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STRUCTURAL BIOLOGY
Cryo-EM structures capturethe transport cycle of theP4-ATPase
flippaseMasahiro Hiraizumi1,2, Keitaro Yamashita1,3, Tomohiro
Nishizawa1*, Osamu Nureki1*
In eukaryotic membranes, type IV P-type adenosine
triphosphatases (P4-ATPases)mediate the translocation of
phospholipids from the outer to the inner leaflet andmaintain lipid
asymmetry, which is critical for membrane trafficking and
signalingpathways. Here, we report the cryo–electron microscopy
structures of sixdistinct intermediates of the human ATP8A1-CDC50a
heterocomplex at resolutions of2.6 to 3.3 angstroms, elucidating
the lipid translocation cycle of this P4-ATPase.ATP-dependent
phosphorylation induces a large rotational movement of the
actuatordomain around the phosphorylation site in the
phosphorylation domain, accompaniedby lateral shifts of the first
and second transmembrane helices, thereby
allowingphosphatidylserine binding. The phospholipid head group
passes through thehydrophilic cleft, while the acyl chain is
exposed toward the lipid environment.These findings advance our
understanding of the flippase mechanism and thedisease-associated
mutants of P4-ATPases.
In eukaryotic cells, the phospholipid com-positions differ
between the outer and in-ner leaflets of the plasma and
organellarmembranes: phosphatidylcholine (PC) andsphingomyelin are
enriched in the outer
leaflet, whereas phosphatidylserine (PS)
andphosphatidylethanolamine (PE) are confined tothe inner leaflet
(1). The maintenance and dis-ruption of the asymmetric composition
affectprocesses, such asmembrane biogenesis, mem-brane trafficking,
signaling, and apoptosis. Threetypes of transporters—scramblase,
floppase, andflippase—have been reported to function as
phos-pholipid translocators (2–4). Scramblases
catalyzebidirectional phospholipid translocations thatdissipate the
membrane asymmetry withoutenergy consumption. In contrast, flippase
andfloppase, which use the energy of adenosinetriphosphate (ATP)
hydrolysis to mediate specificphospholipid translocations against
their con-centration gradients, maintain the asymmetricphospholipid
composition. ATP-binding cassettetransporters function as floppases
that drive theinner-to-outer translocation of lipids, whereas
typeIV P-type ATPases (P4-ATPase) are flippases thatdrive the
outer-to-inner translocation of lipids.The transport by P-type
ATPases occurs essen-
tially according to the Post-Albers mechanism(5, 6), wherein ATP
hydrolysis–coupled phospho-rylation and dephosphorylation within
the cyto-plasmic ATPase domain mediates the transition
between two intermediate states, E1 and E2,which have different
affinities for the substrates,enabling the substrate transport
across themem-brane (fig. S1A). Among the P-type ATPase
familymembers, the P1- to P3-ATPases are ion trans-porters, with
the most representative being theP2-ATPase family, which includes
the sarcoplas-mic reticulumCa2+pump(SERCA),Na+/K+-ATPase,and
H+/K+-ATPase. The P4-ATPase is the onlymember that functions as a
lipid transporter (7).The reaction of the P4-ATPases also follows
thePost-Albers mechanism, but it simply translocatesphospholipids
from exoplasmic leaflet to cyto-plasmic leaflet and does not
require any counter-transported substrate (fig. S1B) (8). The
humangenome encodes 14 P4-ATPase subclasses, whichdiffer in their
lipid selectivities and tissue expres-sion (9). For most
P4-ATPases, heterodimeriza-tion with a CDC50 family protein is
essential forproper expression and flippase activity (10, 11).The
first P4-ATPasemember identified, ATP8A1,
was found in bovine erythrocytes and chromaffingranules as an
aminophospholipid translocase(12, 13). P4-ATPases, includingATP8A1,
are presentin plasma and organellarmembranes and seques-ter PS
lipids from the outer to the inner leafletin resting cells. In
apoptotic cells, P4-ATPasesare cleaved and inactivated by proteases
such ascaspases and calpains. The PS that is subsequentlyexposed on
the cell surface acts as an “eat me”signal to induce phagocytosis
(14, 15). Further-more, the ATP8A1-catalyzed flipping of PS inthe
organellar membrane is necessary for thetransport of recycling
endosomes, membranefission, and cell migration (16, 17). Several
diseasesare associated with P4-ATPases. For example,ATP8B1
mutations cause the liver diseases knownas benign recurrent
intrahepatic cholestasis 1 andprogressive familial intrahepatic
cholestasis 1,ATP10A is associated with type 2 diabetes and
insulin resistance, and ATP11A is associated withcancer (18).
