G protein-membrane interactions II: Effect of G Protein-linked Lipids on Membrane Structure and G Protein-membrane Interactions

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G p o ein-memb ane in e ac ions II: E ec o G P o ein-linked Lipids on Memb ane
S uc u e and G P o ein-memb ane In e ac ions
Jesús Casasa, Mai ane Iba gu ena,b, Ra ael Ál a eza, Sil ia Te ésa,b, Vic o ia Lladóa,
S e ano P. Pio oc, Simona Conciliod, Xa ie Busque sa,b, Da id J. Lópeza,b,* and Pablo
V. Esc ibáa,b
aLabo a o y o Molecula Cell Biomedicine, Depa men o Biology, IUNICS, Uni e si y
o he Balea ic Islands, E-07122 Palma de Mallo ca, Spain.
bLipopha ma The apeu ics, S.L., Pa cBi , 07121 Palma de Mallo ca, Spain.
cDepa men o Pha macy, Uni e si y o Sale no, Via Pon e don Melillo, 84084 Fisciano
(SA), I aly.
dDepa men o Indus ial Enginee ing, Uni e si y o Sale no, Via Pon e don Melillo,
84084 Fisciano (SA), I aly.
*To whom co espondence should be add essed:
Da id J. López, PhD, Labo a o y o Molecula Cell Biomedicine, Depa men o Biology,
Uni e si y o he Balea ic Islands, C a. Valldemossa km. 7.5, 07122, Palma (Spain). Tel.:
+34-97117 33 31; Fax +34-971 17 31 84; E-mail: [email p o ec ed]
Abb e ia ions used: DiI, 1,1’-dioc adecyl-3,3,3’,3’- e ame hylindoca bocyanine pe chlo a e;
DSC, di e en ial scanning calo ime y; GG, ge anylge aniol; GPCR, G p o ein-coupled
ecep o ; GUV, gian unilamella esicle; HII; in e ed hexagonal; LUV, la ge unilamella
esicle; MA, my is ic acid; MOH, my is ic alcohol; PA, palmi ic acid; PC, phospha idylcholine;
PE, phospha idyle hanolamine; POH, palmi ic alcohol; POPC, 1-palmi oyl-2-oleoyl-sn-glyce o-
3-phosphocholine; POPE, 1-palmi oyl-2-oleoyl-sn-glyce o-3-phosphoe hanolamine; TH,
lamella - o-in e ed hexagonal phase ansi ion empe a u e.
This is he accep ed manusc ip o he a icle ha appea ed in inal o m in Biochimica e Biophysica Ac a - Biomemb anes
1859(9) : 1526-1535 (2017), which has been published in inal o m a h ps://doi.o g/10.1016/j.bbamem.2017.04.005. © 2017
Else ie unde CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/)
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ABSTRACT
G p o eins o en bea my is oyl, palmi oyl and isop enyl moie ies, which a o hei
associa ion wi h he memb ane and hei accumula ion in G P o ein Coupled Recep o - ich
mic odomains. These lipids in luence he biophysical p ope ies o memb anes and he eby
modula e G p o ein binding o bilaye s. In his con ex , we showed he e ha ge anylge aniol, bu
nei he my is a e no palmi a e, inc eased he in e ed hexagonal (HII) phase p opensi y o
phospha idyle hanolamine-con aining memb anes. While my is a e and palmi a e p e e en ially
associa ed wi h phospha idylcholine memb anes, ge anylge aniol a o ed nonlamella -p one
memb anes. In addi ion, Gi1 monome s had a highe a ini y o lamella phases, while G and
G showed a ma ked p e e ence o nonlamella p one memb anes. Mo eo e , ge anylge aniol
enhanced he binding o G p o ein dime s and ime s o phospha idyle hanolamine-con aining
memb anes, ye i dec eased ha o monome s. By con as , bo h my is a e and palmi a e
inc eased he Gi1 p e e ence o lamella memb anes. Palmi oyla ion ein o ced he binding o
he monome o PC memb anes and my is oyla ion dec eased i s binding o PE-en iched bilaye .
