Conformational and dynamic plasticity in substrate-binding proteins underlies selective transport in ABC importers

  1. Marijn de Boer
  2. Giorgos Gouridis
  3. Ruslan Vietrov
  4. Stephanie L Begg
  5. Gea K Schuurman-Wolters
  6. Florence Husada
  7. Nikolaos Eleftheriadis
  8. Bert Poolman  Is a corresponding author
  9. Christopher A McDevitt  Is a corresponding author
  10. Thorben Cordes  Is a corresponding author
  1. University of Groningen, The Netherlands
  2. Ludwig-Maximilians-Universität München, Germany
  3. KU Leuven, Belgium
  4. University of Melbourne, Australia
  5. The University of Adelaide, Australia
7 figures, 3 tables and 5 additional files

Figures

Representative SBPs from different structural clusters, categorized by their hinge region.

X-ray crystal structures of PsaA (3ZK7; cluster A), MalE (1OMP; cluster B), OppA (3FTO; cluster C), OpuAC (3L6G; cluster F), SBD1 (4LA9; cluster F) and SBD2 (4KR5; cluster F) are all shown in the …

https://doi.org/10.7554/eLife.44652.002
Figure 2 with 4 supplements
Conformational states of SBPs probed by smFRET reveal multiple active conformations.

(A) Experimental strategy to study SBP conformational changes via FRET. Solution-based apparent FRET efficiency histograms of OpuAC(V360C/N423C) (B), PsaA(V76C/K237C) (C), MalE(T36C/S352C) (D), …

https://doi.org/10.7554/eLife.44652.005
Figure 2—figure supplement 1
Ligand-induced conformational dynamics of SBPs.

Representative fluorescence trajectories (left) and apparent FRET efficiency histograms from all fluorescence trajectories (right) of MalE(T36C/S352C) (A), SBD2(T369C/S451) (B), OpuAC(V360C/N423C) (C

https://doi.org/10.7554/eLife.44652.006
Figure 2—figure supplement 2
OppA uses an induced-fit ligand binding mechanism.

(A) Representative fluorescence trajectories of OppA(A209C/S441C) at different peptide (RPPGFSFR) concentrations; donor (green) and acceptor (red) photon counts. The top panel shows the calculated …

https://doi.org/10.7554/eLife.44652.007
Figure 2—figure supplement 3
Translocation competent conformation(s) of MalE and OppA.

Solution-based apparent FRET efficiency histogram of MalE(T36C/S352C) (A) and OppA(A209C/S441C) (B) in the absence and presence of different cognate substrates as indicated. The OppA substrates are …

https://doi.org/10.7554/eLife.44652.008
Figure 2—figure supplement 4
MalE conformations studied by smFRET.

Solution-based apparent FRET efficiency histogram of MalE(T36C/S352C), MalE(T36C/N205C) and MalE(K34C/R354C) in the absence and presence of different cognate substrates as indicated. Bars are the …

https://doi.org/10.7554/eLife.44652.009
Figure 3 with 2 supplements
Rare conformational states of ligand-free SBPs.

(A) Schematic of the experimental strategy to study the conformational dynamics of ligand-free SBPs. Representative fluorescence trajectories of OpuAC(V360C/N423C) (B), PsaA(V76C/K237C) (C), …

https://doi.org/10.7554/eLife.44652.012
Figure 3—source data 1

Donor and acceptor photon counts, apparent FRET efficiency and most probable state-trajectory of the Hidden Markov Model of the traces in Figure 3.

https://doi.org/10.7554/eLife.44652.015
Figure 3—figure supplement 1
Conformational dynamics of ligand-free and ligand-bound SBPs.

Representative fluorescence trajectories of OpuAC(V360C/N423C) (A), PsaA(V76C/K237C) (B), MalE(T36C/S352C) (C), SBD1(T159C/G87C) (D), OppA(A209C/S441C) (E) and SBD2(T369C/S451) (F) in the absence of …

https://doi.org/10.7554/eLife.44652.013
Figure 3—figure supplement 2
Intrinsic conformational dynamics in the presence of unlabeled protein.

Closing rate (A) and average lifetime of the closed conformation (B) for OppA, SBD1 and SBD2 in the absence of ligand and in the presence of different concentrations of unlabeled protein to scavenge …

https://doi.org/10.7554/eLife.44652.014
Figure 4 with 3 supplements
Substrate-specificity of GlnPQ and SBP conformations induced by non-cognate substrates.

