Binding of natural metabolites to bacterial
transcriptional repressors as tools for
synthetic biology
Actinobacteria of the genus Streptomyces account for ~70% of clinically viable antibacterial compounds.
“Clamp-and-click” binding, where conformational twisting
about the dimer interface occurs upon contact with an ARE.
The TetR-like repressors MmfR & MmyR bind autoregulator response elements (AREs) upstream of the genes they regulate.
There are 3 AREs in S. coelicolor A3(2); upstream of the mmfR/mmfL, mmyB, and mmyR genes.
AHFCA binding is communicated to the DBD via the
transmission helix, reverting the twist and locking the
repressor in a rigid state.
MmfR repression is regulated by the 2-alkyl-4-hydroxymethylfuran-3-carboxylic acids (AHFCAs). These furans are synthesised
in situ by MmfL, MmfH and MmfP. The alkyl chain is variable, determined by fatty acid metabolism.
The MmyR ligand is unknown. This “pseudo-furan” repressor is hypothesised to bind an antibacterial compound based on
other ArpA-like/”pseudo”-ArpA-like regulated systems.
Apo-bound
clamped state
AHFCAs
TetR
Repressor
Binding
Clamping
Promoter
ARE
Gene
RNA Polymerase
24
23
22
21
20
19
18
17
16
15
14
13
12
9
11
8
10
7
6
5
4
3
2
1
24
T
C
A
23
22
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
bits
bits
CG GT GTCTGA CG AT AT
24
23
T
A
22
21
20
TT T
AACGA
19
18
GC
16
13
A
15
C
G
TA
A
14
C
T
12
T
11
7
6
5
4
3
2
GA
10
T
T
AAG A
TT
0
9
1
8
bits
Holo-bound
clamped state
1
Holo-locked
unbound state
2
17
MEME (no SSC) 18.03.2015 08:37
Holo-bound
unclamped state
212.25
AHFCA 4
CH2CH2CH2CH3
198.22
AHFCA 5
CH2CH2CH2CH2CH3
212.25
AHFCA 6
CH2CH2CH2CH(CH3)CH3
226.27
AHFCA 7
CH2CH2CH2CH2CH2CH3
226.27
AHFCA 8
CH2CH2CH2CH2CH(CH3)CH3
240.30
AHFCA 8E
CH2CH2CH2CH2CH2CH2CH3
240.30
AHFCA 9
AHFCA 10
CH2CH2CH2CH2CH(CH3)CH2CH3
CH2CH2CH2CH2CH2CH(CH3)CH3
254.33
254.33
MEME (no SSC) 18.03.2015 08:40
Q130 stabilises the interaction via hydrogen bonding of the
side-chain amide (NH2) to the hydroxyl of Y85. Also in
close proximity is H84, but it’s role is still unclear.
AFHCA binding
Dimer Interface
interaction
Y144
N190
R133
N
Sequence 5’-AATATACCTGCG GGAAGGTATTAT-3’
5’-AAAAAACCTTCG GGAAGGTTTGAC-3’
5’-AACATACCCTCG GGAAGGTATGTT-3’
5’-AAAATACCTTCT CAAAGGAATTAT-3’
5’-AATATACCTGCG CGAAGGTATATT-3’
5’-AAGATACGTTCG TGAACGAATTCT-3’
5’-AATATACCTATG GGAAGGTATATT-3’
5’-TTGAAACCTTCG CGAAGGTTTGAT-3’
5’-AAGAAACTTCCA AGGCGGATCTTT-3’
5’-TGAATACCTCCG CGAAGGAATGAT-3’
Y85
H84
Q
130
Dimerisation 2
A
CH2CH2CH(CH3)CH3
Dimerisation 1
T
G
0
AHFCA 3
LBD Core 3
1
184.19
LBD Core 2
AT GTATCT
MEME (no SSC) 18.03.2015 08:34
2
198.22
CH2CH2CH3
LBD Core 1
TT
CG
CH2CH(CH3)CH3
AHFCA 2
ARE
mmfR-mmfL
mmyB
mmyR
SAV_2301
SAV_mmfR-mmfL
SAV_mmyR-mmfH
SSCL_mmfR-mmfL
SSCL_Qnc_(LNM)
SSCL_mmfH-mmyR
SSCL_ORF-1
AA A ACC CGGGAAGGT T
AA ATAC T C AA G AT T
A ACCT CG GAAGG T T
T
A
AHFCA 1
Transmission
0
T
C
A
mW (g mol-1)
Recognition
1
Unclamping
Molecular formula (R)
Spacer
Release
Ligand
AHFCAs interacts primarily via their carboxylate group with
2 highly-conserved residues; Y85 via it’s side-chain hydroxyl
group and Y144 via it’s backbone amide.
Stabilisation
Ligand
2
HFCAs, as wells other ArpA inducers such as S. coelicolor
butanolide 1 (SCB1), induce little-to-no activity.
E203
Histidine in SAV_MmfR is substituted with phenylalanine.
E206
C
R175
R165
In silico docking of MmfR & orthologues
Ligand
MmfR-AHFCA2
Crystal
18+ clusters containing homologous repressor systems have been identified, including S. avermitilis and S. sclerotialus.