Furthermore, ATP8A1 and ATP8A2have been identified as causative
genes for neuro-logical disorders. ATP8A1 knockout mice
showhippocampus-dependent learning deficits associ-ated with the
exposure of PS on the outer surfaceof the plasma membrane in
hippocampal neu-rons (19–21).As comparedwith the canonical
ion-transporting
P-type ATPases, P4-ATPase has a large transportsubstrate and
thus is expected to use a differentmechanism for substrate
recognition and trans-location (22–24). However, despite
substantialefforts, themolecular mechanism underlying thelipid
flippase activity by the P4-ATPases has re-mained elusive. Here, we
report the cryo–electronmicroscopy (cryo-EM) structures of the
humanATP8A1-CDC50a heterodimer complex in its sixdistinct
intermediates: an apo state (E1), the non-hydrolyzable ATP analog
b,g-methyleneadenosine5′-triphosphate (AMPPCP)–bound state
(E1-ATP),the adenosine diphosphate–inorganic phosphate(ADP-Pi)
analog AlF4
−-ADP–bound state (E1P-ADP), the phosphate-analog AlF4
−-bound tran-sient phosphorylated state (E1P), the BeF3
−- boundphosphoenzyme ground state (E2P), and theAlF4
−-bound dephosphorylation state with the sub-strate phospholipid
(E2Pi-PL), revealing the trans-port cycle along the lipid flipping
reaction.
Overall structure
We performed a cryo-EM analysis of the P4-ATPase lipid
translocator family to elucidate thelipid translocation mechanism
(Fig. 1). We ex-pressed full-length human ATP8A1 and humanCDC50a
together in mammalian human embry-onic kidney–293F cells and
purified the complexin glycol-diosgenin (GDN) micelles (fig. S1C).
SDS–polyacrylamidegel electrophoresis analysis showedhigher
molecular weight bands of CDC50a, prob-ably derived frommultiple
glycosylations (fig. S1D)(25, 26). The purified ATP8A1-CDC50a
complexshowed PS-dependent ATPase activity, with aMichaelis
constantKmof 111 ± 26.4 mMand amaxi-mum rate of reaction Vmax of
99.7 ± 9.50 nmolmin−1 mg−1, as well as weak PE-dependentATPase
activity (Fig. 1C), consistent with previousreports (13, 17). The
ATPase activity was inhibitedby general inhibitors of P-type
ATPases, such asberyllium fluoride (BeF3
−) and aluminum fluoride(AlF4
−) (fig. S1F). The purified ATP8A1-CDC50acomplex was subjected
to cryo-EM single-particleanalyses under several different
conditions; name-ly, without any inhibitors and in the presence
ofAMPPCP, ALF4
−-ADP, BeF3−, and ALF4
− (fig. S1B).The acquired movies were motion-corrected
andprocessed in RELION 3.0 (27), which providedcryo-EMmaps at
overall resolutions of 2.6 to 3.3 Å,according to the gold-standard
Fourier shell cor-relation 0.143 criterion (figs. S4 to S8). The
flexiblecytoplasmic ATPase domain is most stabilizedin the AlF4
−-ADP and BeF3−-bound states, allow-
ing the de novo modeling of almost the entireATP8A1-CDC50a
complex, except for someminordisordered regions (Fig. 1, A and B,
and fig. S9).The overall structure shows the typical P-typeATPase
fold, composed of three large cytoplasmic
RESEARCH
Hiraizumi et al., Science 365, 1149–1155 (2019) 13 September
2019 1 of 7
1Department of Biological Sciences, Graduate School ofScience,
The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,Tokyo, 113-0033,
Japan. 2Discovery Technology Laboratories,Innovative Research
Division, Mitsubishi Tanabe PharmaCorporation, 1000 Kamoshida,
Aoba-ku, Yokohama, 227-0033, Japan. 3RIKEN SPring-8 Center, 1-1-1
Kouto, Sayo-cho,Sayo-gun, Hyogo 679-5148, Japan.*Corresponding
author. Email: [email protected]
(T.N.);[email protected] (O.N.)
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domains (A, actuator; N, nucleotide binding; P,phosphorylation)
and ten membrane-spanninghelices (M1 to M10). CDC50a has two
transmem-brane helices (TM1 and TM2) at the N and Ctermini, an
ectodomain consisting of an antipar-allel b-sandwich (b1 to b8),
and extensions of ~60and 70 amino acids with less secondary
structurein the b3-b4 and b5-b6 loops, respectively, whichare
stabilized by two intrachain disulfide bonds(fig. S3D). The three
N-linked glycosylation sitesof CDC50a, which are important for the
properfolding as well as the membrane trafficking ofthe P4-ATPases,
are clearly visible in the cryo-EMmap (fig. S3, D and E) (11,
28).