Finally, binding o dime s and ime s o lamella -p one memb anes was dec eased by palmi a e
and my is a e, bu i was inc eased in nonlamella -p one bilaye s. These esul s demons a e ha
co/pos - ansla ional G p o ein lipid modi ica ions egula e he memb ane lipid s uc u e and ha
hey in luence he physico-chemical p ope ies o memb anes, which in pa explains why G
p o ein subuni s so o di e en plasma memb ane domains.
Keywo ds: G-p o eins, Memb anes, Palmi oyla ion, My is oyla ion, Cell Signaling, Isop enoids.
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1. INTRODUCTION
Upon agonis -media ed ac i a ion, G p o ein-coupled ecep o (GPCR)-media ed cell
signaling is ampli ied h ough he la ge numbe o G p o ein molecules p esen a he plasma
memb ane compa ed o he numbe o ecep o s [1]. Indeed, one agonis -ac i a ed GPCR can
ac i a e dozens and e en hund eds o G molecules [2]. The e o e, many housands o G
p o eins can be ound in memb ane egions whe e he e is a high densi y o GPCRs. In he
plasma memb ane, p o ein-lipid and lipid-lipid in e ac ions de ine he memb ane lipid s uc u e,
which in u n in luences he ype o p o eins ound in a gi en memb ane egion, as well as he
ac i i y o GPCRs and ela ed signaling p o eins [3, 4].
When a G p o ein is ac i a ed by a GPCR, he G subuni dissocia es om he G dime .
The eleased dime emains in he icini y o he ecep o and i ec ui s GPCR kinases, which
inac i a e GPCRs and egula e o he signaling p o eins [5]. By con as , he G monome
egula es he ac i i y o e ec o p o eins (e.g., adenylyl cyclase, phospholipase C and ion
channels) o en loca ed in di e en memb ane domains, such as lipid a s [6-8]. The
mobiliza ion o each subuni o he co ec memb ane en i onmen la gely depends on hei
p e e ence o ce ain lipids o lipid s uc u es. Howe e , he molecula mechanisms unde lying
G p o ein in e ac ions wi h memb anes, hei mobiliza ion o di e en domains and hei
in luence on memb ane lipid s uc u e emain la gely unknown. In he p esen s udy we used
di e en app oaches o in es iga e he e ec o he co- and pos - ansla ional lipid modi ica ions
o G p o eins on memb ane lipid s uc u e and p o ein-lipid in e ac ions.
The ansmemb ane domains o he GPCR, such as hose o α2-ad ene gic ecep o , inc ease
he HII phase p opensi y o he memb ane [9]. G and G p o eins a e also loca ed
p e e en ially in his nonlamella -p one en i onmen [4], which may pa ly explain why G
p o eins accumula e nea GPCRs. On he o he hand, ce ain G monome s p e e lamella -p one
egions, such as lipid a s, explaining how hey may be mobilized om he ecep o o e ec o
ich memb ane domains [3, 4, 6]. The e o e, memb ane lipid s uc u e plays an impo an ole in
p opaga ing GPCR-media ed signals.
GPCRs equen ly clus e in de ined memb ane egions, whe e Gαβγ p o eins co-localize in
mola excess [10]. In hese egions, G p o eins in e ac wi h he cy osolic lea le o he plasma
memb ane, aided by he my is oyl and palmi oyl moie ies ha a e associa ed wi h he G
subuni , and he isop enyl moie ies associa ed wi h he G subuni [11]. In addi ion o acili a ing
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G p o ein binding o memb anes, hese lipid ancho s may also modi y he lipid bilaye
en i onmen and he G p o ein-memb ane in e ac ions. The e ec o lipid moie ies o G p o eins
on memb ane s uc u e and p o ein-lipid in e ac ions has ecei ed li le a en ion o da e. Thus,
he e we ha e in es iga ed he ole o ce ain lipids on he s uc u al p ope ies o memb anes in
u he de ail. Fo his pu pose, we ha e used model memb anes ha con ain he lamella -p one
phospholipid phospha idylcholine (PC) and he nonlamella -p one phospholipid
phospha idyle hanolamine (PE), es ing hei in e ac ions wi h pu i ied Gi1, G o G
p o eins in he p esence o absence o palmi ic acid (PA), my is ic acid (MA) o ge anylge aniol
(GG). In con as o o he s udies in which poin mu an s we e analyzed wi h o wi hou G
p o ein-ancho ed lipids [12], his app oach enabled us o de e mine he e ec o hese lipids on
wild ype G p o ein-memb ane in e ac ions.
