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Allostery revealed within lipid binding events to
membrane proteins
John W. Patrick
a,1
, Christopher D. Boone
a,1
, Wen Liu
b
, Gloria M. Conover
a
, Yang Liu
b
, Xiao Cong
b,2
,
and Arthur Laganowsky
a,3
a
Department of Chemistry, Texas A&M University, College Station, TX 77842; and
b
Institute of Biosciences and Technology, Texas A&M Health Science
Center, Houston, TX 77030
Edited by Carol V. Robinson, University of Oxford, Oxford, United Kingdom, and approved February 9, 2018 (received for review November 13, 2017)
Membrane proteins interact with a myriad of lipid species in the
biological membrane, leading to a bewildering number of possible
protein−lipid assemblies. Despite this inherent complexity, the iden-
tification of specific protein−lipid interactions and the crucial role of
lipids in the folding, structure, and function of membrane proteins is
emerging from an increasing number of reports. Fundamental ques-
tions remain, however, regarding the ability of specific lipid binding
events to membrane proteins to alter remote binding sites for lipids
of a different type, a property referred to as allostery [Monod J,
Wyman J, Changeux JP (1965) J Mol Biol 12:88–118]. Here, we use
native mass spectrometry to determine the allosteric nature of het-
erogeneous lipid binding events to membrane proteins. We moni-
tored individual lipid binding events to the ammonia channel
(AmtB) from Escherichia coli, enabling determination of their equi-
librium binding constants. We found that different lipid pairs dis-
play a range of allosteric modulation. In particular, the binding of
phosphatidylethanolamine and cardiolipin-like molecules to AmtB
exhibited the largest degree of allosteric modulation, inspiring us
to determine the cocrystal structure of AmtB in this lipid environ-
ment. The 2.45-Å resolution structure reveals a cardiolipin-like mol-
ecule bound to each subunit of the trimeric complex. Mutation of a
single residue in AmtB abolishes the positive allosteric modulation
observed for binding phosphatidylethanolamine and cardiolipin-like
molecules. Our results demonstrate that specific lipid−protein inter-
actions can act as allosteric modulators for the binding of different
lipid types to integral membrane proteins.
native mass spectrometry
|
lipids
|
membrane proteins
|
lipid−protein interactions
|
allostery
Studies over the past four decades have shed light on the crucial
role of specific lipid−protein interactions in modulating
membrane protein structure and function (1–12). Early studies,
dating back to the 1980s, have demonstrated that the lipid envi-
ronment influences the function of the nicotinic acetylcholine
receptor from Torpedo wherein cholesterol plays a critical role in
promoting function (10–12). Phosphatidylinositol 4,5-bisphosphate,
a minor component of the cytoplasmic leaflet, is required for the
activation of all inward rectifying potassium channels (13–16), and
the sensitivity toward this lipid can be modulated by anionic lipids
(17). Additionally, we have recently shown that the activity of the
bacterial water channel aquaporin Z (AqpZ) can be modulated
threefold by cardiolipin, a lipid interaction that was found by native
mass spectrometry (MS) (7). Furthermore, lipids have recently been
shown to allosterically modulate protein activity, and protein−
ligand and protein−protein interactions (18–22). Despite the subset
of examples from the literature presented above, very little is ac-
tually known about how lipids influence, on the molecular level, the
structure and function of membrane proteins, which will advance
our understanding of how lipids participate in a multitude of
physiological processes.
Over the past two decades, native MS has emerged as a pow-
erful biophysical technique to study protein structure, dynamics,
and ligand interactions (23–30). Native MS affords the ability
to analyze intact protein complexes and preserve noncovalent
interactions in the mass spectrometer for analysis (28–32). Al-
though MS has been applied to soluble proteins for nearly three
decades (31), recent advances have led to the ability to preserve
noncovalent interactions and maintain intact, folded membrane
proteins in the gas phase, providing invaluable information
on subunit stoichiometry, nucleotide, drug, peptide, and lipid
binding (7, 33–37). Importantly, the detergent micelle protects
membrane proteins from solution into the mass spectrometer
(38, 39). In the case of membrane protein−lipid complexes,
minimal activation is applied to gently remove the detergent
micelle while preserving both native-like structure and non-
covalently bound lipids (7, 40–45). When using charge-reducing
detergents, such as C
8
E
4
, the minimal activation applied to strip
the weakly bound detergent micelle from the membrane protein
is not sufficient to dissociate lipids bound to membrane proteins,
whereas, at much higher activation, lipids can readily dissociate
from the membrane protein complex (45). Recently, binding
thermodynamics for protein−ligand interactions, including membrane
protein−lipid and protein−protein interactions, have been determined
using native MS coupled to a temperature-controlled source (18, 42).
