus aureus. Fusidic acid can
inhibit protein synthesis by preventing the turnover of elongation
factor G (EF-G) [2]. After EF-G has hydrolyzed GTP to GDP and
completed the translocation step; it undergoes a large
conformational change and falls off from the ribosome, emptying the
position for the aa-tRNA. The structure of EF-G band to the
ribosome reveals that fusidic acid is located in a pocket
surrounded by domain ,Ⅰ Ⅱ and domainⅢ of EF-G and there is no
direct interaction with ribosome. This binding maintains the switch
conformation of Ⅱthe G domain in GTP binding form, thus preventing
the release of EF-G from the ribosome [3].
Staphylococcus aureus, a major antibiotic resistant pathogen
S. aureus is a Gram-positive coccus responsible for many
infections in humans and animals through its toxin production or
invasion. It’s well known for its super adaptability to
antibiotics. In 1947, only after 4 years of penicillin broad usage,
the resistance at this strain was reported. Subsequently resistance
appeared to other drugs like methicillin, tetracycyline, fusidic
acid, erythromycin and vancomycin. In the 1990s, people found
Linezolid, a new class of antibiotic and more efficient than
vancomycin against S. aureus. However, merely 1 year after it was
approve for use in 2000, a linezolid resistant clinical isolate was
found in Israel [4]. That is why understanding the resistance
mechanisms are important and that is also the reason that some
antibiotics like fusidic acid has been valued again for multiple
drug treatment. Due to the high level development of the
resistance, fusidic acid is never recommended as infection
treatment on its own [5].
Staphylococcus aureus antibiotic resistance mechanism
The resistance mechanisms have been intensively studied in S.
aureus. To date, there are four well known routes for different
bacteria to gain resistance against various antibiotics. The first
one, drug modification, works by inactiving the antibiotics. This
mechanism can for example give resistance against Macrolide, and
Lincosamide etc [6]. A second mechanism involves an alteration of
metabolic pathways, which means that once a vital metabolic pathway
is damaged by antibiotics, some bacteria can somehow compensate
this damage
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by changing its metabolic pathway to another one. Some
sulfonamide-resistant bacteria belong to this kind [7]. The third
strategy is to reduce drug accumulation, for example, tetracycline
can be pumped out of the cell by a special membrane protein in some
of the resistant-strains [8]. The fourth one, which is also the
major mechanism used by S. aureus against fusidic acid, works by
changing the target site lowering its affinity to the drug [9]. In
this case, the changed target is S. aureus EF-G. Several mutations
in fusA, the gene coding for EF-G, have been identified both from
in vitro generated and clinically isolated fusidic acid resistant
strains [10]. Further studies shows three positions of these
mutations, V90I, L461K and H457Y/H457Q, could result high-level
resistance to fusidic acid [11]. Since these residues are located
in domainⅠand Ⅲ of EF-G, where fusidic acid binds, it strongly
indicates that the mutations change the binding affinity of fusidic
acid on EF-G.
The identification of FusB, another fusidic acid resistant
determinant
Studies in 1970s identified another fusidic acid resistance
determinant is carried by a 22kb plasmid pUB101, which is also the
host for penicillinase and cadmium resistance genes. In 2000, a
gene named fusb was for the first time recognized causing the
resistance [12]. Interestingly, fusb-caused resistance does not
involve to enzymatic inactivation or modification [9]. Sequence
analysis showed that the protein doesn’t have any membrane location
signals or secretion signals, indicating that it is unlikely to
work by reducing drug accumulation. So, since more than 30 years,
the resistance mechanism is still unclear. However, in 2006, the
fusidic acid resistance protein FusB was identified and
successfully purified. Importantly, FusB was found to interact
specially with S. aureus EF-G in vitro [13]. These results suggest
that FusB works by preventing fusidic acid binding to EF-G or by
somehow helping to unlock the EF-G – fusidic acid ribosome complex.
Unraveling the mechanism behind FusB resistance will undoubtably
enrich our understanding of antibiotics resistance.
FusB and its homologues
FusB is a small protein with a molecular weight of 25kD. Its
homologues could be found in some Gram-positive genera, for
example, Enterococci, Lactococci and Lactobacilli. The majority of
these species were found to be inherently insusceptible to fusidic
acid [14] [15] [16]. Only one of these homologues, located in the
chromosome of Listeria monocytogenes, has been studied previously.
However, it was identified as a fibronectin-binding protein (Fbp)
and its function is to bind to a eukaryotic cell’s fibronectin
facilitating infection of bacteria [17]. Although Fusb and Fbp have
a high sequence identity (43%), they bind to different target and
engage in very different missions. It is noticeable
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that Listeria monocytogenes is one of the strains inherently
insusceptible to fusidic acid and there is no study showing whether
Fbp can only interact with fibronectin or also cause fusidic acid
resistance.
