Structural and functional basis for lipid synergy on the activity of the antibacterial peptide ABC

24 Pages·2016·4.53 MB·English

JBC Papers in Press. Published on August 23, 2016 as Manuscript M116.732107
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M116.732107
The role of lipids on the function of ABC transporters
Structural and functional basis for lipid synergy on the activity of the antibacterial
peptide ABC transporter McjD

Shahid Mehmood 1, #, Valentina Corradi 2, #, Hassanul G. Choudhury 3, 4, 5, 6, #, Rohanah
Hussain 7, Patrick Becker 3, 4, 5, Danny Axford 7, Severine Zirah 8, Sylvie Rebuffat 8, D. Peter
Tieleman 2, Carol V. Robinson* 1, Konstantinos Beis* 3, 4, 5

Affiliations:
1 Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QZ,
United Kingdom
2 Centre for Molecular Simulation and Department of Biological Sciences, University of
Calgary, 2500 University Drive, N.W., Calgary, AB, T2N 1N4, Canada
3 Department of Life Sciences, Imperial College London, South Kensington, London SW7
2AZ, United Kingdom
4 Membrane Protein Lab, Diamond Light Source, Harwell Science and Innovation Campus,
Oxfordshire, OX11 0DE, United Kingdom
5 Rutherford Appleton Laboratory, Research Complex at Harwell, Oxfordshire OX11 0DE,
United Kingdom
6 Current address: Institute for Molecular Bioscience, The University of Queensland, St Do
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Lucia, QLD 4072, Australia n
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7 Diamond Light Source, Harwell Science and Innovation Campus, Oxfordshire OX11 0DE, ad
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United Kingdom fro
8 Communication Molecules and Adaptation of Microorganisms Laboratory, (MCAM, UMR m
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7245 CNRS-MNHN), Sorbonne Universités, Muséum National d'Histoire Naturelle, Centre ttp
National de la Recherche Scientifique, CP 54, 57 rue Cuvier 75005 Paris, France ://w
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Running title: The role of lipids on the function of ABC transporters .jbc
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Keywords: lipid, membrane transport, membrane protein, structure-function, structural y
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biology, ABC transporter, non-denaturing mass spectrometry, molecular dynamics, ue
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delipidation, circular dichroism t o
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# S.M., V.C. and H.G.C. have contributed equally to this work. ril 6
, 20
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* Correspondence to: [email protected] and [email protected] 9

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Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
The role of lipids on the function of ABC transporters
Abstract In recent years, there has been an increase in the
The lipid bilayer is a dynamic environment that numbers of membrane protein structures
consists of a mixture of lipids with different determined at medium to high resolution. Some
properties that regulate the function of of these structures show resolved lipids, which
membrane proteins; these lipids are either most probably are involved in specific
annular, masking the protein hydrophobic interactions since they have survived the
surface, or specific lipids, essential for protein purification and crystallization processes. In
function. In this study, using tandem mass many cases, no correlation between the structure
spectrometry, we have identified specific lipids and function of the specific lipids has been
associated with the Escherichia coli ABC made. For the sarcoplasmic reticulum Na,K-
transporter McjD, which translocates the ATPase (5), Ca-ATPase (SERCA) (6), and
antibacterial peptide MccJ25. Using non- bacterial secondary active betaine transporter
denaturing mass spectrometry, we show that BetP from Corynebacterium glutamicum (7), the
McjD in complex with MccJ25 survives the gas- influence of specific lipids on the activity of
phase. Partial delipidation of McjD resulted in these proteins has clearly been demonstrated.
reduced ATPase activity and thermostability as
shown by Circular Dichroism, both of which ABC transporters are one of the largest
could be restored upon addition of defined E. superfamilies found in eukaryotes, archaea and
coli lipids. We have resolved a prokaryotes. They are powered by the hydrolysis
phosphatidylglycerol lipid associated with McjD of ATP and can be classified as exporters and
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at 3.4 Å resolution, while molecular dynamic importers, the latter found only in bacteria. ABC w
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simulations carried out in different lipid exporters are associated with multidrug lo
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environments assessed the binding of specific resistance in both bacterial and eukaryotic cells. ed
lipids to McjD. Combined, our data show a To-date, there are a few structures of ABC fro
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synergistic effect of zwitterionic and negatively exporters, but none of these structures show h
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charged lipids on the activity of McjD; the well-resolved specific lipids bound to them. ://w
zwitterionic lipids provide structural stability to Moreover, despite the importance of lipids, no w
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McjD whereas the negatively charged lipids are detailed studies have linked their function to a .jb
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essential for its function. specific lipid class. For example, the ABC .o
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exporter TmrAB, from Thermus thermophilus, is b/
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Introduction associated with phosphatidylglycerol (PG) and g
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Biological membranes contain hundreds of lipid lipid A, as revealed by mass spectrometry (8). st o
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types, which differ in acyl chain length and However, electron density in the low-resolution A
p
degree of unsaturation, and head group polarity, electron cryo-microscopy single particle ril 6
size and charge (1). Such a complex lipid reconstruction envelope of TmrAB was assigned , 2
0
environment is essential to maintain the to the detergent micelle (9). In the low- 1
9
structural integrity and function of membrane resolution crystal structure of the eukaryotic P-
proteins, which often have defined binding sites glycoprotein no lipids were observed (10,11),
for lipids (2). Annular lipids are usually defined but non-denaturing mass-spectrometry identified
as the first lipid shell that surrounds the protein tightly associated lipids and a clear dependency
surface spanning the membrane (3). The of ligand and lipid binding (12).