Importantly, binding thermodynamics determined using other bio-
physical techniques, such as isothermal titration calorimetry and sur-
face plasmon resonance, are in agreement with those obtained using
native MS (18, 42, 46). Taken together, protein−ligand equilibrium
Significance
The diverse environment of cellular membranes presents
unique challenges in deciphering the roles that lipids play in
modulating membrane protein structure and function. Here,
we developed a native mass spectrometry approach to monitor
binding of different lipid types to membrane proteins. We
discovered that specific lipid−protein interactions can alloste-
rically modulate the binding of lipids of different types. We
also determined the structure of AmtB bound to cardiolipin,
and mutation of residues involved in binding this lipid abol-
ishes the observed allosteric effect. Our findings are of partic-
ular significance as they contribute to our general knowledge
of how lipids modulate protein structure and function and how
membrane proteins may recruit, through allostery, their own
lipid microenvironment.
Author contributi ons: J.W.P., C.D.B., X.C., and A.L. designed research; J.W.P., C.D.B., W.L., Y.L.,
and A.L. performed research; G.M.C. contributednew reagents/analytic tools; J.W.P., C.D.B., X.C.,
and A.L. analyzed data; and J.W.P. and A.L. wrote the paper with input from the other authors.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Published under the PNAS license.
Data deposition: The atomic coordinates and structure factors have been deposited in the
Protein Data Bank, www.wwpdb.org (PDB ID code 6B21).
1
J.W.P. and C.D.B. contributed equally to this work.
2
Present address: Wolfe Laboratories LLC, Woburn, MA 01801.
3
To whom correspondence should be addressed. Email: alaganowsky@chem.tamu.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1719813115/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1719813115 PNAS Latest Articles
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BIOCHEMISTRYBIOPHYSICS AND
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and thermodynamic parameters that are in agreement with other
biophysical techniques can be obtained by native MS.
One of the greatest modern challenges in membrane protein
structural biology is understanding how the chemically diverse
environment of the biological lipid membrane modulates mem-
brane protein structure and function. Overwhelmingly, there are,
at present, over 40,000 biologically relevant structures in the
LIPID Metabolites and Pathways Strategy structure database
(47). This striking number is over sevenfold more than the pre-
dicted human membrane proteome (48). As a step toward un-
derstanding how this diverse environment influences membrane
protein structure and function, we report a native MS approach
designed to determine the allosteric nature of heterogeneous
lipid binding events to integral membrane proteins. We selected
the Ammonia channel (AmtB) from Escherichia coli, as it has
been previously characterized by native MS, a biophysical tech-
nique that preserves noncovalent interactions in the mass spec-
trometer (28, 29, 31, 32). AmtB forms a trimeric complex of
∼127 kDa with each subunit containing 11 transmembrane helices
(49). Native MS studies have revealed that binding of individual
lipids of similar type both stabilizes the channel and exhibits unique
thermodynamic signatures (7, 40, 42, 45). In particular, from our
previous studies, we identified a specific binding site for phospha-
tidylglycerol (PG), a lipid found to stabilize the channel (7). Mutation
of residues involved in PG binding to AmtB resulted in decreased
stabilization and different thermodynamic signatures compared with
the wild-type channel, supporting the idea of a PG-specific binding
site (7, 42). We build upon these previous findings and methods to
study the molecular interaction of AmtB with a heterogeneous
mixture of lipids—here a mixture of two lipid types or lipid pairs.