MmfR-AHFCA2
-6.8 kcal mol-1
-4.4
Y144
Y144
-4.8
Docking energy - kcal / mol
The prevalence of these regulatory systems in Actinobacteria make them suitable candidates for study, given the “cryptic” gene
clusters they regulate with potential antibacterial compounds of interest.
Y85
Y85
Operator & ligand specificity, low threshold concentration requirement for de-repression, and the potential for modified biligand regulated hetero-dimeric proteins, leaves potential for future synthetic applications.
H84
SAV_MmfR-AHFCA2
-6.4 kcal mol-1
1 kb
ARE
ARE
ARE
SAV % ID S. avermitilis MA-4680
ARE ARE
(50,462 bp)
ARE
1 kb
S. sclerotialus NRRL ISP-5269
ARE ARE
(19,981 bp)
ARE
ARE
40.5 49.8 43.0 % to MmyR 35.0 37.8 35.7 % to MmfL 28.0 30.2 32.4 % to MmfH 39.7 42.0 Y90
-6.8
-7.2
SAV_MmfR-F89H-AHFCA2
-6.8 kcal mol-1
Y150
T153
Y150
H89
in silico ligand design for SAV_MmfR
Prepare selenomethionine-substituted proteins for structure-function studies using MAD X-ray crystallography.
In vitro studies
Genomic DNA
extraction using
FASTDNA® SPIN Kit
for Soil.
Pilot expression,
analysis by SDSPAGE.
Purification via Ni2+
IMAC, concentration
and buffer exchange
via centrifuge
filtration, SDS-PAGE.
850
850
400
400
Transform plasmid
into E. coli BL21 Star
DE3.
pET151/D-TOPO®
cloning &
transformation into
E. coli TOP10.
Plasmid extraction
with GENEJET
Plasmid Miniprep Kit,
verify via PCR &
sequencing.
F
+
R
F
F
+
+
RD1 RS
FD3 FD3 FD3 FS FS
+
+
+
+
+
R RD1 RS R RD1
FS
+
RS
100
SAV_MmyR - PCR - 62°C
F
+
R
F
+
RS
-5.6
-6.6
-5.8
Docking energy - kcal / mol
-6.8
-7.0
-7.2
-7.4
-7.6
-7.8
-8.0
FS
+
R
F
+
RD7
Cleavage of N-Term
tag using TEV
protease & further
purification.
-6.4
Best fit: -7.2
-6.6
-6.8
-7.0
-7.4
-8.4
MmfR
MmfR-H84F
FS
+
RS
FS
+
RD7
CprB-A
CprB-B
CprB-C
CprB-D
!
MmfR
MmyR
…MARQLRA
…MARQLRA
…MARQLRA
…MARQLRA
MmfR-Y85F
MmfR-Y144F
EQTRATIIGAAADLFDRR
EQTRATIIGAAADLFDRR
EQTRATIIGAAADLFDRR
EQTRATIIGAAADLFDRR
MmfR-H84F/Y85F
SAV_MmfR-like_Phyre
GYESTT
GYESTT
GYESTT
GYESTT
LSEIVAHA
LSEIVAHA
LSEIVAHA
LSEIVAHA
GVT
GVT
GVT
GVT
KGALYFH
KGALYFH
KGALYFH
KGALYFH
FAA
FAA
FAA
FAA
KEDLAAIL…!
KEDLAAIL…!
KEDLAAIL…!
KEDLAAIL…!
…HPQQERS IKTRAQILEAASEIFASR GYRGAS VKDVAERV GMT KGAVYFH FPS KESLAIAV…!
MKQARA MRTRDQVLDAAAEEFALH GYAGTN LATVAVRT GMT KGALYGH FPS KKALADEL…!
Conclusions
25 kDa
15 kDa
Ctrl
4.6 kDa
-6.2
-7.2
-8.2
40 kDa
10 kDa
-6.0
MmfR prediction scores: mmyB > mmyR > mmfR/mmfL. EMSA with MmfR implies the specificity is mmyB >
mmfR/mmfL > mmyR, which does not match these predictions. However MmyR did follow this order of specificity.
Optimisation of the HADDOCK algorithm is required.
2000
2000
100
Polymerase chain
reaction.
5000
-6.4
Repressor:operator dockings failed to simulate the dimer-twisting mechanism. One of the monomers still interacted
in the anticipated fashion, although not all chains bind with consistent amino acid specificity. These residues could be
potential future mutagenesis targets. In depth analysis still pending.
If time permits, perform further characterisation of these repressors using site-directed mutagenesis.
SAV_MmfR - PCR - 58°C
Docking energy - kcal / mol
Ligand
Characterise thermodynamic binding profiles of AHFCA/ARE binding for both MmfR/MmyR and their homologues in S.
avermitilis and S. sclerotialus in vitro using SPR, ITC, Trp quenching & SwitchSENSE.
5000
SAV_MmfR_F89H
Ligand
Build in silico models of AHFCA/ARE binding, supporting with experimental data to validate model viability.