interactions established with the protein,
although weak and usually not essential for In order to understand how specific lipids
protein function, allow for decreased exchange modulate the activity of ABC exporters, we have
rates with the bulk lipids, thus promoting the selected the antibacterial peptide ABC
formation of the lipid annulus that mask the transporter McjD from E. coli (13). McjD is
hydrophobic surface of the protein (4). Lower involved in the export of the antibacterial
exchange rates are found for specific lipids, peptide microcin J25 (MccJ25), conferring
tightly bound between transmembrane (TM) immunity to the producing strain towards the
helices or crevices (4). Specific lipids are peptide. MccJ25 is a plasmid-encoded,
associated with the activity of membrane ribosomally synthesized peptide consisting of 21
proteins and their loss during protein purification amino acids with a unique lasso structure
usually results in aggregation or reduced (14,15). It is produced under conditions of
transport and enzymatic activity. nutrient exhaustion and exerts potent
antibacterial activity against closely related
2
The role of lipids on the function of ABC transporters
species. Inside the cell, MccJ25 inhibits the The mass spectrum of McjD solubilized in n-
bacterial RNA polymerase (16,17). The uptake dodecyl-β-D-maltopyranoside (DDM) resulted
of MccJ25 into the target cell is facilitated by the in a very broad peak even under relatively harsh
outer membrane protein FhuA, dependent on the activation conditions (Figure S1). Mass spectral
TonB pathway (18,19), and the inner membrane peaks of this width are usually assigned to the
protein SbmA in E. coli (18,20). We have presence of endogenous lipids co-purified with
previously determined the high-resolution the protein in the DDM micelle. These lipids
structure of McjD (13) and observed electron resist delipidation with DDM implying that they
density that could be attributed to specific lipids. have a relatively high affinity for McjD and
However, the electron density was not strong therefore their interactions are preserved during
enough to model in structures of lipids. We have the purification process. To reduce this
also shown that detergent purified and heterogeneity of lipid binding we carried out a
proteoliposome reconstituted McjD hydrolyzes detergent exchange from DDM into Octyl
ATP with a rate of 35 and 75 nmol/min/mg Glucose Neopentyl Glycol (OGNG). In this
McjD, respectively (13), suggesting that lipids detergent it was possible not to strip the micelle
are important for the ATPase activity. but also to resolve charge states of McjD (Figure
1A). The deconvolution of these charge states
In light of these observations, we have combined showed a mass of 133 kDa, which is in
functional and structural studies to characterize a agreement with the theoretical mass of dimeric
specific class of lipids associated with the McjD. A second series of peaks are assigned to
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function of the ABC exporter McjD. Using non- adducts of mass ~650-800 Da, likely w
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denaturing and tandem mass spectrometry, we phospholipids (discussed later). In addition to lo
a
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have identified specific lipids, phospholipids, a second unexpected series of ed
phosphatidylethanolamine (PE) and PG, that are peaks with adduct masses of ~3.3 kDa was fro
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tightly bound to McjD and have survived the observed. We attribute these adducts to h
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purification and remained associated to the lipopolysaccharides (LPS), in agreement with a ://w
protein in the gas phase. Since the E. coli inner recently published study where it was shown w
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membrane consists mainly of PE, PG and that MscL, an inner-membrane protein, has .jb
c
cardiolipin (CARD), we performed basal and tendency to bind to outer membrane lipids or .o
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ligand-induced ATPase assays in detergent- lipopolysaccharides (21). Given that LPS are b/
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destabilized small unilamellar vesicles with larger than phospholipids or CARD, consisting g
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different lipid compositions, and we showed that of several hydrocarbon chains, their size implies st o
n
both PE and PG rescued the ligand-induced hydrophobic binding to the surface of the A
p
ATPase activity of a delipidated McjD protein. protein. In agreement with this observation, our ril 6
These data were further verified by measuring lasso-peptide binding experiment also shows , 2
0
the melting curves in the different lipids. Our that LPS does not obstruct the binding pocket 1
9
structural data at 3.4 Å resolution revealed one but likely binds to the surface of the protein
resolved PG lipid. MD simulations performed on (Figure 1B). Addition of ATP/Mg2+ did not
McjD embedded in different lipid environments show any displacement of the LPS (Figure S1),
provided molecular insights into the lipid and this is also consistent with LPS binding to
organization in the proximity of the protein. the protein during purification rather than as a
While zwitterionic (PE) and negatively charged substrate, as shown recently for the ABC
lipids (PG) exchange to interact with the transporter TmrAB, which displaces lipid A on
transporter, positively charged residues located addition of ATP/Mg2+ (8). The presence of LPS
on the protein surface provide preferable is therefore likely irrelevant to the function of
interactions sites for PG and CARD. the protein since McjD is known as an
antimicrobial peptide transporter.