(A) Time-dependent uptake [14C]-asparagine (5 μM), [14C]-glutamine (5 μM), [14C]-arginine (100 μM), [14C]-histidine (100 μM) and [3H]-lysine (100 μM) by GlnPQ in L. lactis GKW9000 complemented in …

https://doi.org/10.7554/eLife.44652.016
Figure 4—source data 1

Apparent FRET efficiency histograms of Figure 4D–H.

https://doi.org/10.7554/eLife.44652.020
Figure 4—figure supplement 1
Substrate binding of SBD1 and SBD2 studied by ensemble FRET.

The mean apparent FRET change of SBD1 (top) and SBD2 (bottom) in the presence of 5 mM of the indicated amino acids relative to their absence; measurements were performed in 50 mM KPi, 50 mM KCl, pH …

https://doi.org/10.7554/eLife.44652.017
Figure 4—figure supplement 2
Non-cognate substrate binding by SBD1 and SBD2.

Solution-based apparent FRET efficiency histograms of SBD1(T159C/G87C) (A and C) and SBD2(T369C/S451) (B) in the presence of different ligand concentrations as indicated. Bars are the data and the …

https://doi.org/10.7554/eLife.44652.018
Figure 4—figure supplement 3
PsaA(E74C/K237C) conformational changes probed by smFRET.

Solution-based apparent FRET efficiency histogram of PsaA(E74C/K237C) in the presence and absence of metals as indicated. Bars are the data and solid line a Gaussian fit. The 95% confidence interval …

https://doi.org/10.7554/eLife.44652.019
Opening transition in PsaA dictates transport specificity.

Solution-based apparent FRET efficiency histograms of PsaA(V76C/K237C) in the presence of Mn2+ (A) or Zn2+ (B) and PsaA(D280N) in the presence of Zn2+ (C) upon addition of 10 mM EDTA and incubated …

https://doi.org/10.7554/eLife.44652.021
Figure 6 with 3 supplements
Lifetime of MalE ligand-bound conformations and relation to activity.

(A) Mean lifetime of the ligand-bound conformations of MalE, obtained from all single-molecule fluorescence trajectories in the presence of different maltodextrins as indicated. Data corresponds to …

https://doi.org/10.7554/eLife.44652.022
Figure 6—source data 1

Lifetimes of the high FRET state of the data shown in Figure 6A and Figure 6—figure supplement 2.

https://doi.org/10.7554/eLife.44652.026
Figure 6—source data 2

Donor and acceptor photon counts, apparent FRET efficiency and most probable state-trajectory of the Hidden Markov Model of the traces in Figure 6B–G.

https://doi.org/10.7554/eLife.44652.027
Figure 6—source data 3

Lifetimes of the high FRET state of the data shown in Figure 6—figure supplement 3B.

https://doi.org/10.7554/eLife.44652.028
Figure 6—figure supplement 1
Surface-based smFRET histogram of MalE.

(A) Surface-based apparent FRET efficiency histogram of MalE(T36C/S352C) in the presence of different maltodextrin substrates as indicated. From the probable state-trajectory of the Hidden Markov …

https://doi.org/10.7554/eLife.44652.023
Figure 6—figure supplement 2
Lifetime distribution of the ligand-bound conformations of MalE.

Dwell time histogram of the high FRET (closed ligand-bound conformation) as obtained from the most probable state-trajectory of the Hidden Markov Model (HMM) of all molecules per condition as shown …

https://doi.org/10.7554/eLife.44652.024
Figure 6—figure supplement 3
Conformational changes and dynamics of MalE(A96W/I329W).

(A) Representative fluorescence trajectories of MalE(T36C/S352C/A96W/I329W) in the presence of 10 nM maltose. Fluorescence trajectories: the top panel shows the calculated apparent FRET efficiency …

https://doi.org/10.7554/eLife.44652.025
The conformational changes and dynamics of SBPs and the regulation of transport.

Schematic summarizing the plasticity of ligand binding and solute import via ABC importers. Intrinsic closing of an SBP is a rare event or absent in some SBPs (‘little intrinsic closing’). Ligands …

https://doi.org/10.7554/eLife.44652.029

Tables

Table 1
Dissociation constant KD of substrate-binding proteins.
https://doi.org/10.7554/eLife.44652.003
KD (µM)
Protein*LigandFreely-diffusing proteinSurface-tethered proteinKD WT protein (µM)
OpuAC(V360C/N423C)Glycine betaine3.4 ± 0.43.14–5 (Wolters et al., 2010)
OppA(A209C/S441C)RPPGFSFR7.0 ± 114 ± 5#5 ± 3#
SBD2(T369C/S451)Glutamine1.2 ± 0.2§0.50.9 ± 0.1 (Gouridis et al., 2015)
SBD1(T159C/G87C)Asparagine0.34 ± 0.03§0.30.2 ± 0.0 (Gouridis et al., 2015)
MalE(T36C/S352C)Maltose1.7 ± 0.32.21-2 (Hall et al., 1997a, Kim et al., 2013)
MalE(T36C/S352C)Maltotriose0.6 ± 0.20.90.2-2 (Hall et al., 1997a, Kim et al., 2013)
  1. *. KD could not be determined reliably for labeled PsaA due to background metal contamination.