•  Orthologues generated using Phyre2 (1) based on 1.5 Å crystal structures of MmfR & MmfR:AHFCA-2.
•  Ligand docking performed using AutoDock Tools & AutoDock VINA(2).
•  Repressor:ARE docking performed using HADDOCK(3). Interface residues predicted using CPORT(4),
and DNA structures for docking generated using 3D-DART(5).
Scaled-up
expression in LB
culture; 10, 100, 250,
400 mL etc.
SgnR_Phyre
SSCL_MmfR-like_Phyre
In silico mutagenesis does not correlate in vivo/in vitro
mutagenesis data. Note that ligands probably still bind in
these mutants, but their ability to induce de-repression is
diminished.
Y90
38.4 Aims
Solubility
determination,
analysis via SDSPAGE.
SAV_MmfR-like_Phyre
Luciferase-linked in vivo assays performed by Kathryn
Styles has indicated MmfR:AHFCA affinity as 5>4>2,
which is consistent with the in silico docking data. Data for
AHFCA 1 & 3 is still being validated.
SAV_MmfR-AHFCA_SAVx4(S)
-7.2 kcal mol-1
In silico mutagenesis docking of MmfR
Growth of
Streptomyces
avermitilis &
sclerotialus cultures.
MmfR
H62
43.8 40.2 -6.4
Characterisation of AHFCA specificity using bioassays &
electrophoretic mobility shift assays (EMSA) provided
inconsistent data.
Y63
Y90
32.0 -6.0
-8.0
T153
% to MmfP 1 kb
SSCL % SAV/SSCL ID % ID % to MmfR -5.6
Y122
T153
(19,341 bp)
-5.2
-7.6
SSCL_MmfR-AHFCA2
-6.8 kcal mol-1
Y150
S. coelicolor A3(2)
A: School of Life Sciences
B: Department of Chemistry
2 functional domains: an N-terminal DNA-binding domain
(DBD) and a C-terminal ligand-binding domain (LBD),
linked by the transmission helix.
MmfR:AHFCA-2
1.5 Å
The SCP1 plasmid-encoded, 21-gene biosynthetic cluster of methylenomycin A in S. coelicolor A3(2) encodes 4 transcriptional
regulators; MmyB, MmyJ, MmyR, & MmfR.
Apo-bound
unclamped state
Contact: A.Fullwood@warwick.ac.uk
In silico studies
Introduction
Apo-unbound
state
Alex FullwoodA, Christophe CorreAB,
Richard NapierA, Vilmos FülöpA
Sol.
+
Insol.
IPTG
Ctrl
IPTG
Ctrl
IPTG
Sol.
+
Insol.
Sol.
SAV_MmfR
Ctrl
IPTG
Sol.
SAV_MmyR
One must be wary when using a Phyre2 generated structure, as it may not be necessarily representative of the true structure.
Nevertheless, the methodology could prove insightful when more information – either crystallography or in vivo liquid
chromatography-mass spectrometry (LC-MS), is available. Currently no data for AHFCA 6-10 inducing methylenomycin A
production in S. coelicolor A3(2) is available.
Future in vitro work:
180 kDa
130 kDa
100 kDa
Cleavage of the MmfR N-terminal tag via TEV protease and elution through IMAC to improve protein purity.
70 kDa
55 kDa
Purification of SAV_MmyR, although SAV_MmfR is the priority.
40 kDa
Biacore T200 SPR training to begin 6th-7th July, 2015 – SPR trials likely to
start soon after.
35 kDa
PCR of SSCL_mmfR/mmyR from the genomic
DNA extraction has failed due to excessive nonspecific binding. Synthetic genes from Life
Technologies GeneArt™ DNA-strings are currently
being processed.
25 kDa
2nd set of SwitchSENSE trials: poor experimental design led to failure of 1st trial.
15 kDa
Flow through
Wash
40 mM
imidazole
1. The Phyre2 web portal for protein modeling, prediction and analysis, Kelley LA et al. Nature Protocols 10, 845-858 (2015).
2. O. Trott, A. J. Olson, AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading, Journal of Computational Chemistry, 31, 455-461 (2010).
3. S.J. de Vries, M. van Dijk and A.M.J.J. Bonvin, The HADDOCK web server for data-driven biomolecular docking., Nature Protocols, 5, 883-897 (2010).
4. S.J. de Vries and A.M.J.J. Bonvin, CPORT: a Consensus Interface Predictor and its Performance in Prediction-driven Docking with HADDOCK, PloS One, 6 e17695 (2011).
5. M. van Dijk and A.M.J.J. Bonvin (2009), 3D-DART: a DNA structure modelling server, Nucl. Acids Res., 37 (Web Server Issue):W235-W239 doi:10.1093/nar/gkp287
Elution
200 mM
imidazole
SAV_MmfR
concentration
13.1 mg mL-1
Filtrate
Transformation/expression of the SSCL repressors when gene are available.
Acknowledgements
Acknowledgements
Corre group:
Christophe Corre, Vincent Poon, Kathryn Styles,
Jacqueline Gill & John Sidda (and the rest).
Additional:
Richard Napier, Mussa Quareshy (Napier group),
& Vilmos Fülöp.