To answer the question whether binding of lipids
Results to McjD has an effect on the function and
Mass spectrometry of McjD reveals co-purified stability of the protein, we investigated the
phospholipids and lipopolysaccharides: presence of different lipids in DDM purified and
Following expression and purification of McjD OGNG treated samples, and identified them
from E. coli, we used non-denaturing mass using tandem mass spectrometry (Figure S1 and
spectrometry to determine the oligomeric state Table 1). The tandem mass spectrometry for the
of the protein and presence of any bound lipids. DDM purified McjD revealed a total of 11 lipid
3
The role of lipids on the function of ABC transporters
species of which 2 were negatively charged and whereas PE was able to restore and also shows a
9 were zwitterionic (Table 1). Treatment of slightly elevated ATPase activity, 33.05 ± 1.33
McjD with OGNG resulted in the loss of several nmol/min/mg McjD, relative to the DDM-
zwiterrionic lipids (Table 1). We performed lipidated protein, 28.50 ± 0.39 nmol/min/mg
three replicate analyses of McjD preparations McjD (Figure 2A), which is in agreement with
used for X-ray crystallization and found the loss of PE lipids upon delipidation. Since PE
essentially similar results. Interestingly, we showed slightly elevated basal ATPase activity,
found several species of lipopolysaccharides, we investigated which of its lipid components
ranging from 3.3 to 3.4 kDa, consistent with the were responsible for the increased activity. PE is
denaturing mass spectra of McjD. However, due mostly composed of 1,2-dipalmitoyl-sn-glycero-
to low signal intensity, we were unable to 3-phosphoethanolamine (16:0) (33.5% of the
confirm the presence of LPS. Our lipid total PE composition) and 1,2-dioleoyl-sn-
identification results suggest that McjD has glycero-3-phosphoethanolamine (18:1 or DOPE)
selectivity towards zwitterionic and negatively (34% of the total PE composition) in lipid
charged lipids of varied chain lengths. preparations from E. coli. The mass
spectrometry analysis identified different
Influence of different lipids on the ATPase fragments of PE (Table 1), with variants of 1,2-
activity of McjD dipalmitoleoyl-sn-glycero-3-
The mass-spectrometry lipid identification and phosphoethanolamine (16:1) (9% of the total PE
analysis showed the presence of PE and PG composition) and 18:1. Since not all mixed acyl
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lipids (Table 1). Since these lipids have been variants are commercially available, we decided w
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retained, their roles could be to (i) stabilize the to assess only the effect of pure 16:1 PE and lo
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overall architecture of the protein or (ii) have an 18:1 PE lipids on the basal ATPase activity. In ed
effect on the activity of McjD. We investigated the presence of 16:1 PE or 18:1 PE, the basal fro
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the effect of the different lipid types identified ATPase activity was restored, but neither of h
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by mass-spectrometry on the ATPase activity of them showed an elevated ATPase activity as the ://w
McjD by partially delipidating it using OGNG PE mixture, thus suggesting a synergistic role of w
w
(see Materials and Methods section); McjD the different PE lipids present in the mixture in .jb
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treated using this protocol will be referred to as the McjD activity. We have previously shown .o
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delipidated McjD, and the DDM purified protein that Hoechst33342 (Hoechst) can stimulate the b/
y
as DDM-lipidated. Extended delipidation of ATPase activity of detergent purified McjD, and g
u
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McjD resulted in aggregated/precipitated protein it can also be transported by McjD in st o
n
and total loss of the ATPase activity. Partial proteoliposomes (13). In this study, Hoechst A
p
delipidation of McjD resulted in the loss of stimulated the DDM-lipidated and delipidated ril 6
different chain length PE lipids (Table 1) and McjD by 2.1 and 1.6 fold, respectively, relative , 2
0
retention of PG. The basal ATPase activity of to the basal ATPase activity of the delipidated 1
9
delipidated McjD was 20.77 ± 1.47 McjD (Figure 2A). When delipidated McjD was
nmol/min/mg McjD, which is around 30% less supplemented with lipids, the ATPase activity in
active relative to the DDM-lipidated protein, the presence of Hoechst almost doubled
28.50 ± 0.39 nmol/min/mg McjD (Figure 2A). (calculated relative to the basal ATPase activity
When the delipidated protein was supplemented of delipidated McjD). The ligand stimulated
with detergent-destabilized small unilamellar ATPase activity in the presence of pure PE 18:1
vesicles composed of E. coli polar lipid extract and pure PE 16:1 was not measured since PE did
(PL) (with a molar ratio of McjD to lipid of not show enhanced stimulation compared to the
1:870 mol/mol), the McjD basal ATPase activity other lipids. In the presence of Hoechst, the
was restored to similar levels as for the DDM- slightly higher (2.3 fold) ATPase activity in PL
lipidated protein. Since the E. coli PL extract is a suggests a synergistic role of PE and PG in the
mixture of PE, PG and CARD (6.7:3.2:0.1), the activity and probably stability of McjD. Overall,
ATPase activity of delipidated McjD was in the presence of all the lipids, the ligand-
measured in the presence of different detergent- induced ATPase activity was restored compared
destabilized small unilamellar vesicles to the delipidated protein.
composed of different lipids; we did not measure
the activity in the presence of CARD since it
was not detected in our analysis. PG was not
able to fully restore the basal ATPase activity,
4
The role of lipids on the function of ABC transporters
Influence of different lipids on the structure and structure components of McjD were likely
stability of McjD perturbed before the α-helix content was
affected.