Acco dingly, we ound ha hese lipid moie ies had di e en e ec s on memb ane lipid
s uc u e, and on he in e ac ions o he G p o eins wi h lamella - and nonlamella -p one
memb anes. In summa y, hese esul s show he ole o hese lipid modi ica ions in he complex
in e ac ions be ween G p o eins and memb anes and he possible implica ions in human heal h
a e discussed.
2. EXPERIMENTAL
2.1. Ma e ials
Egg yolk PC and bo ine li e PE we e pu chased om A an i Pola Lipids (Alabas e , AL).
MA, PA and GG we e ob ained om Sigma-Ald ich (Sain Louis, MO). The pu i ied G p o eins
(my is oyla ed Gi1, G and Gi) we e om Calbiochem (Da ms ad , Ge many), he
monoclonal an i-Gi1 an ise um was pu chased o San a C uz Bio echnology (San a C uz, CA)
and he monoclonal an ibody an i-G was om BD Biosciences (F anklin Lakes, NJ). 1,1’-
dioc adecyl-3,3,3’,3’- e ame hylindoca bocyanine pe chlo a e (DiI) and Alexa Fluo 488 C5
maleimide we e p ocu ed om In i ogen (Eugene, OR). ECL Wes e n blo de ec ion sys em
and Hype ilm we e om GE Heal hca e (Pi sbu gh, PA).
2.2. Di e en ial Scanning Calo ime y
DSC measu emen s we e made wi h a Mic ocal MC-2 mic ocalo ime e (Mic oCal Inc.,
No hamp on, MA, USA), as desc ibed elsewhe e [13]. B ie ly, phospholipids we e dissol ed in
chlo o o m: me hanol (2:1, by ol) and d ied unde an a gon lux. Sol en aces we e emo ed
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unde acuum o a leas 3 h a oom empe a u e be o e hyd a ion. Mul ilamella esicles we e
o med by esuspending he lipid ilm in 10 mM HEPES, 100 mM KCl, 1 mM EDTA, pH 7.4,
ollowing o exing a 42ºC. The mix u e was degassed o 5 min and he DSC measu emen s
we e hen ca ied ou om 10 o 50ºC a a scan a e o 1ºC/min. All samples we e subjec ed o
h ee consecu i e scans and calo ime ic ansi ions we e ound o be e e sible. The ansi ion
en halpy and empe a u e alues shown he e co esponded o he means o h ee independen
expe imen s and hey we e ob ained using he so wa e p o ided by he manu ac u e (Mic ocal
O igin).
2.3. 31P-Nuclea Magne ic Resonance
Mul ilamella esicles we e p epa ed by mixing 56 mg o bo ine li e PE wi h deionized
deu e a ed wa e (D2O, 15% w/w) in he p esence o absence o 5 mol% MA, PA o GG. Lipid
suspensions we e hyd a ed and homogenized wi h a pes le- ype minihomogenize (Sigma), and
o exed o homogenei y. The suspensions we e hen subjec ed o 10 cycles o hea ing (60°C)
and eezing (-80°C), and hen equilib a ed be o e da a acquisi ion, as epo ed p e iously [14].
31P-NMR measu emen s we e made in 5 mm ubes on an Ad ance-300 mul inuclea NMR
spec ome e (B uke Ins umen s). Da a we e acqui ed e e y 5ºC be ween 5 and 55ºC,
equilib a ing he empe a u e o 15 min be o e each measu emen . The accumula ed 31P-NMR
ee induc ion decay was ob ained o 128 ansien s using a 4.4 s 90° adio- equency pulse, a
24.3 kHz sweep wid h and 65,000 da a poin s. The delay be ween he ansien s was 2 s and
spec a we e ob ained by scanning om lowe o highe empe a u es.