Results
Our first objective for studying heterogeneous lipid binding
events to AmtB by native MS was to identify lipids such that
multiple binding events for each lipid and combinations thereof
could be resolved in the mass spectrum. Using the experimental
mass spectral resolution from our previous study as a guide (42),
we first considered lipids found in the biological membrane of E.
coli (50) and simulated mass spectra for a number of lipid pairs,
wherein a lipid pair consists of a mixture of two lipids that differ
in chemical composition that can bind to AmtB. From these
simulations, we observed significant peak overlap due to insufficient
mass resolution (SI Appendix,Fig.S1A). We then simulated mass
spectra for nearly 200 commercially available lipid pairs, yielding
greater than 19,000 possible combinations (SI Appendix,Fig.S1B).
From this dataset, we identified as a top candidate a fluorescent-
labeled cardiolipin molecule, TopFluor cardiolipin (TFCDL,
1,1′,2,2′-tetraoleoyl cardiolipin[4-(dipyrrometheneboron difluoride)bu-
tanoyl]) (SI Appendix,Fig.S1C), which had the least peak overlap
with lipids found in the biological membrane. The modified head-
group of TFCDL provides the necessary mass shift such that peaks
can be resolved for this lipid in combination with six other lipids:
phosphatidic acid (PA), phosphatidylethanolamine (PE), PG,
phosphatidylserine (PS), and phosphatidylcholine (PC) containing
1-palmitoyl-2-oleoyl (PO, 16:0–18:1) tails, and cardiolipin (TOCDL,
1,1′,2,2′-tetraoleoyl-cardiolipin). With the exception of TFCDL and
POPC, the remaining five lipids are present in the biological
membrane of E. coli (50). In addition to their physiological rele-
vance, the selected lipids for our study have identical acyl chains,
with the exception of TFCDL and TOCDL, which contain four
oleoyl (18:1) tails (SI Appendix,Fig.S2). TFCDL and TOCDL
differ only in their headgroups, where TFCDL contains a BODIPY
moiety, a fluorophore, covalently attached to the 3′hydroxyl in the
TOCDL phosphoglycerol headgroup (SI Appendix,Fig.S2).
As a step toward understanding integral membrane protein−lipid
interactions in the chemically diverse environment of the lipid bi-
layer, we performed systematic titrations of mixtures of TFCDL
with one of the six “light”lipids (POPA, POPC, POPE, POPG,
POPS, or TOCDL), which are lighter in mass compared with
TFCDL, with detergent solubilized AmtB followed by recording
their native mass spectra at a fixed temperature (42) (Fig. 1Aand
SI Appendix, Fig. S3). The mole fraction for each combination of
lipids bound was determined after deconvoluting the mass
spectra from the titration series (Fig. 1B). Notably, we did not
observe lipid binding patterns consistent with dimers or multi-
mers of lipids binding to AmtB. The free concentration of the
two different lipids was back-calculated, and an equilibrium
coupled binding model (SI Appendix, Fig. S4) was fit to the ex-
perimental mole fraction data, equating to roughly 750 data
points per replicate (SI Appendix, Figs. S5 and S6 and Movie S1–
S6). This allowed for the determination of the equilibrium dis-
sociation constants (K
d
) for AmtB binding up to a total of five
lipids of either TFCDL, light lipid, or combinations thereof (Fig.
1Cand Dand SI Appendix, Fig. S7). We first examined the K
d
values for sequential binding of the light lipid and found the
values to be similar to those from our previous study (42), where
only one lipid type was titrated. The sequential binding of
TFCDL displayed negative cooperativity for the binding of up to
four molecules, followed by positive cooperativity for the fifth
binding event. In contrast, binding of AmtB to both TFCDL and
a light lipid molecule revealed defined and unique states with
higher equilibrium affinity constants for each lipid pair. Mixed
Fig. 1. Native MS enables the determination of
equilibrium binding constants for heterogeneous
lipid binding events to AmtB. (A) Native mass
spectrum of AmtB at a concentration of 2 μMin
the presence of 11 μMTFCDLand11μMTOCDL.
(B) Deconvolution of the mass spectrum shown in
Ausing UniDec (58). The apo and lipid bound states
of TFCDL and TOCDL to AmtB are labeled. (Cand D)
Equilibrium binding constants for AmtB binding
TFCDL and TOCDL, POPA, POPC, POPG, POPS, or
POPE. The legend shown in Cillustrates the number
of TFCDL and TOCDL lipids bound to AmtB per box
and the respective K
d
value plotted. Hatched lines
represent species that were not observed, leading to
erroneous values in the fitting routine. Reported are
the averages from repeated measurements (n=3).