Since the basal and ligand-stimulated ATPase
activity of the delipidated protein was restored in Influence of lipids on the local environment of
the presence of various lipids, we sought to tryptophans
establish if the different lipid environments We also measured the stability of the delipidated
stabilize the overall protein architecture by and lipid supplemented McjD using Differential
measuring the secondary structure and melting Scanning Fluorimetry (nanoDSF) by monitoring
temperature, T , of McjD. The secondary the intrinsic tryptophan fluorescence. McjD
m
structure of delipidated and lipid supplemented contains two tryptophans, Trp167 and Trp247,
McjD was monitored by Synchrotron Radiation which are located in the head-group region of
Circular Dichroism (SRCD). The normalised far the lipid bilayer. Changes in their local
UV-CD spectra of DDM-lipidated McjD showed environment upon denaturation would affect
a typical α-helical structure, with an α-helix their intrinsic fluorescence. The T of the DDM-
m
content of ca. 68% (Figure 2B and Table 2). lipidated McjD was measured to be 53.7 ± 0.3
This is in agreement with our previously °C (Figure 3A, Table 3). The delipidated McjD
published crystal structure in DDM (PDB ID: displayed a biphasic profile (Figure 3B, Table
4PL0), based on the Dictionary of Secondary 3), suggesting that the protein was rather
Structure of Proteins (DSSP) algorithm (22,23) unstable, partially unfolded, at the beginning of
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used to calculate its secondary structure. the experiment. Since the assay started at 20 °C, w
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Delipidation of McjD (in OGNG) resulted in a we could not establish the first unfolding T lo
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lsetrsasn fdo l(d2e6d% s)t ruacntdu rae wsigitnhi fai chaingth erer dcuocntitoennt ionf aβ-- e±v e0n.t5 b u°t Ca s(eFciognudraer y 3TBm) .w aSsu pmpelaesmuerendti nagt 71th.4e ed fro
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helical content (29%) compared to lipidated delipidated protein with PL showed an enhanced h
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McjD (in DDM) (68%) (Table 2). McjD Tm stabilization, 63.2 ± 0.1 °C (Figure 3C, Table ://w
supplemented with PL, PE and PG showed 3) relative to the lipidated protein as well as w
w
similar CD features to that in DDM, with a absence of the biphasic profile (Figure 3B). The .jb
c
comparable a-helix content of 64-68 % (Table two main components of the polar lipid extract .org
2), suggesting that the lipids are capable to PE and PG displayed Tm of 68.5± 0.3 °C and by/
revert the loss of the secondary structure. Slight 54.4 ± 0.2 °C, respectively (Figure 3D and 3E, gu
e
differences in a-helical content in PL may be Table 3). The PE shows a small degree of st o
n
due to the presence of a small ratio of CARD. biphasic profile which could correspond to some A
p
We also measured the thermal stability of McjD partially unstable protein. Finally, the Tm of ril 6
in the different lipid environments by SRCD as a McjD in the presence of 16:1 and 18:1, the two , 2
0
function of secondary structure loss upon components of PE, were 65.7 ± 0.3 °C and 63.2 19
heating. DDM-lipidated McjD has a T of 67.2 ± 0.3 °C (Figure 3F and 3G, Table 3). All the
m
°C and removal of lipids by OGNG destabilizes lipids appear to have rescued/reduced the
McjD dramatically, reducing its T to 48.6 °C biphasic profile that the delipidated protein
m
(Figure 2C and 2D, Table 3). When McjD was displayed, suggesting a possible role in
supplemented with PL, the thermal stability was stabilizing the tryptophans’ local environment.
recovered and its T increased to 65.2 °C (Table
m
3). This trend of stability was also observed in Structurally resolved bound lipids
the presence of PE, whose T (68.9 °C) is the Our current biochemical data show that the
m
closest to that of DDM-lipidated McjD and with ATPase activity of McjD is strongly correlated
PG, which has two transitions, the second one and dependent towards the presence of lipids.
being the higher T of 74.3 °C. PG has another We have previously determined the crystal
m
lower thermal transition of 13.1 °C (Table 3). structure of McjD and we observed some
This trend of thermal denaturation is mirrored in electron density that could correspond to lipids.
the α-helix contents of the McjD in the various The electron density was not continuous and it
lipid conditions. The α-helix component in did not provide us with confidence to build any
McjD in various lipids (PL, PG and PE) gave lipids. A structural study to identify bound lipids
higher T (>70 °C) compared to McjD in DDM on SERCA employed the measurement and
m
(67.2 °C), suggesting that other secondary inclusion of low-resolution diffraction spots in
the refinement and map calculations (6). We
5
The role of lipids on the function of ABC transporters
followed a similar approach and we used the density is likely not associated with lipid A,
same purified McjD batch for crystallization as while it probably corresponds to “well ordered”
for the mass-spectrometry in order to compare detergent-lipid mixture. If lipid A molecules
the identified lipids with the crystal structure. were specifically bound to McjD, we would
We determined the crystal structure of McjD in expect a tighter association with the TMDs as in
the presence of adenosine 5′-(β,γ- the structure of the outer membrane beta-barrel
imido)triphosphate (AMPPNP) and MgCl at 3.4 ferrichrome receptor FhuA (19).