Interaction between ATP8A1 and CDC50a
CDC50a is an essential component for P4-ATPasesand is required
for the proper expression andfolding of ATP8A1 (fig. S1E) (10, 11).
CDC50aand ATP8A1 interact extensively through the
extracellular TM and intracellular regions (fig. S3).In the
extracellular region, the CDC50a ectodo-main covers all of the
extracellular loops of ATP8A1,except for the M1-M2 loop,
interacting in an com-plementary electrostaticmanner: the
extracellularloops of ATP8A1 bear negative charges, whereasCDC50a
bears positive charges (fig. S3, A and B).In particular, Asp961
andGlu1026 of ATP8A1 form asalt bridgewith Arg262 of CDC50a. In
addition, theM3-M4 loop of ATP8A1 extends toward CDC50a,and the two
bulky residues at the tip of the loop,Trp328 andTyr329,
formhydrophobic interactionsinvolving Phe127, Tyr299, Pro300,
Val301, and theN-glycan attached to Asn180 of CDC50a (fig. S3E).In
the TM region, several bulky residues, such asTrp942, Ala947 (M9),
Met1038, Phe1042, and Leu1049
(M10) of ATP8A1, and Phe54, Ile57, Phe61 (TM1),Phe324, Leu325,
Ala328, Tyr329, and Val332 (TM2) ofCDC50, are engaged in the
complex interaction(fig. S3G). Furthermore, we observed a
strong
planar density at the interface between M7 andM10 of ATP8A1 and
TM2 of CDC50a (fig. S3C),which could be assigned to the cholesteryl
hemi-succinate added during solubilization. Therefore,cholesterol
may bind to the same site and facil-itate the heterodimeric
interaction of ATP8A1and CDC50a. In the cytoplasmic region, the
N-terminal tail of CDC50a adopts an unstructuredloop conformation
that extends parallel to theplasmamembrane and interacts with
theM6-M7and M8-M9 loops and the short segment con-necting M4 and
the P domain (fig. S3F). Overall,CDC50a envelops the bulk of the TM
segmentsand forms extensive interactions with ATP8A1,which explains
the chaperone activity of CDC50afor the P4-type ATPases.
Entire transport cycle of P4-ATPase
The cryo-EM structures revealed the clear den-sities of the
inhibitors in their respective maps,
Hiraizumi et al., Science 365, 1149–1155 (2019) 13 September
2019 2 of 7
Fig. 1. Biochemical and cryo-EM studies of the
ATP8A1-CDC50acomplex. (A) Topology diagram of ATP8A1-CDC50a.
Conserved domainsand TM helices are schematically illustrated. In
the cytoplasmic regions,the A, N, and P domains and the C-terminal
regulatory domain are coloredyellow, red, blue, and green,
respectively. M1-M2 and M3 to M10 ofATP8A1 are purple and orange,
respectively, and CDC50a is pink. TheN-glycosylation sites are
shown as sticks. cyto, cytoplasmic side; exo,exoplasmic side. (B)
Overall structure of ATP8A1-CDC50a complex.
Cryo-EM maps (top) and ribbon models (bottom). The same color
schemeis used throughout the manuscript. (C) Phospholipid-dependent
ATPaseactivity of ATP8A1. Data points represent the mean ± SEM of
three to sixmeasurements at 37°C. By nonlinear regression of the
Michaelis-Mentenequation, ATP8A1-CDC50a in GDN micelles has a Km of
111.0 ± 26.4 mMfor
1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and
amaximal ATPase activity of 99.7 ± 9.5 nmol min−1 mg−1. POPE,
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine.
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bound at the catalytic site of ATP8A1 and sta-bilizing the
ATPase domain in different conforma-tions (Fig. 2), whereas CDC50a
adopted almostthe same conformation in all of these states
[rootmean square deviation (RMSD) (Å) = 0.24 to 0.66].
Most notably, the TM region of ATP8A1 remainsstructurally rigid
throughout the transport cycle,probably because of the tight
association withCDC50a, in contrast to other P-type ATPases, suchas
SERCA (figs. S10 and S11 and movie S1).