Aim of the study and result
With the aim of structure determination and biochemical studies
of FusB and the FusB-EF-G complex, we have successfully cloned FusB
and S. aureus EF-G into different constructs. FusB, EF-G and the
FusB - EF-G complex were purified by immobilized metal ion affinity
chromatography (IMAC) and size exclusion chromatography. The
purified proteins were subjected to screens for crystallization.
FusB with a short polyhistidine-tag (His-tag) has been
crystallized. The crystals diffract to 3.9 Å resolution and belong
to space group P21212 with cell dimension 129, 187, 93; 90, 90, 90.
The crystals are being further optimized. We also study the in
vitro interaction between FusB and EF-G. Due to the inner filter
effect of FusB, binding affinity could only been poorly determine
to 1μM using the fluorescence spectroscopy method. Surprisingly,
further study of the biniding shows that FusB can not bind to the
digested fragments of EF-G domainⅠtogether with domain Ⅱ. Together
with the sequence alignment analysis and structure analysis, domain
Ⅴ is believed the key domain interacting with FusB.
Material and Method
Plasmid pUB101 extraction
A Single S. aureus colony carrying the FusB resistance
determinant was picked and grown in 15ml LB medium containing 0.5%
glycine at 37℃ overnight without shaking. The cells were spun down
at 4000rpm for 15mins and re-suspended in lysis buffer (100ul TE
pH7.0, 100μl 300μg/ml lysostaphin, Sigma; 100μl 100mg/ml lysozyme,
Sigma; 45μl 20mg/ml proteinase K, Sigma) incubating at 37℃ 60mins.
Lysis buffer from QIAprep® Spin Miniprep Kit (Qiagen) was added
into the cell lyse and then followed the kit protocol to extract
the plasmid.
TA cloning
Cleavable His-tagged FusB (FusB_LN) TA cloning
Primers for cloning were designed to be 24bp (fusB_f1) for the
forward direction, and 28bp (fusB_b1) for reverse direction
(Table1). Because of the low GC content of the gene (25%), a silent
mutation A6G was directed by the forward primer to increase the GC
content in order to prevent any formation of
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secondary structure. PfuUltraTM High-Fidelity DNA Polymerase
(Stratagene) was used for amplification. The PCR reaction was
programmed as following; double-stranded DNA was denatured at 95℃,
for 5mins, followed by 35cycles of amplification. Each cycle
contains, 1min denaturing at 95℃, 1min annealing at 56.8℃, 1min
extension at 72℃. The program was ended with extra 10min at 72℃.The
amplification product was detected on 1% agarose gel and purified
by QIAquide® Gel Extraction Kit (Qiagen). A 3’ adenine overhang was
added by Taq polymerase (Invitrogen) at 72℃ for 10mins. Ligation of
the gene to the plasmid was conducted by pEXP5-NT/TOPO TA
Expression Kit (Invitrogen). The plasmids carrying the gene were
transformed into TOP10 competent cells following kit protocol.
Non-cleavable His-tagged FusB (FusB_SN) TA cloning
The same protocol was followed as above. The Amplification was
using forward primer fusB_f2 and backward primer fusB_b1 (Table1).
The amplified gene was cloned into the pEXP5-CT (Invitrogen)
vector.
Non His-tagged EF-G (EF-G_NoT) TA cloning
Plasmid pET-30 was constructed carrying S. aureus EF-G from
collaborator Suparna Chandra Sanyal. The template was then used as
the template for further cloning. The same protocol was used as
above except, forward primer EF-G_f1 and backward primer EF-G_b1
(Table1). The annealing temperature was optimized to be 59℃. Table
1. Primers
Name Sequence fusB_f1 ATGAAGACAATGATTTATCCTCAC fusB_f2
ATGGCTCATCATCATCATCATCATGGTATGAAGACAATGATTTATCCTCAC fusB_b1
CACAAACATAGTTAATTCCTTAATCTAG EF-G_f1 ATGGCTAGAGAA
TTTTCATTAGAAAAAACT EF-G_b1 AAGCCCGGTTATTCACCTTTATTTTTC
Transformation and large-scale Expression
His-tagged FusB transformation and large scale expression
100μl BL21(DE3) competent cells were taken directly from a -70℃
refrigerator and thawed on ice, 10μl 20ng/μl plasmid was added.
After incubation on ice for 10mins, cells were heat shocked for 90
seconds at 42℃ and immediately placed back on ice for additional
2mins. 150μl of pre-warmed SOC medium (Invitrogen) was added and
incubated for 1 hour at 37℃ with shaking at 225rpm. 200μl medium
containing transformed cells was plated on a LA plate with 100mg/ml
ampicillin (Sigma) and incubated overnight at 37℃. A single colony
was picked and inoculated to 10ml 2×TY medium (1.6% tryptone,
1%
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yeast extract, 0.5% NaCl) containing 50mg/ml ampicillin at 37℃
overnight. The small culture was then inoculated into 1L medium and
incubated at 37℃. IPTG (Sigma) was added to a final concentration
of 1mg/ml when the OD600 of the culture reached 0.6 and the culture
was then continued at 30℃ for overnight. The culture was shaked at
100rpm. Cells were harvested by centrifugation at 4000rpm for
30mins and stored at -20℃.