2
Å resolution including low diffraction data to 60
Å resolution (Table 4). The overall structure is Molecular simulations in different lipid
identical to our previously published one; McjD environment
is in a nucleotide-bound outward occluded To investigate the structural organization of the
conformation. Inspection of electron density lipid bilayer around McjD, we performed CG
maps revealed the presence of elongated positive MD simulations of the transporter in four lipid
|Fo|-|Fc| electron density at 2.0 s near the elbow environments, i.e. pure palmitoyloleoyl PE
helix and between transmembrane helices (TMs) (POPE), pure palmitoyloleoyl PG (POPG), a
3 and 4 (Figure 4A and 4B) of one subunit; the mixture of 70% POPE and 30% POPG, and a
electron density was assigned as a PG molecule. mixture of 67% POPE, 23% POPG and 10%
We excluded the presence of DDM, as its alkyl CARD. This choice of lipids allowed us to study
chain is not long enough to occupy the electron the interactions between the protein and some of
density. Among all the lipids that were identified the hallmark components of the E. coli inner D
o
in the mass-spectrometry, only 1,2-di-(9E- membrane. w
n
octadecenoyl)-sn-glycero-3-phospho-(1'-sn- loa
d
glycerol) (PG (18:1/18:1) (9.7% of the total PG Membrane organization in the proximity of the ed
composition)) was long enough to fit in the protein. fro
m
electron density. In addition, the cleft of the We first estimated the number of lipids in the h
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resolved lipid electron density is positively proximity of the protein based on the number of ://w
charged (Figure 4C), thus further suggesting that phosphate head groups within a distance cut-off w
w
a negatively lipid like PG is most likely to of 0.7 nm (24). The number of lipids in contact .jb
c
occupy this site. However, we cannot exclude with the protein converges for all the simulation .o
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that the lipid might be PE, since a PE head group systems within the first ca. 5 µs (Figure S3). All b/
y
can also be refined within the electron density. the systems are characterized by a higher gu
e
In the refined structure, the PG lipid (18:1/18:1) number of lipids around the protein in the lower st o
n
is weakly associated with residues from TMs 3 leaflet of the membrane, correlating with the A
p
and 4 (Figure 4B); Arg186 is within hydrogen wider shape of transporter in the cytosolic side. ril 6
bond distance with the glycerol moiety of PG For the systems with 100% POPE and 100% , 2
0
(18:/18:1), which is further stabilized by weak POPG lipids (Figure S3), the number of lipids in 1
9
electrostatic interactions with Thr185. The 9E- the upper and lower leaflet differs by about 4
octadecenoyl acyl chain makes hydrophobic and 2 molecules, respectively. In the binary
contacts with Ile155, Ile158 and Phe174. We mixture (Figure S3), both the number of POPE
also observed some weak electron density and POPG lipids decreases when compared to
between the elbow helix and TM5 that may the pure bilayers. In the ternary mixture (Figure
correspond to either the phosphoethanolamine S3), the CARD molecules also establish contacts
head group of PE or to tightly bound maltoside with the protein, with a significant difference
head groups of DDM. As previously described, between lower and upper leaflets, thus altering
the non-denaturing mass spectrometry and lipid the profile of POPE and POPG lipids within the
analysis also identified an LPS molecule. Some same cut-off. To interpret these changes in the
positive |Fo|-|Fc| electron density at 2.5 s is number of lipids in contact with the protein, we
present between the elbow helix and TM 4 of the calculated the time-averaged lipid densities for
opposite monomer. The observed electron the phosphate head groups of each lipid type.
density is in close proximity to charged residues Figure 5 shows such densities calculated for
from TM 4 that could bind the sugar moieties of each system during the last 10 µs of the
the lipid A molecule. However, because the acyl simulations, together with the average number of
chains are not stabilized by hydrophobic lipids molecules found within the contact cut-off
contacts as they point away from the distance during the last 10 µs. The first and the
transmembrane domains (TMDs), this electron
6
The role of lipids on the function of ABC transporters
second panels of Figure 5A to 5D represent the showed higher contacts than POPE, and this
lipid densities (calculated on the phosphate head behaviour was observed also in the binary
groups only) in the upper and lower leaflet for system (Figure S5). However, for the ternary
each system, respectively. In both the 100% PE bilayer, the presence of CARD molecules
and 100% PG systems, in the proximity of the decreases the number of contacts of the POPG
protein, the lipid density shows the same molecules to a higher extent than the number of
organization, and in the binary and ternary contacts of the POPE molecules (Figure S5 and
mixtures, the density of different lipid types Figure 5, panel 4). This might suggest a
overlap. This is an indication that, although competition between POPG and CARD
POPE lipids are the most abundant species in molecules stronger than between POPE and
our simulation setup, lipid exchange occurs with CARD. Overall, the positively charged residues
regards to the interactions established by lipids in proximity of both the elbow helices are
of different type with the protein. In fact, associated with higher number of contacts in the
changes in the number of POPE and POPG simulation systems tested in this study. The
molecules are observed for the binary and combination of (i) positive charges to coordinate
ternary mixture when compared to the pure the binding of the lipid phosphate group, and (ii)
bilayers (Panel 4 of Figure 5, and Figure S3). grooves on the protein surface to accommodate
A focused view on the lipid-binding site the lipid tails might be correlated with the
identified via protein crystallization, in isolation and crystallization of McjD with PG
combination with the densities calculated on the lipids.