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species containing both TFCDL and POPA generally possessed a
higher K
d
than those for sequential binding of the lipid compo-
nents alone. Moreover, POPC, POPG, and TOCDL demon-
strated a preference for binding multiple light lipids and one
TFCDL. In the case of POPE, the lowest K
d
observed occurred
for a species containing four TFCDL and one POPE molecule.
In contrast, the lowest K
d
observed for POPS occurred when four
POPS and one TFCDL were bound to AmtB.
To gain further insight into the trends observed above, we
calculated the coupling factor (α), which informs the magnitude
of positive, neutral, or negative modulation of ligand affinity
(51), for each quadrant in our equilibrium coupled model (Fig.
2Aand SI Appendix, Fig. S7B). Given the sensitivity of our native
MS approach, we determined coupling factors that spanned a
range of values, including positive, neutral, and negative allo-
steric modulation (Fig. 2A). The allosteric modulation for the
TOCDL and TFCDL pair displayed largely neutral allosteric
modulation, which is anticipated given the cardiolipin-like nature
of TFCDL and, in essence, behaves as a homogeneous lipid pair.
POPG, which is essentially half of a CDL molecule, demon-
strated a pattern similar to TOCDL where no significant allo-
steric modulation was observed. POPA contained the largest
number of αvalues less than 1, indicating negative allostery or a
decrease in ligand affinity. For POPA, there is a twofold re-
duction in K
d
when binding a mixture of TFCDL and POPA
compared with binding of four POPA molecules. The largest
positive allosteric modulation we observed was for POPE and
TFCDL, which steadily increased along the direction of binding
one POPE and multiple TFCDL molecules, peaking at a total of
five bound lipids in this study (Fig. 1).
Coinciding with the calculation of coupling factors, we inspected
individual mass spectra recorded for the titration of different lipid
pairs. AmtB in the presence of TFCDL at a 4:1 molar ratio
(TFCDL:AmtB) binds up to four TFCDL molecules (Fig. 2B). The
addition of POPC at a molar ratio of 3:1.5:1 (TFCDL:POPC:
AmtB) did not enhance TFCDL binding (Fig. 2B). In contrast,
AmtB in the presence of 3:1 TFCDL:POPE resulted in a dramatic
enhancement of TFCDL binding, with up to four TFCDL mole-
cules bound to AmtB (Fig. 2 Band C). In summary, native MS
captures snapshots of solution equilibria and clearly demonstrates
the potent allosteric effect observed for the POPE and TFCDL pair
binding to AmtB.
We next performed a principal component analysis (PCA)
on the average coupling factors for different lipid pairs
binding to AmtB, to identify patterns (Fig. 2D) (52). The lipid
pair that exhibited the largest allosteric modulation, TFCDL and
POPE, was separated the farthest from the five other light lipids
by the first principal component (PC1). In contrast, the
remaining lipids clustered around −0.5 for PC1 but were sepa-
rated by the second principal component (PC2) into three
groups: POPA; POPC and POPG; and TOCDL and POPS.
POPA displayed the greatest negative allosteric modulation,
providing an explanation for its separation from the other groups
on the PC2 axis.
To test the specificity of TFCDL and POPE binding to AmtB,
we acquired native mass spectra of AqpZ, a tetrameric integral
membrane protein from E. coli, bound to TFCDL and mixtures
of lipids (SI Appendix, Fig. S8). AqpZ in the presence of TFCDL
at a 3:1 molar ratio (TFCDL:AqpZ) bound up to two TFCDL
molecules. The addition of POPE at a molar ratio 3:3:1 (POPE:
TFCDL:AqpZ) did not enhance TFCDL binding as observed for
AmtB. The mass spectrum for AqpZ in the presence of 3:3
TFCDL:POPC was similar to the mixture for TFCDL and
POPE. These results provide additional support for the positive
allosteric modulation observed for AmtB binding TFCDL
and POPE.