2.4. Molecula dynamics
Two all-a om lipid bilaye s we e used o symme ic memb ane models con aining 1-
palmi oyl-2-oleoyl-sn-glyce o-3-phospha idylcholine: 1-palmi oyl-2-oleoyl-sn-glyce o-3-
phospha idyle hanolamine (POPC:POPE) (6:4; mol a io) and POPC. The POPC memb ane was
made wi h 98 POPC molecules, 33 Na+ ions and 33 Cl- coun e ions, and 11,301 wa e
molecules. The POPC:POPE memb ane was made wi h 58 POPC and 40 POPE molecules, 28
Na+ ions and 28 Cl- coun e ions, and 9,825 wa e molecules. In bo h cases, he wa e densi y
was o 0.997 g/mL. Simula ions we e pe o med using he YASARA p og am [15] a 310 K and
1 a m unde a NPT ensemble, coupling he sys em o a Be endsen he mos a and ba os a [16]
combined wi h a con ol o sol en densi y as implemen ed in he so wa e Yasa a. The
AMBER03 o ce ield was used and he geome y o he molecules was op imized by he semi-

6
empi ical AM1 me hod, using he COSMO sol a ion model [17]. Pa ial a omic cha ges we e
calcula ed using he same le el o heo y as he Mulliken poin cha ge app oach [18].
Elec os a ic in e ac ions we e calcula ed wi h a 10.48 Å cu -o , and he long- ange elec os a ic
in e ac ions we e handled by he pa icle mesh Ewald (PME) algo i hm [19] using a six h-o de
B-spline in e pola ion and a g id spacing o 1 Å. The leap og algo i hm was used in all
simula ions wi h a 1.25 s s ep ime o in amolecula o ces and a 2.5 s s ep ime o
in e molecula o ces.
Fi e ypes o molecules (MOL_SET) we e included o bo h memb ane sys ems: GG, MA,
PA, my is ic alcohol (MOH) and palmi ic alcohol (POH). The lipid bilaye s we e assembled and
elaxed, educing he box dimension un il he Van de Waals ene gy o he sys em s a ed o
inc ease, and he s uc u al pa ame e s o he memb anes we e hen compa ed using he
expe imen al da a [20]. To a oid a om-a om bumps and abno mal non-co alen in e ac ions, he
size o he MOL_SET molecules was ini ially educed o 20% o he o iginal size and he non-
co alen in e ac ions o 10% o hei no mal alue [21]. The MOL_SET molecules we e hen
placed a 160 di e en posi ions ac oss he memb ane using a so wa e specially designed o
his pu pose [22]. The size and ene gy cons an s we e hen g adually inc eased un il hey eached
no mal alues h ough cycles o s eepes descen minimiza ion, and a cycle o annealing was
unde aken un il he speed o he as es a om d opped below 500 m/s. Fo each memb ane/lipid
sys em, he minimum in po en ial ene gy was hen hea ed o 310 K and equilib a ion dynamics o
50 ns we e comple ed. The la e al p essu e p o ile was calcula ed as desc ibed [23-25] and he
esul a e exp essed as he a e age o 5 snapsho s o he las 5 ns o simula ions.
F ee ene gy o inse ion was es ima ed by means o me adynamics calcula ions [26] using he
Desmond p og am [27] unde pe iodic bounda y condi ions. Simula ions we e pe o med in an
iso he mal-isoba ic ensemble (1 a m, 310 K) wi h Lange in ba os a and he mos a . The
me adynamics simula ions we e ca ied ou a e equilib a ion. The ee ene gy p o ile G(z)
associa ed wi h lipid molecule ansloca ion h ough he lipid bilaye was calcula ed along he z-
componen o he dis ance ec o joining he memb ane and he lipid molecule cen e o mass (z-
dis ). Fo his s udy, he dis ance be ween one headg oup oxygen o each molecule o MOL_SET
and he cen e o he memb ane was chosen as collec i e a iable. A second collec i e a iable
was he dis ance be ween he e minal ca bon o each molecule o MOL_SET and he cen e o
he memb ane. The ime in e al be ween he addi ion o wo Gaussian unc ions, τ, as well as
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he Gaussian heigh , w, and Gaussian wid h, δ, we e uned o op imize he a io be ween
accu acy and compu a ional cos . We used: τ = 100 s, w = 0.2 kJ/mol, δ = 0.5 Å. The ee
ene gy o he inse ion o lipid molecules in he memb ane was calcula ed as he di e ence
be ween he minimum ene gy o he molecule inse ed in memb ane and he ee ene gy in wa e .