None His-tagged EF-G (EF-G_NoT) transformation and large-scale
expression
The same protocol was used as above except the following:
competent cells were BL21-AITM One Shot® Chemically Competent cells
(Invitrogen); when OD600 of the 1L cell culture reached 0.5, the
culture was immediately moved to 4℃ for 30mins with a shaker speed
of 70rpm. The culture was induced by IPTG (Sigma) and L-arabinose
(Sigma) at final concentration of 1mg/ml and 0.2% respectively and
incubated at 16℃ overnight.
His-tagged EF-G transformation and large-scale expression
Same protocol used as for EF-G_NoT purification except that
kanamycine (Sigma) was used for LA plates and 2×TY medium was used
at a concentration of 50mg/ml.
Protein purification
His-tagged FusB purification
Cell pellet was re-suspend in 10ml IMAC A buffer (Table2) with
1/2 pill Complete Protease Inhibitor Cocktail Tablets (Roche).
Cells were lysed by sonication and debris was spun down at
18,000rpm for 30mins. Supernatant was transferred into a filter
column together with 2ml Ni SepharoseTM (GE healthcare) and
equilibrated for at 4℃ for 1 hour. The matrix was washed with 30ml
IMAC A buffer, followed by 150ml IMAC A (high salt) buffer.
His-tagged FusB was then eluted with 16ml IMAC B buffer. Eluted
fractions were poured together and concentrated to 5ml in a
VIVASPIN 6 (Sartorius Stedim) with membrane cut-off 10,000 at
4000rpm. Concentrated fractions were further purified by size
exclusion column HiLoad 16/60 Superdex75 TM (GE healthcare) with
gel filtration buffer (Table2) using AKTA Purifier (GE healthcare).
Protein concentration was measured spectrophotometrically using
extinction coefficient ε280 = 26610 by NanoDrop ®, and concentrated
to 12mg/ml.
His-tagged EF-G purification
The same procedure as with His-tagged FusB was uesd except; IMCA
A/B buffer (Table2), extinction coefficient ε280 = 52510; and size
exclusion column was HiLoad 16/60 Superdex200 TM (GE
healthcare).
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TEV protease purification
His-tagged TEV protease was purified using Ni SepharoseTM using
a previously published protocol [18]. Table2 Protein purification
buffer
Buffer FusB EF-G IMAC A 50mM Na-phosphate, 300mM NaCl,
20mM Imidazole, 5mM BME pH7.0
50mM Tris-HCl, 200mM NaCl, 20mM Imidazole, 5mM BME, pH7.5
IMAC A (high salt)
50mM Na-phosphate, 600mM NaCl, 20mM Imidazole, 5mM BME,
pH7.0
50mM Tris-HCl, 600mM NaCl, 20mM Imidazole, 5mM BME, pH7.5
IMAC B 50mM Na-phosphate, 300mM NaCl, 500mM Imidazole, 5mM BME,
pH7.0
50mM Na-phosphate, 200mM NaCl, 500mM Imidazole, 5mM BME,
pH7.5
Gel filtration buffer
20mM Tris-HCl, 300mM NaCl, 5mM BME, pH8.3
20mM Tris-HCl, 200mM NaCl, 5mM BME, pH7.5
FusB – EF-G complex purification
The same protocol was used as for FusB purification until the Ni
SepharoseTM was equilibrated with the supernatant of FusB cell
lyses. The Sepharose was washed by 20ml FusB IMAC A buffer, and
then equilibrated with EF-G cell lyses supernatant (The debris was
spun down at 18,000rpm 30mins) for 1 hour. Then followed the same
procedure for FusB purification to wash, elute and further purify
by size exclusion column HiLoad 16/60 Superdex200 TM.
Digested S.a EF-G and FusB binding test
100μl 10μg/ml trypsin (Jena Bioscience) was added into 200μl
12mg/ml S.a EF-G and incubated at room temperature for 1 hour.
Meanwhile, 100μl 16mg/ml FusB was equilibrated with 100μl Ni
Sepharose in a filter tube at room temperature. The digestion
reaction was quenched by adding 1/20 pill Complete Protease
Inhibitor Cocktail Tablets (Roche). Digested EF-G was poured into
the filter tube and incubated together with FusB and Ni Sepharose
for 5min. Flow though was collected after centrifugation 4000rpm
for 1min. The digested fragments that couldn’t bind to nickel or
FusB were further washed with 500μl FusB IMAC A buffer for 6 times.