To gain structural insight into the potent allosteric effect ob-
served for TFCDL and POPE, we set out to determine the
crystal structure of AmtB in this lipid environment. Although we
screened different ratios of AmtB:TFCDL:POPE, the best dif-
fracting crystals grew in a 1:3:1.5 molar ratio. The crystals were
light brown, in accord with an enrichment of TFCDL and
quenching of the fluorophore (SI Appendix,Fig.S9A). The 2.45-Å
resolution structure of AmtB is composed of one subunit in the
asymmetric unit cell (Fig. 3). One TFCDL lipid was resolved in
the electron density, which had strong positive density (σ>10) in
the difference density map (F
o
−F
c
) after molecular replacement
(SI Appendix,Fig.S9F). TFCDL is located on the periplasmic
facing side of AmtB, and, through crystallographic symmetry, the
lipid is bound to each subunit within the trimeric assembly (SI
Appendix,Fig.S9B), which is distinct from the site determined for
PG (7). We did not observe electron density for POPE, likely
owing to the small molar ratio present in solution. The overall
structure of AmtB bound to TFCDL displayed an all-atom RMSD
of 0.5 ±0.1 Å compared with the other AmtB structures deposited
in the Protein Data Bank (PDB). Despite this similarity, structural
rearrangements were observed for a number of residues that form
distinct contacts with TFCDL. Hydrogen bonds were formed to
both the sidechain and backbone amide of N73 from the phos-
phate moiety on the TFCDL phosphoglycerol bridge (Fig. 3B). An
Fig. 2. Different lipid pairs binding to AmtB exhibit
distinct coupling factors. (A) Coupling factors (α)for
AmtB binding xlight lipid and yTFCDL species (α
x,y
).
Reported are the mean and SEM from repeated
measurements (n=3). (B) The 15
+
charge state re-
gion is shown for AmtB titrated with TFCDL (Top),
TFCDL and POPC (Middle), and TFCDL and POPE
(Bottom). The mole ratio of TFCDL to AmtB is 3:1,
and POPC and POPE are at a mole ratio of 1.5 and 1,
respectively, to AmtB. The number of TFCDL and
POPC or POPE bound is shown as in Fig. 1B.(C) Mole
fraction for TFCDL bound to AmtB determined from
deconvolution of mass spectra shown in B.(D) PCA
of the coupling factors for different lipid pairs
binding to AmtB.
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additional hydrogen bond is formed to H156 by the sn2ester
linkage of TFCDL where the indole ring is precisely positioned
through interaction with the sidechain of E357 on a neighboring
helix. This H156·E357 interaction is unique among the other
21 structures for AmtB currently in the PDB. In comparison with
the structure of AmtB bound to PG (PDB 4NH2), E357 rotates
∼150° and shifts ∼3.6 Å toward H156 (SI Appendix, Fig. S9E).
Three of the four 18:1 tails of TFCDL were resolved and are
arranged within the transmembrane spanning surface of AmtB,
forming an interface area of 655 Å
2
for the AmtB·TFCDL mo-
lecular interaction (Fig. 3C). The boron-dipyrromethane (BODIPY)
moiety attached to the headgroup of TFCDL is positioned away
from the protein such that the neutral moiety is packed against
the lipid tails, where it does not form direct contacts with AmtB
(SI Appendix, Fig. S9D). The covalent modification of TFCDL
removes the ability for an additional hydrogen bond donation
that would occur in CDL; however, the position of this ester
group is not in proximity to form any potential hydrogen bonds
with AmtB.
In an effort to gain insight into the allosteric mechanism for
TFCDL and POPE, we analyzed AmtB containing the His156 to
Ala mutation (AmtB
H156A
), a residue that interacts with TFCDL
and forms a distinct interaction with E357. Equilibrium binding
constants for AmtB
H156A
interacting with TFCDL, POPC, or
POPE were determined in a similar fashion to the lipid pairs
investigated for the wild-type channel. Interestingly, the K
d
for
TFCDL in the presence of POPC or POPE significantly in-
creased, nearly tripling in some cases (SI Appendix, Table S2).