2.5. G p o ein binding o la ge unilamella esicles (LUVs)
LUVs con aining di e en mola a ios o PC:PE we e p epa ed in he p esence o absence o
5 mol% PA, MA o GG. The lipids we e dissol ed in chlo o o m/me hanol (2:1) and mixed a
he app op ia e olumes. The sol en was e apo a ed unde a gon lux and sol en aces we e
emo ed unde acuum o a leas 3 h. Lipid ilms we e hyd a ed in 10 mM HEPES, 100 mM
KCl, 0.1 mM EDTA, pH 7.4 a 42°C o 1 h, wi h igo ous o exing e e y 15 min. The lipid
suspension was submi ed o i e eeze/ haw cycles and was sonica ed in a p obe- ype sonica o
om B anson (Danbu y, CT) o 10 s a 15 W. The LUVs we e hen incuba ed o 1 h a 37°C
wi h pu i ied Gαβγ (300 ng), G dime s (100 ng) o Gi1 monome s (150 ng) in a o al olume
o 200 l. The binding o Gαβγ o memb anes was ca ied ou in he p esence o GDPS (50
M) and ha o he Gi1 monome s in he p esence o GTPS (50 M). Unbound G p o eins
we e hen sepa a ed om memb ane-bound G p o eins by cen i uga ion o 1 h a 25°C a
100,000 × g. Finally, he memb ane pelle s we e esuspended in elec opho esis loading bu e
(84 mM T is-HCl, pH 6.8, 4% SDS, 1% 2-me cap oe hanol, 5% glyce ol, 0.01% b omophenol
blue) and boiled o 5 min. In all expe imen s, my is oyla ed G subuni s and ge anylge anyla ed
G subuni s we e used.
Immunoblo and quan i ica ion o bound G p o eins- Immunoblo ing was pe o med as
desc ibed elsewhe e [28]. B ie ly, samples om he binding expe imen s we e esol ed on 10-
20% g adien SDS-polyac ylamide gels and he p o eins we e hen ans e ed o ni ocellulose
memb anes. The memb anes we e blocked wi h PBS con aining 5% non- a d y milk, 0.5%
bo ine se um albumin and 0.02% Tween-20 (blocking solu ion), and hey we e hen incuba ed
wi h an i-Gi1 (1:1000 dilu ion in esh blocking solu ion) o de ec Gi1 and G, o an i-G
(1:1000) o de ec G. An obody binding was de ec ed wi h a ho se adish pe oxidase-linked
an i-mouse IgG (1:2000) in esh blocking solu ion, which was isualized by ECL. The
immuno eac i e bands on he ilms we e quan i ied by image analysis and he binding o G
p o eins o pu e PC liposomes in he absence o o he lipids was conside ed as he con ol alue
(100%). Fo each a io o PE, he ela i e e ec o PA, MA and GG on G p o ein binding was
8
compa ed o he same memb ane wi hou any lipid moie y and indica ed in pa en heses in Table
1.
2.6. Con ocal mic oscopy
Gian unilamella esicles (GUVs) we e p epa ed using he elec o o ma ion me hod [29, 30].
Fo his pu pose, lipid solu ions con aining 0.3 mM o al lipid supplemen ed wi h 0.4 mol% DiI
we e p epa ed in chlo o o m: me hanol (2:1; : ). Th ee µl o he lipid mix u e we e added o he
su ace o pla inum elec odes and sol en aces we e emo ed unde acuum o 60 minu es.
Pla inum elec odes we e co e ed wi h 400 µl o 25 mM HEPES, pH 7.4, p e iously hea ed a
50ºC. The pla inum wi es we e connec ed o an elec ic wa e gene a o a 50ºC unde he
ollowing AC ield condi ions: 500 Hz, 0.22 V o 5 min; 500 Hz, 1.9 V o 20 min and inally
500 Hz, 5.3 V o 90 min. A e GUV o ma ion, he chambe was placed on a Leica TCS SPE
in e ed con ocal luo escence mic oscope (Ba celona, Spain).