    †. Population of the closed conformation P in the presence of a ligand concentration L was determined using solution-based smFRET. The KD=L (1-P)/P for a one-binding site model. Data corresponds to mean ± s.d. of duplicate experiments with the same protein sample.

  2. #. Figure 2—figure supplement 2

    ¶. The KD values of wildtype (WT) proteins are obtained from the indicated references.

Table 2
Steady-state anisotropy values.
https://doi.org/10.7554/eLife.44652.004
Anisotropy
Alexa555Alexa647Cy3BAtto647N
Free dye0.250.200.080.08
OpuAC(V360C/N423C)NANA0.170.11
OppA(A209C/S441C)0.250.19NANA
SBD1(G87C/T159C)0.270.19NANA
SBD2(T369C/S451)0.260.20NANA
MalE(T36C/S352C)0.290.24NANA
PsaA(V76C/K237C)0.280.22NANA
  1. NA: not applicable. Data correspond to mean (s.d. below < 0.01) of duplicate experiments, using the same labeled protein sample.

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene(Escherichia coli)MalENAUniProt:
P0AEX9
AntibodyMouse anti-hisQiagenRRID:AB_2714179(1:200)
Strain,
strain background (Streptococcus pneumoniae)
D39National Collection of Type CulturesNCTC:7466Capsular serotype 2
Strain, strain
background
(Streptococcus
pneumoniae)
D39 ∆psaAThis paperReplacement of psaA with the Janus cassette (∆psaA::Janus)
Strain, strain background (Streptococcus pneumoniae)D39 ∆czcDThis paperReplacement of czcD with the Janus cassette (∆czcD::Janus)
Strain, strain background (Streptococcus pneumoniae)D39 ΩpsaAD280NThis paperReplacement of ∆psaA::Janus with psaA D280N (∆psaA::psaAD280N)
Strain, strain background (Streptococcus pneumoniae)D39 ΩpsaAD280N∆czcDThis paperReplacement of ∆psaA::Janus with psaA D280N; replacement of czcD with the Janus cassette (∆psaA::psaAD280N∆czcD::Janus)
Strain, strain background
(Lactococcus lactis)
NZ9000NIZO food
research
Strain, strain
background
(Lactococcus lactis)
GKW9000DOI: 10.1038/
nsmb2929
Lactococcus lactis NZ9000 with glnPQ gene deleted
Strain, strain
background
(Escherichia coli)
K12OtherProvided by Tassos Economou, KU Leuven
Strain, strain
background
(Escherichia coli)
BL 21 DE3OtherProvided by Tassos Economou, KU Leuven
Recombinant
DNA reagent
pET20bMerckCat#:69739–3
Recombinant
DNA reagent
pNZglnPQhisDOI: 10.1047/
jbc.M500522200
Expression plasmid for GlnPQ
Recombinant
DNA reagent
SBD1-T159C/G87CDOI: 10.1038/
nsmb2929
Expression plasmid for SBD1(T159C/G87C)
Recombinant
DNA reagent
SBD2-T369C/S451CDOI: 10.1038/
nsmb2929
Expression plasmid for SBD2(T369C/S451C)
Recombinant
DNA reagent
pCAMcLIC01-PsaADOI: 10.1038/
nchembio.1382
Expression plasmid for PsaA
Recombinant
DNA reagent
pCAMcLIC01-PsaAD280NDOI: 10.1038/
nchembio.1382
Expression plasmid for PsaA(D280N)
Recombinant
DNA reagent
pNZOpuCHisDOI: 10.1093/
emboj/cdg581
Expression plasmid for OpuAC
Recombinant
DNA reagent
pNZcLIC-OppADOI: 10.1002/pro.97Expression plasmid for OppA
Recombinant
DNA reagent
PsaA-V76C/K237CThis paperExpression plasmid for PsaA(V76C/K237C) from the pCAMcLIC01-PsaA construct
Recombinant
DNA reagent
PsaA-E74C/K237CThis paperExpression plasmid for PsaA(E74C/K237C) from the pCAMcLIC01-PsaA construct
Recombinant
DNA reagent
PsaA-D280N/V76C/K237CThis paperExpression plasmid for PsaA(D280N/V76C/K237C) from the pCAMcLIC01-PsaAD280N construct
Recombinant
DNA reagent
MalE-T36C/S352CThis paperProgenitors: PCR, E. coli gDNA; pET20b vector