Equilibrium binding affinities for POPC and POPE bound to
AmtB
H156A
were not affected by the H156A mutation, as these
values were similar to the wild-type protein. Moreover, the po-
tent allosteric modulation observed for the wild-type protein
binding TFCDL and POPE was completely abolished for
AmtB
H156A
(Fig. 4).
Discussion
Allostery is a biological phenomenon that underlies the regula-
tion of macromolecular structure and function and has been
identified in numerous biological processes, such as cellular
signaling, transcriptional control, and disease (53–56). Here, we
have shown that this biological phenomenon extends to lipid−
protein interactions using AmtB as a model membrane protein
system. We observed differences for AmtB binding to lipid pairs
that differ only in their headgroup composition (Fig. 1). The six
light lipids, which differ from each other in molecular weight by
only a few daltons (SI Appendix, Fig. S2), display markedly dif-
ferent equilibrium binding constants that can be captured using
native MS. In addition, PCA of the coupling factors (Fig. 2D)
reveals four distinct patterns for the six different lipid pairs,
providing additional support for the specificity of lipid binding
events to AmtB. The allostery observed for TFCDL and POPE
appears to be specific, as binding enhancement for this lipid pair
to AqpZ, a different integral membrane protein, was not ob-
served. These findings elucidate the preferential binding of lipids
to AmtB and, more importantly, highlight the role of allostery as
the underlying mechanism of action that modulates the affinity
of lipid binding.
The largest positive allosteric modulation observed was for
POPE and TFCDL. This observation is most intriguing, as PE
accounts for around 75% of the total lipid composition in E.
coli (57). POPC, which differs from POPE by three methyl
groups, shared some similarity to POPE, albeit with a de-
creased magnitude of allosteric modulation (Fig. 1). In com-
parison with POPE, the additional methyl groups on the
choline headgroup of POPC could impose steric restrictions
that weaken or abolish the interaction of the polar headgroup
with AmtB. These observed allosteric results demonstrate the
highly specific nature of lipid−protein interactions and illus-
trate the exquisite sensitivity of native MS for uncovering spe-
cific protein−lipid interactions.
Fig. 3. Crystal structure of AmtB Bound to TFCDL. (A) Top view of the
periplasmic face of AmtB (Left) and parallel view (Right) to the trans-
membrane portion of the trimeric AmtB·TFCDL
3
assembly. Structure is
shown in surface representation, with secondary structure shown in the top
view. TFCDL is shown as a surface representation, with the headgroup
(phosphoglycerol bridge) colored light red and the acyl chains in white.
(B) Molecular interactions formed between TFCDL and AmtB. Hydrogen
bonds are shown as yellow dashed lines. (C) A large interacting surface (dark
blue) is generated by TFCDL bound to AmtB. TFCDL is shown as sticks and
colored as in A. The BODIPY moiety of TFCDL has been omitted for clarity
but is shown intact in SI Appendix, Fig. S9.
Fig. 4. Mutation of His156 to Ala in AmtB (AmtB
H156A
) diminishes TFCDL
binding and abolishes the allosteric effect for binding TFCDL and POPE.
(A) Mass spectra for AmtB
H156A
with TFCDL, TFCDL and POPC, and TFCDL
and POPE as labeled. (B) Zoomed regions for the AmtB
H156A
15
+
charge
state illustrating reduced POPE and TFCDL binding compared with WT
AmtB. The mole ratio of TFCDL to AmtB
H156A
is 3:1. POPC and POPE were
analyzed at a mole ratio of 1.5 and 1 to AmtB, respectively. Shown as de-
scribed in Fig. 2B.
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Complementary to our native MS results, the crystal struc-
ture of AmtB bound to TFCDL provides molecular details on
specific lipid−protein interactions and insight into the mecha-
nism of allostery (Fig. 3). Interestingly, the BODIPY moiety
attached to the headgroup of TFCDL does not form direct
contacts with AmtB. Instead, the fluorophore is positioned
away from the protein such that the neutral moiety is packed
against the lipid tails (SI Appendix,Fig.S9B). This result im-
plies that TFCDL is an effective mimic of CDL, and the ob-
served allosteric effect reported here for TFCDL and POPE is
anticipated to hold if TFCDL is substituted with other CDL
molecules. This is further supported by AmtB
H156A
,which
harbors a point mutant engineered to disrupt CDL binding,
that exhibits reduced binding affinity to TFCDL and abolishes
the allosteric effect for the TFCDL·POPE pair (Fig. 4). Given
the impact of the H156A mutation, we speculate that H156
fluctuates between two states observed crystallographically: (i)
interacting with E357, as in our crystal structure, or (ii) not
interacting with E357, as found in other AmtB structures. Lipid
binding could allosterically modulate the H156·E357 interaction,
which could order the specific CDL binding site and enhance the
apparent binding affinity, providing a plausible mechanism for the
allostery observed.