The GVMD so wa e om he Beckman Ins i u e (Uni e si y o Illinois) was used o localize
ee cys eine esidues on he su ace o Gi1, G and Gi in o de o be used o p o ein
labeling (see Fig. S1) [31]. In b ie , ee cys eine esidues we e labeled wi h Alexa 488 C5-
maleimide by mixing 10 µl o 0.8 µg/ml p o ein wi h 0.5 µl Alexa Fluo 488 (10 µg/ml s ock
solu ion) o 10 min a RT. Fluo escen ly-labeled G p o eins we e added o GUVs a a inal
concen a ion o 15 ng/ml. The binding o Gαβγ o memb anes was ca ied ou in he p esence o
GDPS (50 M) and ha o he Gi1 monome s in he p esence o GTPS (50 M). The
exci a ion wa eleng h o DiI was 532 nm and he emission was collec ed a 555-750 nm; he
exci a ion o Alexa Fluo 488 was 488 nm and he emission 500-533 nm. The binding o
luo escen ly labeled G p o ein subuni s o he GUVs was measu ed using he so wa e p o ided
wi h he mic oscope. The luo escence signal so ounding he lipid memb ane was used as
backg ound.
2.7. Da a analysis
The da a shown co espond o mean ± SEM alues om he numbe o expe imen s
indica ed. One-way ANOVA ollowed by a Bon e oni es o wo- ailed - es was used o
s a is ical e alua ion. Di e ences we e conside ed s a is ically signi ican a p< 0.05.
3. RESULTS
3.1. E ec s o PA, MA and GG on memb ane lipid s uc u e
9
In he empe a u e ange s udied (15 - 45ºC), DSC showed a lamella - o-in e ed hexagonal
(HII) phase ansi ion peak o bo ine li e PE a 22.4ºC (Fig. 1A). The p esence o MA and PA
inc eased he lamella - o-in e ed hexagonal phase ansi ion empe a u e (TH) alue o 31.2ºC
and 28.5ºC, espec i ely. By con as , when GG is added no ansi ion is obse ed in he s udied
ange o empe a u es, al hough 31P-NMR expe imen s indica ed ha his lipid dec eased he TH
alue, sugges ing ha i a o ed he occu ence o nonlamella phases (Fig. 1B) [32]. When
assessed by 31P-NMR, PE o ganized in o lamella phases a empe a u es below 20°C; be ween
20ºC and 25ºC, bo h lamella and HII phases co-exis ed, and a highe empe a u es (≥ 30°C), PE
molecules adop ed HII phases. 31P-NMR scans also showed ha MA and PA inc eased he TH,
whe eas GG dec eased i abou 10ºC (Fig. 1B).
The binding ee ene gies o GG, PA, POH, MA and MOH o POPC:POPE and POPC
memb anes we e calcula ed by compu a ional analysis (Fig. 2 and S2). MOH and POH we e
s udied o isola e he e ec o he acyl chain and o compa e hei e ec on lipid memb anes wi h
GG, which sha es he same alcohol headg oup. MA and PA, which a e nega i ely cha ged a pH
7.4, exhibi ed g ea e binding ene gies han GG, POH and MOH, which lack o ne cha ge. On
he o he hand, he binding o GG o POPC:POPE memb anes was ~6 kcal/mol highe han ha
o POPC bilaye s, indica ing a p e e ence o GG moie ies o HII-p one domains. This di e ence
is due o he balance be ween he hyd ophobic ma ch o he isop enyl chain in he memb ane and
he hyd ogen bonds be ween he hyd oxyl g oup and wa e molecules. The di e ences in ee
ene gies o MA binding o POPC and POPC:POPE memb anes we e la ge han hose o GG,
al hough he o me p e e ed lamella (POPC) memb anes. In addi ion, MA showed an e en
highe p opensi y o associa e wi h lamella memb anes han PA. These esul s sugges ha GG
apidly seg ega es o memb ane domains ich in he nonlamella p one phospholipid PE, while
PA and MA p e e lamella -p one memb ane domains. In addi ion, his binding beha io also
con ibu es o explain he memb ane mic odomain p e e ence o he G-con aining G p o eins
(G and G complexes wi h an isop enyl moie y) and G monome s con aining MA and/o
PA.
The p esence o each ype o lipid has an impo an e ec on he lipid memb ane o ganiza ion.