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o
full lipid molecules is shown (Figure 5, third w
n
panel). Time-averaged lipid densities for the Discussion lo
a
d
three lipid types (POPE, POPG and CARD) used In this study, we have evidence for the ed
in the simulations are found in the region synergistic role of specific lipids on the fro
m
corresponding to the crystallographic binding modulation of the structure and activity of the h
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site, and in particular in the region surrounding ABC exporter McjD. The tandem mass ://w
the elbow helix (residues 4-16) and the spectrometry of McjD has identified various w
w
hydrophobic groove formed by TM3, TM4 and zwitterionic PE lipids, negatively charged PG .jb
c
TM6, which accommodates one of the lipid tails lipids and LPS associated with McjD. These .o
rg
of the crystallographic lipid. The lipid density lipids are tightly associated with McjD, since b/
y
identified around the elbow helix can be linked they have survived the detergent extraction from g
u
e
to the presence of a cluster of positively charged the membrane, the purification process and the st o
n
residues, such as Lys4, Lys6, Arg186, and gas phase, and they likely represent specific A
p
Arg311 (Figure S4), which could facilitate the lipids. Using non-denaturing mass-spectrometry, ril 6
binding of negatively charged lipid head groups we have shown that McjD can bind the lasso , 2
0
(POPG and CARD) or the binding of antibacterial peptide MccJ25 without 1
9
zwitterionic lipids (POPE) via the phosphate interference from the lipids or LPS. From the
moiety of their head group. various lipids identified, we sought to explore
the role of each class of lipids on the structure
Protein-lipid interactions. For each residue of and function of McjD. Delipidation of McjD
the TMDs, we calculated, for each frame of the resulted in loss of PE lipids with retention of PG
trajectory, the total number of contacts (Table 1), suggesting a functional role of
established with the phosphate head group of the negatively charged lipids. Partial delipidation of
lipids, where a contact is defined when the McjD reduced its basal ATPase activity by 30%,
phosphate head group is within 0.7 nm of any which could be restored with the addition of
bead of a given protein residue (Figure S5). This different classes of lipids, such as PL extract and
analysis showed preferable interaction sites with PE (including PE 16:1 and PE 18:1), while
some of the TMD residues and highlighted how addition of PG did not restore the basal ATPase
positively charged residues strongly interact activity of McjD. These data suggest that
with the lipid head groups (Figure S5). The zwitterionic lipids are able to provide the correct
majority of such residues is located on the hydrophobic environment where the transporter
intracellular side of the membrane (Figure S4). can bind and hydrolyze ATP effectively.
Similar interaction patterns were found for both However, the basal ATPase activity only
POPE and POPG in all the simulation systems. measures the futile hydrolysis of ATP and it is
Overall, for the two pure bilayer systems, POPG only a measure of ATP turn over. We have
7
The role of lipids on the function of ABC transporters
previously demonstrated that the ATPase is reversible upon addition of detergent-
activity of McjD can be stimulated in the destabilized small unilamellar vesicles
presence of the antibacterial lasso peptide composed of PL, PE or PG lipids. The data are
MccJ25 and Hoechst (13). The ligand Hoechst also in very close agreement with the lipid
stimulated the ATPase activity of McjD in the exchange (PE-PG-CARD) observed in MD
presence of all the different lipids. Interestingly, simulations.
McjD supplemented with PG lipids appears to
have a higher relative ligand induced ATPase The structurally resolved lipid in our McjD
activity compared to the other lipids. In this structure was assigned as PG (18:1/18:1), and it
study, we did not observe significant stimulation probably acts as a specific lipid. We can
or inhibition of the basal ATPase activity by speculate that the PG stabilizes the transporter in
lipids as shown for the Na,K-ATPase (5,25). the nucleotide-bound outward occluded state in
From a molecular point of view, this could addition to the reported salt-bridges (13); the
suggest preferential interactions between McjD lipid is found between TMs 3 and 4 that are part
and negatively charged lipids. of the bundle of helices that undergoes
conformational changes during the transport
Our MD simulations indicate that a belt of cycle. Lipids are known to modulate the
positively charged residues distributed on the structure of membrane proteins and for their role
surface of McjD at the protein-lipid head group in stabilizing a given conformational state
interface favours interactions with the negatively (29,30). Structures of SERCA in different
D
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charged lipid molecules, POPG and CARD, thus conformations with resolved lipids have been w
n
reducing the number of contacts with the POPE reported (reference (31) lists all the SERCA lo
a
d
lipids (Figure 5, Figure S3-S5). However, states with different lipids), where lipids are ed
zwitterionic lipids, like POPE, with their small found in state-dependent and state-independent fro
m
and polar head group, also play structural and sites (four distinct sites, namely A-D). In the low h
ttp
functional roles in the bacterial inner membrane, calcium affinity state E2, PE lipids occupy sites ://w
as they are known to facilitate the insertion of A and B, where lipids in site A have been w
w
proteins in the lipid bilayer (26), to affect protein proposed to stabilize this state by keeping TMs 2 .jb
c
function, as shown for the E. coli lactose and 4 apart. Transition from E2 to the high .o
rg
permease LacY (27), and to be localized in the calcium affinity state E1 results in a structural b/
y
annular region of membrane proteins together conformation along TMs 2 and 4 and g
u
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with PG lipids (28). Indeed, for McjD, the lipid displacement of the PE lipid (31); in the E1 st o
n
organization, described in terms of lipid density state, PC lipids are found in sites C and D. A
p
(Figure 5), identifies both zwitterionic and ril 6
negatively charged lipids in the proximity of the All our data suggest a synergistic role of PE and , 2
0
protein, and highlights lipid exchange in the PG lipids that are reflected during the transport 1
9
binary and tertiary mixtures. cycle from an inward- to an outward-facing
conformation, measured as a function of
Our thermostability measurements in the substrate induced ATPase activity, similar to the
different lipids by nanoDSF and SRCD further stabilization of SERCA at the different states;
suggest a synergy between zwitterionic and specific lipids might interchange in specific
negatively charged lipids. The different melting surface grooves to facilitate this transition, as
temperatures measured by the two techniques shown by the elevated ATPase activity upon
suggest a more complex relationship between addition of PL lipids. Crystal structures of McjD
the stabilization of the overall structure of McjD in different conformations with resolved lipids
by lipids and the local environment of the two can provide further support for the role of lipids
tryptophans, Trp167 and Trp247, in the presence on its activity.