In closing, we demonstrate that lipid−protein interactions
can, indeed, allosterically modulate remote binding sites for
lipids of different type. In our study, all lipid pairs contained a
TFCDL, and we foresee that higher mass resolution instru-
ments (35) will enable the opportunity to study natural lipid
pairs, such as POPE with other PO-type lipids, as well as more
complex lipid mixtures. It is likely that interaction with addi-
tional lipids, beyond two different lipids and/or five total lipids
bound, will reveal additional insight into protein−lipid inter-
actions, such as amplification of the observed allosteric effects
and the adoption of biologically useful conformations. We
anticipate allosteric coupling of lipid−protein interactions to
be a general phenomenon observed in other membrane pro-
teins. Lastly, our data provide compelling evidence that allo-
steric protein−lipid interactions could be utilized in the
biological membrane to effectively recruit a defined lipid
microenvironment.
Methods
Protein Expression and Purification. The Ammonia channel (AmtB) from E. coli
was expressed (7, 42) and purified (18) as previously described. Detailed
methods are provided in SI Appendix, Supporting Methods.
Preparation and Titration of Phospholipids. Phospholipid samples were pre-
pared as previously described (42). Lipid stocks were diluted such that both
light and heavy lipids were mixed with AmtB to yield samples for native MS
analyses (SI Appendix, Supporting Methods).
Native MS Analysis. Native MS was performed on a Synapt G1 HDMS in-
strument (Waters Corporation) equipped with a 32k RF generator. Additional
details are provided in SI Appendix, Supporting Methods.
Automated Data Processing. To handle the large volume of data as well as
interpretation of complex mass spectra, we developed a pipeline for auto-
mated data processing. In brief, MS data files were deconvoluted in batch
using UniDec software (58) with the following settings: no smoothing, m/z
range 6,000 to 12,500, charge range 10 to 25, mass sampling of 10 Da, and
peak FWHM of 10. A custom Python script was written to import the zero-
charge mass spectrum outputted from UniDec and extract the intensities for
apo and various lipids bound species followed by conversion to mole frac-
tion. For each titration experiment, the free light lipid can be back calcu-
lated as follows:
½Lfree =½Ltotal −½Ptotal X
m
j=0X
n
i=1
iFPLiHj,[1]
and the free concentration of the heavy lipid can be calculated as
½Hfree =½Htotal −½Ptotal X
m
j=1X
n
i=0
iFPLiHj ,[2]
where Frepresents the mole fraction of a particular species. The total pro-
tein ([P]
total
) was determined with the DC Protein Assay kit (Bio-Rad) using
BSA as the standard. The extracted mole fraction data and free con-
centration of lipids were saved and used for determination of equilib-
rium binding constants.
Determination of Lipid Equilibrium Binding Constants. Analysis of MS data
obtained for AmtB and AmtB bound to different lipid species was performed
using UniDec software packages (58) and in-house scripts written in Python
programming language. The intensities of AmtB (P) and AmtB bound to light
(L) or heavy (H) lipid species were converted to mole fraction for a given lipid
titration. Apparent equilibrium binding constants (K
A
) for protein−lipid spe-
cies were obtained using the following equilibrium coupled binding model
(SI Appendix,Fig.S4). For protein binding one light or heavy lipid,
P+L
⇔
KA;L1
PL KA,L1=
½PL
½P½L,[3]
or binding to multiple ligands,
PLn−1+L
⇔
KA;Ln
PLn KA,Ln=
½PLn
½PLn−1½L,[4]
where nis the number of bound light lipids, and K
A,Ln
represents the
equilibrium association binding constant for the nth lipid binding to AmtB.