Everything binding to Ni Sepharose was eluted by 200μl FusB IMAC B
buffer at 4000rpm for 1 min. Imidazole in the elution was diluted
by a concentrator with FusB gel filtration buffer. Samples in
different stages were checked by both SDS and native PAGE.
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Crystallization of FusB and complex
FusB was screened by Easy Xtal JCSG+ (Molecular Dimensions),
MorpheusTM (Molecular Dimensions) and Structures Screen (Molecular
Dimensions). Drops mixed with 0.3μl FusB (12mg/ml) and 0.3μl
reservoir buffer (80μl) were prepared as a sitting-drop vapor
diffusion experiment using a crystallization robot and incubated
both at 277K and 293K. Hits from the Morpheus screen were repeated
manually with 2μl hanging drops (50% FusB) against 400μl reservoir
buffer. Morpheus screen C8, the only successfully repeated
condition was optimized by grid-screening with different
combination of pH (from 7.1 to 7.7) and precipitant concentration
(from 32% to 39%). Further optimization was initially screened by
ADDitTM Additive screen (Emerald BioSystems) in sitting drops each
contains 0.3μl 12mg/ml FusB, 0.3μl buffer (Morpheus C8, pH7.1, 32%
precipitant) and 0.1μl Additive. Some promising conditions with
less precipitate were re-checked by 2μl hanging drops with additive
concentration 2.5×10-4M, 5×10-3M and 2×10-3M. Easy Xtal JCSG+,
MorpheusTM, Structures screen and Protein Complex Suite (Qiagen)
were used for complex screening by sitting drops 0.3μl 15mg/ml
complex mixed with 0.3μl reservoir buffer (80μl).
Data collection
Before vitrified by liquid nitrogen, FusB crystals were soaked
with different concentrations of glycerol. Some of them were
dehydrated by changing the reservoir buffer with 50% precipitant
and incubate for two days. Crystals were checked at ID23-1 station
of European Synchrotron Facility in Grenoble, France. Two data sets
were collected with resolution beyond 5 Å. One of them was
collected by using 10% intensity of the beam. 725 images were
collected with oscillation range 0.4 degree, each image 0.79s.
Another one was using the same beam intensity but collected 340
images with oscillation range 0.25 degree, each image 0.581s.
Results
FusB and EF-G plasmid construction
The fusB gene (661bp) was successfully amplified (Figure.1) and
cloned into the pEXP-NT vector. fusB gene with added His-tag
sequence (688bp) and fusA which encodes EF-G, (2090bp) were cloned
into the pEXP-CT vectors. Plasmids with insert of correct site were
then sent for sequencing. All plasmids were confirmed to have the
right sequence except EF-G which contained a point mutation E242G.
This mutation was shown to come from the template and
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since it does not affect the activity, we decided to keep going
with it.
Figure.1. Amplified PCR products used for TA cloning run on 1%
agarose gel. 200bp ladder (O’ranger) was used for reference.
Tev protease His-tag cutting test
In order to increase the possibility of crystallization, the
presumably unstructured His-tag was cut off after protein
purification. We purified TEV protease and FusB_LN separately. To
test the home made TEV protease activity, a series of FusB mixed
with different concentration of TEV were incubated for different
time at 4℃ and 25℃. The digestion was then checked by SDS-PAGE.
(Figure.2). From the SDS-PAGE we can see that after digestion of
His-tagged FusB (28.8kD) by TEV protease, a smaller band
corresponding to FusB without tag (26kD) appears. Later large scale
purification shows that after TEV digest, FusB could not bind to
the nickel column, indicating that the His-tag has been cut off.
The experiment shows that even at protein concentration 1:200 (TEV:
FusB), most His-tag has been successfully chopped off at 25℃
overnight. Later experiment, the radio 1:10 mg/ml (TEV:FusB) was
used to make sure the reaction went to completeness.
Figure.2. TEV protease cutting test. 1mg/ml FusB was incubated
overnight with different concentration of TEV protease at both 4℃
and 25℃ and then examined by SDS-PAGE (20%
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PhastGelTM Homogeneous, GE Healthcare) using PhastSystemTM
(Pharmacia). The “n/a” mean no TEV protease was added.
FusB binds to S. aureus EF-G
To test if FusB could bind to S.a EF-G, purified FusB and S.a
EF-G were mixed at different molar ratios in FusB gel filtration
buffer (20mM Tris-HCl, 300mM NaCl, 5mM 2-Mercaptoethanol) and
incubated at room temperature for 10mins. The complexes were
examined by native-PAGE. (Figure.3 B) From the SDS-PAGE (Figure.3
A), we can see that the molar ratio of these two protein roughly
agree with the calculation. Since FusB has a very high pI (8.9,
calculated by ExPASy Proteomics Server), it did not run into the
native-PAGE. S.a EF-G alone shows two bands with similar
contribution. This indicates S.a EF-G could have two different
conformations in the native-PAGE buffer. After mixing with FusB,
the S.a EF-G bands disappear. Instead, a single band at a higher
position shows up. Mass spectrometry analysis later showed that it
contained both EF-G and FusB. With molar ratio (FusB:EF-G) 1:2,
excess EF-G was found together with the complex band, suggesting
that the complex is formed by these two proteins in a 1:1 molar
ratio.