of detergent and/or lipids. Despite the
differences, both techniques are in close Materials and Methods
agreement about the stabilizing role of lipids on Protein purification and crystallization
the local or overall structure. The SRCD of the McjD was purified and crystallized as before
delipidated McjD confirmed the structural with no modifications (13). The same purified
stabilization role of PE lipids and subsequent protein preparations were used for mass
reduced ATPase activity due to loss of helical spectrometry, ATPase assays, thermostability
content (i.e. structural integrity and stability) that measurements and crystallization in order to
8
The role of lipids on the function of ABC transporters
have an identical sample to correlate all the data. Bio-Spin columns (Bio-Rad). For OGNG
Three different protein preparations were used detergent exchange samples were diluted 200
for the functional studies. The protein fold in 200 mM ammonium acetate
concentration was determined by UV280 and supplemented with 0.16% OGNG and incubated
BCA protein assay. In brief, 10 mg/ml purified for 30 min at 4 ˚C. Prolonged incubation
McjD (in 20 mM Tris pH 7.5, 150mM NaCl and initiated protein aggregation/precipitation. The
0.03% DDM) was incubated with 10 mM OGNG diluted samples were centrifuged at
AMPPNP and 2.5 mM MgCl at room 12000g for 15 min to remove any possible
2
temperature for 30 min prior to crystallization. aggregation/precipitation and then concentrated
Crystals were grown at 293 K using the vapour using ultrafiltration device (Amicon) with 100
diffusion method by mixing protein and kDa cutoff to 2.0 mg/ml final protein
reservoir solution at 1:1. Crystals were grown concentration.
from a precipitant solution containing 100 mM
sodium citrate pH 5.5, 45 mM NaCl, 28% PEG MccJ25 in complex with McjD
400 and 1% nonyl-glucopyranoside. The crystals MccJ25 lasso peptide was added to OGNG
were directly frozen into liquid nitrogen and solubilised McjD sample at room temperature
diffraction screening and data collection were incubated for 10 min. Peptide stock solution was
performed at Diamond Light Source prepared in DMSO at 20 mM concentration. The
synchrotron. stock solution was diluted to 100 µM
concentration in 200 mM ammonium acetate
D
o
MccJ25 purification buffer supplemented with 0.16% OGNG. The w
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MccJ25 was produced from cultures of E. coli final concentration of peptide and protein was 8 lo
a
d
K12 MC4100 harboring the plasmid pTUC202 µM in reaction mixture. ed
and purified as previously described (32). fro
m
Non-denaturing mass spectrometry h
ttp
Tandem mass spectrometry of McjD Non-denaturing mass spectra were acquired on a ://w
McjD protein was digested with trypsin (1:20 previously modified Q-TOF2 mass spectrometer w
w
units) for 15 hours at 37 °C in a thermomixer for the transmission of high-mass and also .jb
c
(Eppendorf). The digest was dried in a modified for high collision energy in collision .o
rg
SpeedVac until complete dryness and re- induced dissociation (CID) cell (33,34). Samples b/
y
dissolved in 35% acetonitrile aqueous solution. were introduced into the ion source via in-house g
u
e
The tryptic digest mixture was loaded onto a prepared gold-coated capillaries under static st o
n
pre-equilibrated C18 column (Acclaim PepMap nanospray. Q-ToF was operated under following A
p
100, C18, 75 µm × 15 cm; Thermo Scientific) instrumental parameters: capillary voltage 1.4– ril 6
and separated with a linear gradient of 35–100 % 1.7 kV, cone voltage 150 V, extractor 4 V, CID , 2
0
acetonitrile at a flow rate of 300 nl/min. The energy 220-350 V, source backing pressure 4.5– 1
9
nano-flow reversed-phase liquid 6 mbar.
chromatography (DionexUltiMate 3000 RSLC
nano System, Thermo Scientific) was directly ATPase assays in detergent-lipid mixtures
coupled to an LTQ-Orbitrap XL hybrid mass ATPase activities were measured in 0.1 % DDM
spectrometer (Thermo Scientific) via a dynamic solution using a coupled assay as before
nanospray source. Typical MS conditions were (Enzcheck, Molecular Probes) (13). The inactive
spray voltage of 1.6 kV and capillary E506Q mutant, which retains 2% ATPase
temperature of 160 °C. activity relative to the wild type McjD, was used
The LTQ-Orbitrap XL was set-up in negative to subtract background degradation of ATP (13).