The structures obtained under three condi-tions, namely without
inhibitors, with AMPPCP,and with AlF4
−-ADP, describe the conformationalchanges upon ATP binding and
autophospho-rylation, which correspond to the E1, E1-ATP,
andE1P-ADP conformations, respectively, in the Post-Albers scheme
(Fig. 2A). The densities of the Nand A domains are only weakly
visible in the E1state, indicating the highly flexible motion of
thesedomains without any ligand (E1 in Fig. 2 and fig.S4). The
particles were then classified according tothe densities of the N
and P domains, and thesedomains were modeled into the class with
thestrongest densities, which probably representsthemost likely
arrangement in theE1 state (class 2in fig. S4). The particles of
the AMPPCP-boundstate can be classified into three similar
confor-mations, wherein the N and P domains adoptslightly different
orientations (fig. S5). The com-parison of these classes indicated
that ATP bindingat the N domain induces the mutual approach
oftheNandPdomains. The density for theAMPPCPis most clearly visible
within the class where thesedomains are proximal and bridged by
AMPPCP(E1-ATP in Fig. 2B and fig. S5D): The adenine
ringinteractswith Phe534 of theNdomain,whereas thephosphate group
interacts with Asp409 and Thr411
(the DKTGmotif), Asn789, and Asp790 at the phos-phorylation site
of the P domain, in cooperationwith a Mg2+ ion. The AlF4
−-ADP–bound state issimilar to the E1-AMPPCP conformation, but
theNand P domains are more tightly bridged by ADPand AlF4
− (E1P-ADP in Fig. 2 and fig. S6), capturedin the phosphoryl
transfer intermediate (E1P-ADP).Overall, ATP binding and the
subsequent
phosphoryl transfer reaction induce the proximalarrangement of
the N and P domains, which isaccompanied by a slight outward shift
of the Adomain by ~6.5 Å (E1, E1-ATP, and E1P-ADP inFig. 2A, fig.
S10, and movie S1). The phosphoryl-ation reaction is mediated by
the motions of theATPase domain and does not require any changesin
the TM region. The TM segments of ATP8A1adopt almost the same
conformation throughoutthe transition (fig. S11), which is
consistent withthe substrate-independent autophosphorylationof
P4-type ATPases (8).The two phosphate analogs, BeF3
− and AlF4−,
occupy the phosphorylation site in a similar man-ner, but their
coordination geometries are slightlydifferent (E1P, E2P, andE2Pi-PL
in Fig. 2). BeF3
− iscovalently attached to the carboxylate side chainof Asp409,
in coordination with a Mg2+ ion, andcaptures the phosphoenzyme
ground state (E2Pin Fig. 2B). The A domain is tightly fixed to
thephosphorylation site (E2P in Fig. 2A and fig. S7)through the
backbone carbonyls of Asp189 andGly190 in the conservedDGETmotif
(residues 189to 192) (E2P in Fig. 2B). The N domain is pushedapart
from the P domain and no longer has accessto the phosphorylation
site, thus representing theADP-insensitive E2P state (8). The
particles of theAlF4
−-bound state could be separated into twodifferent classes (fig.
S8), and both showed clearAlF4
− density at the phosphorylation site. In thefirst class, the
bound AlF4
− does not mediate anyinterdomain interactions, and the
catalytic domains
Hiraizumi et al., Science 365, 1149–1155 (2019) 13 September
2019 3 of 7
Fig. 2. Entire transport cycle of ATP8A1-CDC50a. (A) The six
different intermediates ofATP8A1-CDC50a during the phospholipid
translocation cycle are shown, arranged clockwise asin the
Post-Albers reaction cycle: E1, E1-ATP, E1P-ADP, E1P, E2P, and
E2Pi-PL.The bound inhibitors areshown in space-filling model
representations. (B) Comparison of the phosphorylation sites in
eachintermediate. AMPPCP and ADP are shown as sticks, and AlF4
− and BeF3− are shown as spheres.
Densities are shown as green mesh, contoured at 3.5s.
Single-letter abbreviations for the aminoacid residues are as
follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I,
Ile; K, Lys; L, Leu;M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser;
T, Thr; V, Val; W, Trp; and Y, Tyr.
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adopt a conformation similar to the AlF4−- ADP
bound state (E1P-ADP and E1P in Fig. 2B), likelyrepresenting the
transient phosphorylated state(E1P) immediately after the ADP
release. In thesecond class, AlF4
− mediates the interaction be-tween the N and A domains through
the DGETmotif in a similar manner to the BeF3
−-boundstate, but the A domain is rotated by ~22° aroundthe
phosphorylation site, as compared with theBeF3
−-bound state (Fig. 3A). This allows the re-positioning of the
carboxyl side chain of Glu191 intheDGETmotif to provide a catalytic
base for thedephosphorylation reaction (E2PandE2Pi-PL inFig.2B)
(8), therebymimicking the dephosphorylationtransition–like
intermediate (29). The rearrange-ment of the A domain accompanies
the swing-outmotion of the TM1-TM2 segment, which is di-rectly
connected to the A domain, consequentlycreating a large cleft
between the M1-M2 andM4-M5 segments, in which the clear density of
aglycerophospholipid is observed (Fig. 3, B to D).Therefore, this
second AlF4
− class structure repre-sents the substrate-bound E2Pi state
(E2Pi-PL).The rearrangement of the A domain is likely tobe coupled
to the binding of the substrate lipid,as it occupies the cleft and
pushes out theM1-M2segment (figs. S10 and S11), which explains
thesubstrate-dependent dephosphorylation of P4-ATPases (Fig. 1C)
(8).