Figure.3. Binding test of FusB and S.a EF-G. the two proteins
were mixed at different molar ratio (FusB:EF-G; 1:1, 1:2, 2:1) and
then run on both (A) 20% Homogeneous, SDS-PAGE (GE healthcare) and
(B) 8-25% gradient native-PAGE (GE healthcare) by PhastSystemTM
(Pharmacia). Low molecular maker is from Amersham Bioscieces.
FusB does not bind to E. coli EF-G
To check if FusB and E. coli EF-G could also form a complex,
FusB was mixed with E. coli EF-G and checked by native-PAGE. From
Figure.4 we can see that FusB and S. aureus EF-G could form a
complex. E. coli EF-G alone runs as a single bind slightly higher
than S. aureus EF-G. Since both proteins have similar
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molecular weight and shape, this is probably because E.coli EF-G
has higher PI (5.24) compared to S. aureus EF-G (4.93). No change
is observed when FusB is added, indicating that FusB does not bind
to E. coli EF-G. This means the FusB interact specifically with S.
aureus EF-G. Because E. coli EF-G and S. aureus EF-G’s sequence
identity is quite high (59%), and they presumably have very similar
over all structure, most likely the binding determinant is based on
some surface-exposed residues.
Figure.4. Binding test of FusB and E. coli EF-G compared to FusB
and S.a EF-G. FusB was incubated with the two EF-Gs separately in
FusB gel filtration buffer (20mM Tris-HCl, 300mM NaCl, 5mM
2-Mercaptoethanol) for 10mins and then run on a 8-25% gradient
native-PAGE (GE healthcare).
Complex purification
To increase the chance of complex crystallization, an EF-G
construct without His-tag was designed for complex purification.
FusB_SN was used to fish out EF-G. By doing so, the complex with
just one short His-tag can be purified directly and there is no
need to calculate the two protein concentrations separately for
mixing. Here we compare the size exclusion (HiLoad 16/60
Superdex200 TM, GE healthcare) results from the EF-G purification
and complex purification (Figure.5 A). There are two peaks for
complex purification. One comes earlier (69.81ml) than EF-G monomer
(74.37ml) peak, which is conformed by SDS-PAGE to be the complex
(Figure.5 B). Another peak that comes later than EF-G is excess
FusB and there is no peak around the EF-G monomer elution volume.
All these proteins have larger molecular weight calculated from the
elution volume than expected theoretically, possibly because they
have elongated shapes.
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Figure.5. (A) Complex purification comparing with EF-G
purification by size exclusion column HiLoad 16/60 Superdex200 TM
(GE healthcare) The first peak form EF-G purification is EF-G
dimmer or precipitation (B) SDS-PAGE results from different
peaks
FusB and S.a EF-G affinity test by fluorescence spectroscopy
The fluorescence spectroscopy method was used to determine the
binding affinity between FusB and S.a EF-G. However, due to the
inner filter effect [19] of FusB, protein-protein interaction could
only be poorly determined to 1μM (data not shown).
Digested S.a EF-G and FusB binding test
To investigate which part of S.a EF-G in interacting with FusB,
S.a EF-G was first digested by chymotrypsin, trypsin, subtilisin
and papain (Floppy-Choppy, Jena Bioscience) separately and then
checked by SDS-PAGE. Among these four proteases, trypsin and papain
could cut EF-G into two distinguishable bands with molecular weight
around 60kD and 25kD (Figure6 A). Based on this result, His-tagged
FusB was incubated with the trypsin-digested S.a EF-G in Ni
SepharoseTM. After washing out the fragments which could bind
neither nickel
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nor FusB, everything was eluted by 500mM imidazole. The samples
were run on both SDS and native-PAGE and checked by mass
spectrometry. Digested S.a EF-G had 5 distinctive bands on SDS-PAGE
(Figure.6 B). The top band is full-length EF-G not digested by
trypsin. The same band can also be found in previous
test-experiments (Figure.6 A). Three bands with molecular weight
around 55kD, 50kD and 45 kD were found both in digested EF-G and in
the flow-through of the Ni SepharoseTM, meaning that they bind
neither to FusB nor to nickel. Mass spectrometry analysis showed
that they all belong to S.a EF-G domainⅠand domain Ⅱ (Figure.6 B).