Recombinant
DNA reagent
MalE-T36C/N205CThis paperProgenitors: PCR, E. coli gDNA; pET20b vector
Recombinant
DNA reagent
MalE-K34C/
R354C
This paperProgenitors: PCR, E. coli gDNA; pET20b vector
Recombinant
DNA reagent
MalE-T36C/S352C/
A96W/I329W
This paperProgenitors: PCR,
E. coli gDNA;
pET20b vector
Recombinant
DNA reagent
OpuAC-V360C/
N423C
This paperExpression plasmid
for OpuAC(V360C/N423C)
from the pNZOpuCHis
construct
Recombinant
DNA reagent
OppA-A209C/
S441C
This paperExpression plasmid
for OppA(A209C/
S441C) from the
pNZcLIC-OppA
construct
Sequence-
based reagent
PrimersMercksee Supplementary File 2
Peptide,
recombinant
protein
RPPGFSPFRMerckCat#:B3259peptide sequence:
RPPGFSPFR
Peptide,
recombinant
protein
RDMPIQAFCASLO ApSpeptide sequence:
RDMPIQAF
Peptide,
recombinant
protein
SLSQSKVLPVPQCASLO ApSpeptide sequence:
SLSQSKVLPVPQ
Peptide,
recombinant
protein
SLSQSKVLPCASLO ApSpeptide sequence:
SLSQSKVLP
Chemical
compound,
drug
Glycine BetaineMerckCat#:B3501
Chemical
compound,
drug
CarnitineMerckCat#:94954
Chemical
compound,
drug
MaltoseMerckCat#:63418
Chemical
compound,
drug
MaltotrioseMerckCat#:851493
Chemical
compound,
drug
MaltotetraoseCarbosynth
Limited
Cat#:OM06979
Chemical
compound,
drug
MaltopentaoseMerckCat#:M8128
Chemical
compound,
drug
MaltohexaoseSanta Cruz
Biotechnology
Cat#:sc-218665
Chemical
compound,
drug
MaltoheptaoseCarbosynth
Limited
Cat#:OM06868
Chemical
compound,
drug
MaltodecaoseCarbosynth
Limited
Cat#:OM146832
Chemical
compound,
drug
MaltooctaoseCarbosynth
Limited
Cat#:OM06941
Chemical
compound,
drug
Beta
Cyclodextrin
MerckCat#:C4767
Chemical
compound,
drug
MaltotetroitolCarbosynth
Limited
Cat#:OM02796
Chemical
compound,
drug
MaltotriitolMerckCat#:M4295
Chemical
compound,
drug
3H-AsparagineAmerican
Radiolabeled
Chemicals
Cat#:ART 0500–250 µCi
Chemical
compound,
drug
14C-GlutaminePerkinEllmerCat#:NEC451050UC
Chemical
compound,
drug
14C-HistidinePerkinEllmerCat#:NEC277E050UC
Chemical
compound,
drug
14C-ArginineMoravekCat#:MC 137
Chemical
compound,
drug
3H-LysinePerkinEllmerCat#:NET376250UC
Chemical
compound,
drug
Alexa555Thermo Fisher
Scientific
Cat#:A20346
Chemical
compound,
drug
Alexa647Thermo Fisher
Scientific
Cat#:A20347
Chemical
compound,
drug
Cy3BGE HealthcareCat#:PA63131
Chemical
compound,
drug
ATTO647NATTO-TECHCat#:AD 647 N-45
Software,
algorithm
Dual-Channel-
Burst-Search
DOI: 10.1021/
jp063483n
Software,
algorithm
LabView data
acquisition
DOI: 10.1371/journal.
pone.0175766
Provided by
Shimon Weiss,
UCLA
Software,
algorithm
Hidden Markov
Model
DOI: 10.1109/
5.18626
Software,
algorithm
OriginOriginLabRRID:SCR_002815
Software,
algorithm
MATLABMathWorksRRID:SCR_001622

Additional files

Supplementary file 1

P-values of two-way Kolmogorov-Smirnov test on the solution-based smFRET data.

https://doi.org/10.7554/eLife.44652.030
Supplementary file 2

Primer sequences of all protein constructs used in this study.

https://doi.org/10.7554/eLife.44652.031
Supplementary file 3

Apparent FRET efficiency values of solution-based measurements.

https://doi.org/10.7554/eLife.44652.032
Supplementary file 4

Statistics of confocal scanning experiments of immobilized molecules.

https://doi.org/10.7554/eLife.44652.033
Transparent reporting form
https://doi.org/10.7554/eLife.44652.034

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