A similar derivation is made for binding mnumber of heavy lipids. For
heterogeneous lipid binding events, the binding to light lipid is first cal-
culated using Eqs. 1and 2followed by binding to mnumber of heavy
lipids,
PLnHm−1+H
⇔
KA;LnHm
PLnHm KA,LnHm=
½PLnHm
½PLnHm−1½H.[5]
The total protein ([P]
total
) in the system can be expressed as follows:
½Ptotal =X
n
i=0X
m
j=0
½PLnHm.[6]
After substitution of Eqs. 1–3into Eq. 4and rearrangement, the mole
fraction of P bound to nlight lipids and/or mheavy lipids can be
calculated,
βPLnHm =½Hm½Ln∏
n
j=0
KA,Li ∏
m
j=0
KA,LnHj,[7]
FPLnHm =βPLnHm
Pn
j=0Pm
i=0βPLiHj
.[8]
The equilibrium association constants for each species PL
n
H
m
were de-
termined by global fitting of the equilibrium coupled binding model to the
experimental mole fraction (F
PLnHm
) data through minimization of the
pseudoχ
2
function,
χ2= X
n
i=0X
m
j=1X
d
k=1FPLiHj,expk−FPLiHj,calck2,[9]
where nand mare the number of light and heavy bound lipids, and dis the
total number of data points.
The coupling factors (α
x,y
) for an xnumber of light and ynumber of heavy
lipids bound were calculated using the calculated equilibrium association
constants (K
A
),
∝x,y=
KHyLx
KHy−1Lx
.[10]
AmtB Crystallization and Structure Determination. AmtB in [50 mM Tris,
pH 7.4 at room temperature, 130 mM sodium chloride, 10% (wt/vol) glycerol
and 0.5% (wt/vol) C
8
E
4
] was concentrated to 12 mg/mL. Initial cocrystallization
trials were carried out using Mosquito LCP (TTP Labtech) crystallization robot
in hanging drop plates at 20 °C for 1 mg/mL to 12 mg/mL (8 μMto95μM) of
AmtB combined with POPE and TFCDL in molar excess of 1:1.5:3, 1:3:3, and
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BIOCHEMISTRYBIOPHYSICS AND
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1:3:1.5, respectively. The best diffracting crystals grew within 2 d to 3 d at
10 mg/mL (79 μM) AmtB mixed with 1:1.5:3 molar excess of POPE and TFCDL
in crystallization solution 0.1 M Tris, pH 8.0, 0.3 M magnesium nitrate,
22% (wt/vol) PEG 8000 at a 1:1 sample to mother liquor ratio. No
cryoprotection was needed, as determined by screening on an in-house source.
Single crystals were mounted with CrystalCap HT Cryoloops (Hampton Re-
search) before being flash frozen. Diffraction data were collected at the Ad-
vanced Photon Source (Argonne National Laboratory) on beamline 24-ID-E.
The data were integrated, merged, and scaled using X-ray detector software
(59) in space group H32 to a resolution of 2.45 Å. Initial phases were found
using molecular replacement using the high-resolution, apo form of AmtB
(PDB 1U7G) (49). Model refinement and building was performed using Phenix
(60) and Crystallographic Object-Oriented Toolkit (61) programs, respectively.
All structural figures were rendered in PyMOL (62). The atomic coordinates
have been deposited into the PDB with the accession number 6B21.
ACKNOWLEDGMENTS. We thank Dr. David Russell (Texas A&M University) for
useful discussion. This work was supported by new faculty startup funds from the
Institute of Biosciences & Technology, Texas A&M Health Science Center, and the
Department of Chemistry, Texas A&M University, and support from National
Institute of General Medical Sciences (NIGMS) of the National Institutes of Health
(NIH) (DP2GM123486). Part of this work was conducted at the Northeastern
Collaborative Access Team beamlines, which are funded by the NIGMS from
NIH (P41 GM103403), and NIH Office of Research Infrastructure Programs
High-End Instrumentation Grant (S10OD021527). This research used resources
of the Advanced Photon Source under Contract DE-AC02-06CH11357.
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