The larger band (Figure.6 B, bind “1”) contains fragments “a”
(Figure.6 D) at the N-terminus of the protein. Taking into account
the molecular weight and the fact it could not bind to nickel, this
suggests it contains the main part of domainⅠand domain Ⅱ but not
the His-tag from the N-terminus. Since band “1” was not further
digested into smaller pieces as suggested by PeptideCutter (ExPASy
Proteomics Server) and it ran as a clear band on the native-PAGE
(Figure.6 C), probably its structure was not destroyed by the
digestion. Therefore, we conclude that domainⅠand domain Ⅱof S.a
EF-G can not bind to FusB just by themselves. Band “4” (Figure.6 B)
with a molecular weight similar with FusB (26kD), became a very
pale band in flow though meaning it could have some interaction
with nickel or FusB. Mass spectrometry analysis shows that it
containing peptides fragments of EF-G domainⅢ (only C-terminal
part), domainⅣand domainⅤ (Figure.6 D, with blue label). Because
this part of EF-G only has 3 histidines spread out in the sequence,
it is unlikely to bind the Ni SepharoseTM, indicating that it
probably interacts with FusB. According its size from SDS-PAGE,
most likely it contains the whole part of domainⅣ, domainⅤand few
residues from the C-terminus of domain Ⅲ. Another evidence is that,
although bind “5” (Figure.6 B) is identified as FusB, two peptide
peaks, 1837.79 (Figure.6 D, “b”) and 1979.82 (Figure.6 D, “c”)
which do not belong to FusB, were detected and recognized parts of
domainⅣ and domainⅤ. These results indicate that the FusB mainly
interact with S.a EF-G with the domains that doesn’t have any
interaction with Fusidic acid.
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15
Figure.6 (A) S.a EF-G was digested by chymotrypsin, trypsin,
subtilisin and papain at room temperature for 30mins and 60mins.
(B) Different samples from the fragment binding test run on a 20%
Homogeneous SDS-PAGE (GE healthcare). (C) Different samples from
the fragment binding test run on 8-25% gradient gel (GE
healthcare). (D)The mass spectroscopy results of different bands on
SDS and native PAGE. S.a EF-G sequence is colored by different
domains. Fragments labeled yellow are the ones found in bands 1, 2
and 3 except fragment “a” just found in band 1. Fragments labeled
gray were detected from band 4. Two peptides marked “b”and “c”also
recognized belonging to S.a EF-G from band 5, which is mainly
FusB.
Crystallization and crystallography
FusB_LN was first purified and screened with three commercial
crystallization kits, Easy Xtal JCSG+ (Molecular Dimensions),
MorpheusTM (Molecular Dimensions) and Structures Screen (Molecular
Dimensions) using a crystallization robot. No protein crystal was
found even after two months.
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16
The second batch of FusB_SN was purified and screened using the
same screens. In MorpheusTM, 6 conditions with crystals were found
after 1 month (Table.3). The crystals from condition C8 were
successfully reproduced in manually prepared hanging drops. The
crystals were tested for diffraction at a synchrotron beamline.
However, the X-ray results showed that these crystals diffracted to
low resolution (7Å). To improve the crystals, the crystallization
condition was first optimized by grid-screening with different
combinations of pH and precipitant concentrations. Combinations of
pH7.1, 32% precipitant and pH7.7, 36% precipitant were used for
further tests because they gave crystals in different shapes
(Figure.7 A, B). ADDit screen was used for further optimization.
Crystal could grow in the present of low concentration hexamine
cobalt trichloride, magnesium chloride hexhydrate, guanidine HCl
and N-acetyl-L-cysteine (Figure.7 C, D, E, F), but these additives
failed to improve the resolution. Selenomethionyl protein was
produced using the methionine pathway inhibition method [20] for
multi-wavelength anomalous diffraction (MAD). After 1 month, some
tiny crystals were found but they were not good enough for
analysis. The complex with FusB_SN and EF-G without His-tag was
subjected to the JCSG+, MorpheusTM, Structure and ProComplex
screens but no crystals appeared. Table3 A. Hits formulation from
MorpheusTM screen
Well Buffer system Mix of additives Mix of precipitants
B8 0.1M MOP/HEPES-Na pH7.5 0.03M of each halide
C8 0.1M MOP/HEPES-Na pH7.5 0.03M of each NPS
G8 0.1M MOP/HEPES-Na pH7.5 0.03M of each carboxylic acid
A12 0.1M bicine/Trizma base pH8.5 0.03M of each divalent
cation
C12 0.1M bicine/Trizma base pH8.5 0.03M of each NPS
G12 0.1M bicine/Trizma base pH8.5 0.03M of each carboxylic
acid
12.5% w/v PEG 1000, 12.5%
w/v PEG 3350, 12.5 v/v MPD
Table3 B. Component of MorpheusTM additives mix
Name Component
Halides Sodium fluoride, Sodium bromide, sodium iodide
NPS Sodium nitrate, Sodium hydrogen phosphate, ammonium
sulfate
Divalent cation Magnesium chloride, Calcium chloride
Carboxylic acid Sodium formate, Ammonium acetate, trisodium
citrate, Sodium potassium L-tartrate, Sodium
oxamate
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17
Figure.7 (A) Crystals in MorpheusTM C8, pH7.1, 32% precipitant
(B) Crystals in MorpheusTM C8, pH7.7, 36% precipitant. (C) FusB
co-crystallized with hexamine cobalt trichloride. The yellow
crystal in the central is the cobalt crystal with the colorless
protein crystal covering it. (D) FusB crystal grew with the present
of 5×10-3M guanidine HCl. (E) FusB crystal grown with the present
of 5×10-3M magnesium chloride hexhydrate. (F) FusB crystal grew
with the present of 5×10-3M N-acetyl-L-cysteine. The obtained data
sets were processed by XDS. Systematic absences from data set A
indicates that the most probable space group is P21212 with cell
dimensions a=187.516, b=99.537, c=129.703, α=β=γ=90°. Self-rotation
analysis with polarrfn shows a 4-fold axis 3° away from a
crystallographic 2-fold. The crystal has a severe anisotropy
problem (Figure.8) which means the resolution is direction related.
