Download Structural and functional analyses of the outer membrane

Structural and functional analyses of the
outer membrane transport of aromatic
hydrocarbons by the biodegrader
Pseudomonas putida F1
Anne Doble
Pseudomonas putida F1; a versatile biodegrader
• Gram-negative, non-pathogenic, soil-dwelling, metabolically highly versatile
• Isolated from a polluted stream in Urbana (Illinois) through growth on
ethylbenzene
• Capable of growth on range of mono-aromatic hydrocarbons as sole carbon
source (e.g. BTEX compounds)
• A valuable biodegrader for the bioremediation of contaminated groundwater
BTEX compounds (Benzene, Toluene, Ethylbenzene &
Xylene):
• Volatile mono-aromatic hydrocarbons (MAHs)
• Widely used in chemical/petroleum industry
• Classed as “priority” soil & groundwater pollutants
• Highly toxic/carcinogenic
Bioremediation: “The use of either naturally
occurring or deliberately introduced
microorganisms to consume and break down
environmental pollutants, in order to clean a
polluted site”
Uptake of hydrophobic compounds by Gramnegative bacteria
• FadL OM protein family;
• Facilitate passive transport of hydrophobic
compounds
• Exemplified by E. coli FadL LCFA transporter
L3
L4
•
•
•
•
Monomeric 14-stranded β-barrel
Globular N-terminal ‘plug’ domain
Lateral opening in barrel wall
Hydrophobic pocket from L3 & L4 loops;
substrate binding
• Hydrophobic passage leading from
Current
uptake surface
model: to lateral opening
extracellular
Ligand-gated lateral diffusion of substrates
through barrel wall into OM outer leaflet
E. Coli FadL Crystal structure
FadL schematic; cut-through view
EM. Hearn et al., Nature 458, 367-370 (March 2009)
Project Aims
• Delineate mode of aromatic hydrocarbon uptake by
the P. putida F1 FadL proteins
• Applications…..
• Understand how biodegradative organisms may be used
more effectively for bioremediation (e.g. by increasing
substrate uptake range)
• Broader implications; understanding the uptake of
hydrophobic antibiotics.
Toluene uptake in Pseudomonas putida F1
TodX
• Toluene metabolic genes encoded on F1-specific genomic island
• tod operon (toluene degradation); toluene  pyruvate & acetyl CoA
• todX encodes OM protein; involved in toluene uptake?
• F1 Genome encodes 3 FadL-family proteins; F1FadL, TodX & CymD
PtodX
PtodST
X
F
C1
C2 B
A
D
E
G
I
H
S
T
P. putida
F1 genome
CH3
CH3
CH3
todC1C2BA
CH3
todD
O2
O2
Toluene
Toluene cisdihydrodiol
OH
3-Methylcatechol
Pyruvate,
acetyl-CoA
O
OH
OH
OH
todF  todI
todE
COOOH
(Meta-cleavage pathway)
CH3COO-
TCA cycle
P. putida F1 TodX structure solution
• 20-23% sequence ID to E.coli & F1 FadL
• Structurally similar to E. coli FadL (lateral wall opening, N-terminal ‘plug’ domain), but……
• Smaller lateral opening in barrel wall
• Hydrophobic lumen; continuous 4.5-5 Å channel through N-terminal hatch?
?
Does the ‘lateral diffusion’ model of substrate uptake apply to the
uptake of toluene by TodX?
TodX (cut-away surface view)
TodX
EcFadL
EM. Hearn et al., PNAS. 105 8601-8606 (June 2008)
P. putida F1 in vivo toluene growth assays
• F1 FadL protein mutants show diminished growth on LOW LEVELS toluene (sole
carbon source)
• Growth recovered with complementation with Tn7-todX & Tn7-cymD (genomic
insertions)
F1-3
F1 – WT
F1-1 – ΔtodX
F1-2 – ΔtodX ΔcymD
F1-3 – ΔtodX ΔcymD
ΔF1fadL
F1-3 Tn7
-EcfadL
F1-3
Tn7-F1fadL
F1-3 Tn7
-todX
F1-3
Tn7-cymD
Site-directed mutagenesis of TodX
• Target regions of TodX potentially important for function:
• Assess protein function using in vivo growth assays and X-ray crystallography studies
S2/S3 strand lateral opening
N-terminal hatch channel
Extracellular
OM
TodX
(WT)
Periplasm
S3 S2
(surface cut-through view)
Functionality of S2/S3 lateral opening mutants
• S2/S3 strand mutants designed to close up lateral wall opening
• Both Tn7-todX S2 & S2/S3 mutant variants unable to complement growth of F1-3
(ΔtodX/cymD/fadL) mutant on toluene
• Solution of mutant crystal structures confirmed flattening of S2 & S3 strands
WT TodX
F1-3 (ΔtodX/cymD/fadL)
F1 (WT)
0
0
1
1
S3
4
2
S3 3
0 – Parent strain
1 – Tn7-todX
4
S2
TodX S2/S3
TodX S2
2
S2
3
2 – Tn7-cymD
WT TodX (orange), S2 (blue) & S2/S3 (purple)
mutants: cartoon overlay
3 – Tn7-todX S2
S3
S2
4 – Tn7-todX S2/S3
S3
Surface view of lateral opening regions
S2
Functionality of N-terminal hatch mutants
• Site-directed mutants designed to block continuous channel through N-terminal hatch domain
• Tn7-todX hatch mutants still able to complement growth on toluene
• Crystallisation & structure-solution of hatch mutant variants ongoing.
F1-3 (ΔtodX/cymD/fadL)
Key
1 = Parent strain
1
5 2
Ala-28 4Met
3
2 = Tn7-todX
Ala-28  Gln
F100
*3 = Tn7-todX A28M
Y9
Q83
*4 = Tn7-todX A28Q
A28
*5 = Tn7-todX A28W
TodX N-term.
channel
6WT
= Tn7-todX
Y9C/F100C
constriction site
*7 = Tn7-todX Y9W/F100W
12
Ala-28 
6
7 Trp
Tyr-9  Trp
Phe-100  Trp
TodX N-term. channel block mutants –
theoretical structures
Putative toluene-binding site
• Mutate aromatic residues  abolish toluene binding?
• Solve structure of TodX:toluene complex
(Surface view)
Hydrophobic
Hydrophilic
Conclusions & ongoing targets
• TodX (and CymD?) are required for toluene acquisition by P. putida F1 at
low toluene concentrations
• Functional redundancy between the FadL proteins?
• Growth assays and structural studies with site-directed mutant variants of
TodX show:
• S2/S3 strand lateral opening is important for TodX function
• Presence of hatch channel in N-terminal plug domain unnecessary for function?
• Importance of S2/S3 strand structure supports ‘lateral diffusion’ model of
substrate uptake in TodX
• Ongoing work:
• Solve structure of N-terminal hatch channel mutants of TodX
• Solve structure of TodX in complex with toluene; identify toluene binding site(s)
• Assess basis of TodX substrate specificity; TodX L3 & L4 loop structure, TodX Nterminus, CymD structure solution
Acknowledgements
Newcastle University:
• Professor Bert van den Berg & lab:
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•
•
•
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Javier Abellon-Ruiz
David Bulmer
Amy Glenwright
Monisha Pathania
Michael Zahn
• Arnaud Baslé
Diamond Light Source Ltd. (UK
Synchrotron)
National Institutes of Health