Phospholipid recognition
Given that the substrate lipids (such as PS or PE)were not added
during purification, it is likely
that endogenous phospholipid contained in theGDN micelles is
specifically bound to ATP8A1in the AlF4
−-bound state (Fig. 3, A to D, and fig.S12A). ATP8A1 shows PG-,
PE-, and PS-dependentATPase activity, with the highest preference
for PS(13, 17), and the size and shape of the head groupdensity are
in good agreement with those of theserine moiety (fig. S1H).
Therefore, we modeledPS into the density. PS is recognized within
theopen cleft, in which the phosphate group is co-ordinated by the
backbone amide groups of Ile357
and Ser358 in the conserved PISL motif at the un-wound kink of
M4 and further stabilized by theGln88, Asn353, andAsn882 side
chains (Fig. 3, C andE), whereas the attached acyl chains are
exposedto the bulk lipid environment and partly accom-modated in
the hydrophobic pocket formed bythe conserved residues in TM2 and
TM4, such asVal103, Pro104, Phe107 (M2), Val361, Val365
(M4),Val883, and Leu891 (M6) (Fig. 3C). In the currentcryo-EMmap,
the acyl chains aremost visible nearthe attached glycerol moiety,
and PS moleculeswith shorter acyl chains showed weaker
ATPaseactivity (fig. S1G), indicating that the acyl chains,as well
as the hydrophilic head group, are specif-ically recognized in the
substrate binding pocketof ATP8A1.The head group of PS is
situatedwithin a small
cavity on the extracellular half of the cleft andis surrounded
by hydrophilic residues, such asGln88, Asn352, Asn353, and Asn882
(Fig. 3C), withwhich the serine moiety forms hydrogen
bondinginteractions. Consistently, mutational studies have
shown the importance of the uncharged polarresidues Gln88,
Gln89, Asn352, and Asn353 for PSselectivity (22–24). Such
interactions explain thehead group preferences of ATP8A1, which has
weakselectivities for PE and PG, with head groups thatcan form
similar hydrogen bonding interactions,and no selectivity for PC,
with headmethyl groupsthat cannot form such hydrogen bonding
inter-actions. The PC selective P4-ATPases have nonpolarresidues,
such as Ala and Gly, at the correspond-ing positions (fig. S2),
also supporting the pro-posal that the residues constituting this
exoplasmiccavity primarily define the head group selectivity.
Lipid translocation pathway of ATP8A1
In the P2-ATPases, conformational changes inthe cytoplasmic
ATPase domains are coupled torearrangement of the core TM helices
that con-stitute the cation binding sites (Fig. 4A) (30,
31).Especially in SERCA,Glu309 of the conserved PEGLmotif, located
in theunwoundM4kink, constitutespart of the ion binding sites,
enabling coupling be-tween ion binding and release and
rearrangementof theATPasedomain. InATP8A1, theM4 segmentis
similarly kinked at the PISL motif, but the ionbinding sites are
lost by the substitution with hy-drophobic residues Ile357, Leu854,
and Val977 (Fig.4B). Although the PS binding site partially
over-laps the Ca2+ binding site in SERCA (site II), thearrangement
of the surrounding residues remainsalmost unchanged throughout the
transport cycle.It has been suggested that the P4-ATPases use
adifferent translocation pathway for a large lipid
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2019 4 of 7
Fig. 3. Phospholipid recognition. (A) Structural comparison of
the E2P and E2Pi-PL states, showing the large rearrangement of
M1-M2 and the N and Adomains upon phospholipid binding. (B and C)
Phospholipid binding site, viewed (B) parallel to the membrane
plane and (C) as a close-up of the headgroup. Residues within 4 Å
of the bound phospholipid are shown as sticks. Hydrogen bond
interactions are shown as black dashed lines. (D) Cryo-EMdensity
showing the bound endogenous phospholipid (green mesh, 2.5s). (E)
Residues constituting the hydrophobic gate are shown. The
putativetranslocation pathway is indicated by an orange arrow.