In this case, dimension b and c are diffracted to 4.5Å; dimension a
can diffract beyond 4 Å and data completeness will dramatically
fall at high resolution shell (Table.4) thus, further optimization
of the crystal is needed to solve this problem. Table4. Data
collection statistics of FusB crystals
Data set A Data set B Resolution 50-3.69 Å 50-3.8 Å
Highest space group determined
P21212 P2122
Cell parameters a=187.5, b=99.5, c=129.7,
α=β=γ=90° a=185.7, b=97.9, c=127.8,
α=β=γ=90° Unique reflections 20840 18626
Completeness
96.8% (5.0 Å) 70.0% (4.17Å) 53.2% (3.68 Å) 77.5% (total)
90.8% (5.11 Å) 68.0% (4.05Å) 44.3% (3.81 Å) 78.7% (total)
1/σ 9.11 10.14
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18
Figure.8 Data set B was analysised by diffraction anisotropy
server: http://www.doe-mbi.ucla.edu/~sawaya/anisoscale.
Discussion
Structure determination of FusB and FusB - EF-G complex
Further optimization of the FusB crystal is needed to get higher
resolution and to minimize the anisotropy problem. First thing to
try is to crystallize FusB without His-tag. Since long-tagged FusB
can not be crystallized, it is a hint that the His-tag might
disturb the crystal packing. Second, we know that FusB has a high
pI (8.9) and high lysine content (13%), proteins of this kind are
usually not easy to crystallize, because high charge and lysine on
protein surface is not good for protein packing, which often
requires hydrophobic interaction and low surface entropy [21]. FusB
crystals could also suffer from this. The easiest thing to try is
to change buffer pH. Theoretically, when the pH is near pI, the net
charge on the surface of protein is minimized. Buffer screen showed
that FusB is stable in different buffers from pH 6.0 to 10.0, thus,
a pH screen from 8.5 to 9.5 is worth trying. Lysine methylation is
often used as a standard rescue method when the lysine content is
high. After methylation, the lysines on the protein surface are
“neutralized” and often cause a drop of pI and facilitate the
crystallization. Based on the same principle, mutation of the
lysine on protein surface could also help. Since no homologue
structure is known for FusB, it is hard to decide which lysine to
mutate; leaving lysine methylation the best way to try. We noticed
that FusB tend to bind nucleic acids during purification. This
means that it probably prefer an environment with the presence of
nucleic acid,
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19
thus by adding some ATP or ADP could help to stabilize the
protein. For the FusB – EF-G complex, lysine methylation is also
worth trying because it can change protein surface property. In
order to increase the crystallization probability and to conform
which domain of EF-G can bind to FusB, we are now constructing
hybrid EF-Gs with different domains from E. coli and S. aureus
EF-G. If a certain domain is found to bind to FusB, a complex of
FusB and that single domain could also be tested for
crystalization.
The binding target of FusB
The binding experiment of FusB with protease digested EF-G was
aiming to find out where FusB binds. This will help us to
understand the resistance mechanism and maybe facilitate the
complex crystallization by designing some small binding partner.
The overall structure of EF-G is conserved among different species.
EF-G can be divided into 5 different domains (Figure.9 A), where
domainⅠand domain Ⅱ are quite similar to EF-Tu (another bacterial
elongation factor) [22]. Domain Ⅲ acts as a linker of domainⅠ,Ⅱ and
domainⅣ,Ⅴ. The structure of ribosome with EF-G trapped by fusidic
acid reveals the binding of fusidic acid involves domainⅠ,Ⅱ and
domain Ⅲ. When EF-G is locked on the ribosome, domains Ⅲ and Ⅳ are
relatively buried inside; therefore they are not easy for FusB to
access (Figure.9 B, C). Since our result showed that domainⅠand Ⅱ
together can not bind to FusB, leaving only domainⅤ. There are some
other evidences also supporting that domainⅤ is responsible for the
binding. First, the mass spectroscopy identified two peptide
fragments belong to domainⅣ and domainⅤ eluted together with FusB.