ion mode and in data-dependent acquisition All ATPase activity data were measured at 25
mode to perform five MS/MS scans per MS ºC. The McjD concentration was 1.5 µM for all
scan. Survey full-scan MS spectra were acquired experiments. ATPase measurements were
in the Orbitrap (m/z 350 − 2,000) with a performed at 1mM ATP. For ligand induced
resolution of 60,000. ATPase activity assays, Hoechst33342 was
dissolved in water, and was added to a final
OGNG treated McjD concentration of 100 µM. All the lipids were
The McjD protein samples solubilized in DDM purchased from Avanti Polar Lipids. Powder
were buffer exchanged to 200 mM ammonium lipids were rehydrated in a buffer consisting of
acetate supplemented with 0.02% DDM using 20 mM Tris pH 7.5, 150 mM NaCl at a
9
The role of lipids on the function of ABC transporters
concentration of 1mg/ml. The opaque lipid The Tm was calculated by fitting the data to a
stocks were bath sonicated for 10-15 minutes modified Boltzmann sigmoidal equation (1) to
until they were clear. The small unilamellar take into account the sloping baselines.
vesicles were destabilized by the addition of
0.2% DDM. Delipidated McjD was added to a (1) Y=(Bottom+m2*X)+((Top+m1*X)-
final concentration of 0.1 mg/ml in each lipid (Bottom+m2*X))/(1+exp((V50-X)/Slope))
mixture to give a molar ratio of McjD to lipid of
1:870 (mol/mol). The protein/lipid/detergent where: Y is fluorescence ratio 350 nm / 330 nm,
mixtures were incubated at room temperature for X is temperature degrees Celsius, V50 is the Tm,
30 min and immediately used in the ATPase Top and bottom denote the fluorescence before
assays. All lipids and protein/lipid/detergent and after transition, Slope describes the
mixtures were freshly made. All measurements steepness of the curve, m1 and m2 are the
were performed in triplicate (mean ± SEM, n = gradient of the sloping top and bottom baselines
3). respectively.

SRCD measurements Data collection
SRCD experiments were performed using a Diffraction data to 3.4 Å resolution were
nitrogen-flushed Module B end-station collected on I24 at Diamond Light Source at a
spectrophotometer at B23 Synchotron Radiation wavelength of 0.968 Å using a Pilatus3 6M
CD Beamline at the Diamond Light Source detector and a modified beam stop in order to be D
o
(35,36). McjD samples at 0.1mg/ml were able to measure the low resolution data (6); the w
n
prepared in 20 mM Tris pH 7.5, 8 mM NaCl usual beam stop measuring 800 µm diameter loa
d
containing 0.03 % DDM or lipids PE, PG or PL. was replaced with a smaller one measuring 700 ed
Using sample volumes of 30 µl, scans were µm x 600 µm, in order to maximise the chance from
acquired using an integration time of 1 sec, of recording low angle (low resolution) http
pathlength of 0.02 cm and 1.2 nm bandwidth. reflections. The diffraction data were processed ://w
For thermal stability studies, McjD samples using xia2 (39). Further processing was ww
were incubated at 5 °C and spectra obtained over performed using the CCP4 suite (40). The .jb
c
a range of temperatures starting at 5 °C and resolution of the data and anisotropy analysis .org
increasing incrementally to 90 °C, with 2 min were evaluated by half-dataset correlation b/
y
equilibration time followed by a return to 20 °C coefficient in Aimless (41). The space group was gu
e
in one step of 15 min equilibration time (Figure determined to be P212121 with two copies of st on
S2). Results obtained were processed using McjD in the asymmetric unit. The data A
p
CDApps (37) and OriginLab with Tm calculated collection statistics are summarized in Table 4. ril 6
using Boltzmann equation. Secondary structure , 2
0
estimation from CD spectra was carried out Structure solution and refinement 19
using CDApps using Continll algorithm (38). The structure was determined by molecular
replacement in Phaser (42) using our previously
Melting temperature assays published McjD structure (PDB ID: 4PL0) (13)
The melting temperature, Tm, of McjD in as search model. All model building was
detergent and lipid/detergent mixtures was performed in Coot (43). Initial refinement was
measured by nano Differential Scanning carried out in REFMAC5 (44) and at later stages
Fluorimetry (nanoDSF) using a Prometheus in Buster (45). The structure was refined with
NT.48 instrument (NanoTemper Technologies restraints against the high resolution structure
GmbH). 10 µl of protein mixture at 0.1 mg/ml in 4PL0.
different lipids, prepared as above, was loaded After rigid body and restrained refinement extra
into nanoDSF Grade capillaries. Intrinsic electron density corresponding to two AMPPNP
tryptophan fluorescence at emission wavelengths molecules and two MgCl ions were identified.
2
of 330 and 350 nm was measured continuously Additional unaccounted positive |Fo|-|Fc| and
with a temperature gradient of 20-95 ºC 2|Fo|-|Fc| electron densities were observed at the
increasing at a rate of 1 ºC/min. All surface of the protein (Figure 4A) that
measurements were performed in triplicate correspond to lipid molecules. Strong |Fo|-|Fc|
(mean ± SEM, n = 3). The 350/330 nm ratio of electron density was observed between TMs 3
tryptophan fluorescence was calculated and data and 4, and the elbow helix that corresponds to
were analyzed and plotted in GraphPad PRISM.
10