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substrate (9), and according to previous muta-tion studies on
the yeast and bovine P4-ATPases,the residues associated with the
head group selec-tivity are mapped along the hydrophilic cleft
be-tween theM1-M2andM3-M4 segments (fig. S12B).In the PS-bound
structure, the head group entersfrom the exoplasmic leaflet and
stays occludedin the middle of the pathway by the side chain
ofIle357 in the PISL motif. The mutation of Ile357 tobulky
residues, such as Met and Phe, drasticallyreduced the lipid
transport activity, whereas themutations to smaller residues only
moderatelyaffected the transport activity (22), suggesting
thatIle357 constitutes a central hydrophobic gate forthe lipid
translocation, together with other resi-dues on the M1-M2 segment,
such as Phe81 andIle108 (Fig. 3E). Because the exoplasmic end
isalso closed by the M1-M2 loop (fig. S12B), thecurrent structure
probably represents a partiallyoccluded state. Although PS binding
induces aslight reorientation of the Ile357 side chain towardthe
M1-M2 segments (Fig. 4B), the translocationof the hydrophilic head
group requires furtherrearrangement of the central gate residues,
whichis probably coupled with the phosphate releasefrom the P
domain. By analogy to the E2P-to-E2transition inSERCA, thephosphate
release “unlocks”the A domain and allows a further outward shiftof
the M1-M2 segment, thus inducing the open-ing of the central
hydrophobic gate (32). Previousstructures of P2-ATPases revealed
several lipidbinding sites; for example, the E2 structure ofSERCA
stabilized by thapsigargin and an inhib-
itor, 2,5-di-tert-butyl-1,4-dihydroxybenzene (BHQ)(33), showed
PE binding between theM2 andM4segments at the intracellular
leaflet, correspond-ing to the putative exit of the lipid
translocationpathway in ATP8A1 (fig. S13A).
Furthermore,phospholipids are anchored to the positivelycharged
residues at the protein-lipid interfaceand interplay with the
protein conformationalchanges during the transport cycle in
SERCA(34). The ATP8A1-CDC50A complex has clustersof positively
charged residues at both the en-trance and exit of the
translocation pathway,which may play important roles in lipid
trans-location (fig. S12C).
C-terminal autoregulatory domain
In the BeF3−-stabilized E2P state, we observed an
extra density extending through the cytoplasmiccatalytic domains
(Fig. 5, A and B), which we as-signed as the C-terminal
autoregulatory domain(residues 1117 to 1140) (35, 36), consisting
of theconserved GYAFS motif (residues 1119 to 1123)and a short
helical domain (residues 1131 to 1137),although the ~50–amino acid
linker connectedto the M10 helix was disordered. The
regulatorydomain interacts with the N domain, and theGYAFS motif is
specifically recognized by a shortloop region of the N domain
(residues 533 to539) (Fig. 5B). Notably, Phe1122 occupies the
ATPbinding site and stacks with Phe534. The densitiesof the
C-terminal residues are only visible in theBeF3
−-stabilized E2P conformation and are com-pletely disordered in
the other conformations,
including the ligand-unbound E1 state. This sug-gests that the
regulatory domain specifically sta-bilizes ATP8A1 in the E2P
conformation, in whichthe N domain is somewhat farther apart (Fig.
5Cand fig. S10).The C-terminal regulatory domain has differ-
ent effects between the yeast and mammalianP4-ATPases. In the
yeast Drs2p flippase, it exertsan autoinhibitory effect on ATPase
activity (37).However, in the mammalian ATP8A2 flippase, itmediates
a rather complicated regulation mode.The partial truncation of the
GYAFS motif andthe short helical domain results in decreasedATPase
activity, whereas the complete loss of theC-terminal residues,
including the disordered loopregion, restores the ATPase activity
to the samelevel as the wild-type enzyme (38), indicating thatthe
GYAFS motif and the short helical domainobserved in the current
cryo-EM map positivelymodulate the enzymatic reaction. We
hypoth-esize that the regulatory domain keeps the Ndomain apart
from the A domain in the E2P stateand thus facilitates the
rotational rearrangementof the A domain that is required for PS
binding.The conformation of the N domain in the E1 andE1-ATP states
would sterically prevent this rota-tional motion (Fig. 5D).
Mechanism of the P4-type ATPase
The current cryo-EM structures revealed sixdifferent
intermediates of ATP8A1, namely, E1,E1-ATP, E1P-ADP, E1P, E2P, and
E2Pi-PL, dem-onstrating the transport cycle of the lipid
flippase
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2019 5 of 7
Fig. 4. Comparison of the phospholipidbinding sites. (A) Ca2+
binding site of SERCAin the Ca2+ binding state (right: PDB ID 1T5S)
andthe H+ binding state (left: PDB ID 3B9R),viewed from the
cytoplasmic side. Residuesinvolved in Ca2+ and H+ transport are
shownas ball-stick representations. Hydrogen bondsare shown as
black dashed lines and the boundCa2+ are pale blue spheres. (B)
Phospholipidbinding site of ATP8A1 in the unbound state(right:
E1-ATP) and the phospholipid-bound state(left: E2Pi-PL) from the
same viewpoint as in(A). Residues involved in phospholipid
translocationand other residues corresponding to thosecoordinating
H+ and Ca2+ in SERCA are shown asball-and-stick representations.