Second, the multiple alignment between S. aureus, L. monocytogenes,
and E. faecalis EF-G (all have FusB homologues and intrinsically
resistance to fusidic acid) and E. coli EF-G. shows that if we
split domainⅤ into two parts, the one facing to the ribosome is
very conserved, however, the other part exposed to the environment
is only conserved in S. aureus, L.monocytogenes and E.faecalis
(Figure.9 G, H). Actually, there are only four places where E. coli
EF-G has low similarly with the other three species and two of them
belong to domainⅤ. This indicates the face of domainⅤ exposed to
the environment could possibly be the FusB binding determinant. If
this assumption is right, how can the binding of domainⅤ cause the
resistance when the fusidic acid binds to other parts of the
protein? A previous study observed a movement between domain Ⅲ,Ⅳ,Ⅴ
and domainⅠ,Ⅱ [23] suggesting that domainⅤ and domainⅢ are
functionally closely related. For instance, a movement of domainⅤ
could change the conformation of domainⅢ. Interestingly, some
mutations, F652S, Y654N, A655V, located on domainⅤ and were found
to be related to fusidic acid resistance [10]. Furthermore, they
all
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20
belong to the conserved part of domainⅤ and facing towards the
ribosome (Figure.9 A, G). This means the changes on domainⅤ itself
could cause fusidic acid resistance.
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21
Figure.9 (A) S.a EF-G domains were depicted in different colors.
(PDB: 2XEX) the mutations on domainⅢ and domainⅤ are highlighted
(B) Side view of EF-G binding on the ribosome, showing that domains
Ⅰ, Ⅱ and Ⅴ are more exposed. (PDB: 2WRI, 2WRL) (C) Top view of EF-G
binding on the ribosome (D) EF-G is extracted from the ribosome,
showing the binding side of fusidic acid (E) fusidic acid (F)
Multiple alignment showing that domain Ⅲ is very conserved among S.
aureus, L.monocytogenes, E.faecalis and E. coli. Mutations that
cause fusidic acid resistance are labled. (G) Multiple alignment of
domianⅤ, two pieces of sequences are only conserved in S. aureus,
L.monocytogenes and E.faecalis. (H) S.a EF-G with highlighted
sequences which only conserved among S. aureus, L.monocytogenes and
E.faecalis,
Hypothetical resistance mechanisms
As discussed previously, the mechanism of FusB mediated fusidic
acid resistance doesn’t fall into any of the four classic
strategies bacteria used to against antibiotics. Since FusB can
bind to S. aureus EF-G and rescue the translation but not E. coli,
this binding is probably the key for the resistance. Assuming the
FusB binds to EF-G domainⅤ, there are two possible FusB induced
resistance mechanisms. The first one is due to changes in the
fusidic acid binding environment. This mechanism is like some point
mutations in domainⅠ and domainⅢ (P88A, V90I, L461K and
H457Y/H457Q) that may affect the fusidic acid binding [3] [24].
Another possible mechanism is that FusB could facilitate the
release of the EF-G from the ribosome and fusidic aicd drop off
afterwards.
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22
As mentioned before, we found that FusB tend to bind nuclear
acids in vitro (data not shown). A binding experiment of FusB with
ribosome in presence and in absence of EF-G and fusidic acid will
clarify if FusB can interact with the ribosome. Since we don’t know
if FusB will constantly bind to EF-G in vivo or it needs to be
re-cycled by other factors, the ribosome could be a potential
candidate competing with EF-G. We also plan to test FusB in an in
vitro translation assay to see how it will affect the translation
in presence and in absence of the fusidic acid. It will tell
whether FusB can rescue the in vitro translation and at what cost.
Because FusB may interfere the normal functions of EF-G or the
ribosome, by comparing the translation rate with and without FusB,
we can investigate if there is any side effect of this
resistance.
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23
Acknowledgement
First and foremost I’d like to thank my supervisor Maria Selmer.
For the chance you gave me and your patience to guide me into
structure biology. Everyday I had in your group is fantastic
adventure. I remember although sometimes the experiment seems going
no where, you always have faith in me and encourage me until the
break though comes. I’ll also thanks to our group member Cha San
Koh, Kristina Bäckbro, Chen Yang and Avinash Punekar, for all your
kindly help and suggestions. Especially Cha San, She always came
first when I needed help. Thanks to Alwyn Jones, Lars Liljas and
Terese Bergfors, who taught me the course crystallography. Thanks
to Celestine Chi, and Per Jemth for the help of fluorescence
spectroscopy test. Thanks to Suparna Chandra Sanyal for the S.a
EF-G vector. Thanks to Diarmaid Hughes for the extraction of pUB101
plasmid.
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24
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