Hydrogenbonds are shown as black dashed lines. Cryo-EMdensity
showing the side chain of Ile357
in unwound M4 kink (green mesh, 3.0s).
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reaction (Fig. 6). Although ATP8A1 shares a sim-ilar ATP
hydrolysis–dependent catalytic reactionwith the ion-transporting
P2-type ATPases, such asSERCA (30, 31), Na+/K+-ATPase (39), and
H+/K+-ATPase (40), there are notable differences in theirtransport
mechanisms and substrate transportingpathways (fig. S14). These
canonical ion-transportingP-type ATPases undergo extensive
rearrangementsof the TM region, especially in the M1 to M6
seg-ments that constitute the ion translocating path-
way (fig. S14A). In contrast, ATP8A1maintains thestructural
rigidity of the TM region throughoutthe transport cycle, as the
core TM segments (M3to M10) could be superimposed well in all of
theintermediates [RMSD (Å) = 0.30 to 0.69]. Conse-quently, ATP8A1
has a distinct pathway for thelipid head group, between the M1-M2
and M3-M4 segments, and the lipid translocation is es-sentially
accomplished by the mobile segmentsof M1-M2 (fig. S14B).
The critical rearrangement in SERCA occursduring the E1P-to-E2P
transition, in which theA-domain rearrangement toward the
phospho-rylation site induces the opening of the “luminalgate”
composed of the M1 to M4 segments andalters the affinity for the
substrate ions (fig. S15A)(31). Although theAdomain ofATP8A1
undergoesa similar rearrangement during this transition,the
conformational change is limited to the M1and M2 segments in the
region proximal to the A
Hiraizumi et al., Science 365, 1149–1155 (2019) 13 September
2019 6 of 7
Fig. 5. ATP8A1 autoregulation by the C-terminal domain. (A)
Inthe BeF3
−-stabilized E2P state, an extra density is observed around
thecytoplasmic catalytic domains, corresponding to the C-terminal
auto-regulatory domain. The density is shown as a green mesh,
contouredat 3s. (B) Close-up view of the interaction between a
short loopregion (residues 533 to 539) in the N domain and the
regulatorydomain. An atomic model of the GYAFS motif and a short
helicalregion in the regulatory domain are modeled into the
density.
(C and D) Arrangements of the N and A domains and the
regulatorydomain, shown for the E2P (C) and E1-ATP (D) states,
viewed fromthe cytoplasmic side. The N and A domains of the E2Pi-PL
state aresuperimposed in a transparency representation. The
regulatorydomain keeps the N domain apart from the A domain and
thus facilitatesthe rotational movement of the A domain around the
phosphorylationsite in the E2P state (C), whereas the similar
rearrangement ishindered by the N domain in the E1-ATP state
(D).
Fig. 6. Proposed mechanism of phospholipid trans-location.
Schematic model of the phospholipidtranslocation cycle by
ATP8A1-CDC50a, accordingto the Post-Albers mechanism. The model is
depicted withthe same colors as in Fig. 1A. ATP binding induces
theproximal arrangement of the N and P domains, by bridgingthese
domains and slightly forcing out the A domain.After the phosphoryl
transfer reaction, ADP is releasedfrom the N domain, and the A
domain approaches theN domain and interacts with it, through the
DGETmotif, to form the E2P state. The C-terminal regulatorydomain
penetrates between the P and N domains andstabilizes the E2P state.
The rearrangement of the Adomain induces flexibility in the M1-M2
segments, thusallowing phospholipid binding at the interface
between theM1-M2 and bulk TM segments. Phospholipid bindinginduces
further rearrangement of the A domain, therebyfacilitating the
dephosphorylation reaction (E2Pi-PL).Ile357 constitutes a
hydrophobic gate that occludes themiddle of the translocation
pathway. Phospholipidtranslocation to the cytoplasmic leaflet is
probably coupledto the phosphate release at the P domain, allowing
thefurther outward shift of the M1-M2 segment (E2-PL).The
translocated phospholipid laterally diffuses to thecytoplasmic
leaflet, and the enzyme adopts the E1 confor-mation, ready to
initiate another reaction cycle.
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domain, and the luminal side remains unchanged(fig. S15B). This
rigidity is probably achieved bythe tight association with CDC50a,
which holdsthe M3 to M10 segments of ATP8A1 on both theluminal and
cytoplasmic sides. Most notably, theloop connectingM3-M4 and the
cytoplasmic endof M4 is constrained by the interaction withCDC50a,
which probably hinders theM3 andM4rearrangement (figs. S3, E and F,
and S15B). Thedeletion of the CDC50a N-terminal tail,
whichinteracts with the cytoplasmic end of the M4segment, decreased
the flippase activities of P4-ATPases (25). Therefore, the rigidity
of the TMsegments is important for the transport activityof
P4-ATPases. Although the phosphorylation-induced A-domain
rearrangement in ATP8A1causes only minor changes on the luminal
side,the density of the M1-M2 segment near the Adomain ismore
disordered in the E2P conforma-tion (fig. S16), suggesting higher
flexibility in thelinker region. This flexibility may facilitate
thesubsequent binding of the phospholipid betweentheM1-M2 andM3-M4
segments by allowing theswing-out motion of the M1-M2 segment, as
ob-served in the AlF4
−-stabilized dephosphorylationtransition-like state. Overall,
the P4-ATPases haveevolved a distinct mechanism for the lipid
trans-location, while sharing the similar rearrangementof the
cytoplasmic domains with the canonicalion-transporting P-type
ATPases.
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ACKNOWLEDGMENTS
We thank H. Hirano for assistance in generating the movie,H.
Nishimasu for fruitful discussions, T. Nakane for assistance
withthe single-particle analysis, and the structural biophysics
team atMitsubishi Tanabe Pharma Corporation, especially H. Kishida,
fortechnical advice about model building. We also thank the
staffscientists at the University of Tokyo’s cryo-EM facility,
especiallyK. Kobayashi, T. Kusakizako, H. Yanagisawa, A.
Tsutsumi,M. Kikkawa, and R. Danev. Funding: This work was
supportedby a MEXT Grant-in-Aid for Specially Promoted Research
(grant16H06294) to O.N. Author contributions: M.H. prepared
thecryo-EM samples and performed the functional analyses. M.H.and
T.N. collected and processed the cryo-EM data and built
thestructures. K.Y. assisted data processing and structure
refinement.M.H., T.N., and O.N. wrote the manuscript. T.N. and
O.N.supervised the research. Competing interests: M.H. is agraduate
student at Mitsubishi Tanabe Pharma Corporation andis supported by
the company with nonresearch funds. Thecompany has no financial or
other interest in this research.Data and materials availability:
Cryo-EM density maps havebeen deposited in the Electron Microscopy
Data Bank under theaccession codes EMD-9931 (E1 class1), EMD-9932
(E1 class2),EMD-9933 (E1 class3), EMD-9935 (E1-ATP class1),
EMD-9934(E1-ATP class2), EMD-9936 (E1-ATP class3),
EMD-9937(E1P-ADP), EMD-9938 (E2P class1), EMD-9939 (E2P
class2),EMD-9940 (E2P class3), EMD-9941 (E2Pi-PL), and
EMD-9942(E1P). Atomic coordinates have been deposited in the
ProteinData Bank under IDs 6K7G (E1 class1), 6K7H (E1 class2),
6K7J(E1-ATP class1), 6K7I (E1-ATP class2), 6K7K (E1P-ADP),
6K7L(E2P-class2), 6K7M (E2Pi-PL), and 6K7N (E1P). The raw
imageshave been deposited in the Electron Microscopy Public
ImageArchive, under accession code EMPIAR-10303.
SUPPLEMENTARY MATERIALS
science.sciencemag.org/content/365/6458/1149/suppl/DC1Materials
and MethodsFigs. S1 to S16Table S1References (41–58)Movie S1
10 June 2019; accepted 6 August 2019Published online 15 August
201910.1126/science.aay3353
Hiraizumi et al., Science 365, 1149–1155 (2019) 13 September
2019 7 of 7
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Cryo-EM structures capture the transport cycle of the P4-ATPase
flippaseMasahiro Hiraizumi, Keitaro Yamashita, Tomohiro Nishizawa
and Osamu Nureki
originally published online August 15, 2019DOI:
10.1126/science.aay3353 (6458), 1149-1155.365Science
, this issue p. 1149Sciencelipids bind differently, powering
translocation.requires for function, CDC50. ATP binding and
autophosphorylation of ATP8A1 drive a cycle of conformations in
which
electron microscopy structure of six intermediates of the human
flippase ATP8A1 bound to the partner protein it−the cryo report et
al.ATPases that are important in processes such as membrane
trafficking, signaling, and apoptosis. Hiraizumi
against a concentration gradient from the outer to inner or
inner to outer leaflets, respectively. Flippases are P4-typeknown
as flippases and floppases use the energy from adenosine
triphosphate (ATP) hydrolysis to translocate lipids
The membranes of eukaryotic cells have different lipid
compositions in their inner and outer leaflets. EnzymesFlipping a
lipid
ARTICLE TOOLS
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