Indeed, POPC memb anes displayed an al e ed s ess p o ile a e he addi ion o any o G
p o ein lipids, MA, PA and GG (Fig. 3A), indica ing ha egions ich in G p o eins may unde go
s uc u al lipid egula ions ha could con ibu e o con ol he localiza ion and ac i i y o ce ain
16
lamella -p one egions, and G and G o nonlamella -p one mic odomains, whe e hese
p o eins can pa icipa e in p oduc i e in e ac ions wi h speci ic signaling e ec o s. Finally, bo h
MA and PA egula e memb ane luidi y, which could modula e he ac i i y o GPCRs and o he
memb ane p o eins [49].
I has been shown ecen ly ha memb ane lipid composi ion and i s s uc u al egula ion
in luences physiological p ocesses such as blood p essu e, pla ele agg ega ion, cell p oli e a ion
and apop osis, as well as unde lying he mechanism o ac ion o ce ain d ugs [50-52]. The
egula o y e ec o memb ane lipid composi ion on he localiza ion and ac i i y o pe iphe al
and in eg al p o eins can be pa ly explained by changes in he lipid bilaye la e al p essu e [53]
o luidi y [54]. T ea men wi h lipids o lipid-in e ac ing molecules can egula e he composi ion
and s uc u e o memb anes, e e sing impo an pa hological al e a ions such as cance ,
hype ension o obesi y [32, 50]. This no el he apeu ic s a egy, called “memb ane-lipid
he apy”, is based on he egula ion o he ac i i y o impo an signaling p o eins by modula ing
he eo ganiza ion o memb ane mic odomains [55] and he subsequen p o ein-lipid in e ac ions
[50, 56]. By con as o he gene al opinion ha in e en ions on memb anes could a ec a la ge
numbe o p ocesses, his app oach has been shown o be highly speci ic [52, 57], u he
demons a ing ha he s uc u e- unc ion ela ionships o memb ane lipids can be inely
egula ed. Thus, he p esen s udy sheds u he ligh on he molecula mechanisms go e ning
pha maceu ical and nu aceu ical he apies a ge ing memb ane lipids.
ACKNOWLEDGMENTS - FUNDING
This s udy was suppo ed by he Spanish Minis e io de Economía y Compe i i idad g an
BIO2010-21132, BIO2013-49006-C2-1-R, RTC-2015-3542-1 and RTC-2015-4094-1,
co inanced by FEDER unds om he EU (“Una mane a de hace Eu opa”), by he Go e n de les
Illes Balea s (G ups compe i ius and Resea ch Excellen G an ) and he Ma a hon Founda ion.
JC and RA we e suppo ed by p edoc o al ellowships om he Minis e io de Ciencia e
Inno ación and om he Minis e io de Educación, Cul u a y Depo e, espec i ely. ST, MI and
DJL hold a To es-Que edo con ac om he Spanish Minis e io de Economía y
Compe i i idad. VL is suppo ed by a pos doc o al con ac om he Asociación Española
Con a el Cánce .

17
18
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22
Table 1 Binding o G p o eins o LUVs in he p esence o absence o PA, MA and GG
Binding o Gi, G and G o LUVs o PC and PE a a ious mola a ios, in he p esence o
absence o 5 mol% PA, MA o GG. The binding o hese G p o eins o PC:PE (10:0, mol:mol)
memb anes in he absence o PA, MA o GG was conside ed 100%. Resul s a e exp essed as
a e age alues ± SD o 3 expe imen s. - es s we e used o de e mine s a is ical signi icance * p
< 0.05, ** p < 0.01, *** p <0.001. The da a in pa en heses a e he pe cen change (posi i e o
nega i e) wi h espec o he co esponding con ol model memb ane (same PC:PE a io),
conside ing he binding o each G p o ein o each memb ane composi ion wi hou PA, MA o
GG as 0% change.
Gi1
PC:PE
Con ol
PA
MA
GG
10:0
100.0± 2.6 (0)
123.6 ± 7.4* (23.6)
109.3 ± 7.1 (9.3)
86.8 ± 7 (-13.2)
8:2
89.5 ± 3.9 (0)
98.4 ± 11.4 (9.9)
64.8 ± 8.1* (-27.6)
74.3 ± 12.8 (-17)
6:4
68.7 ± 2.7 (0)
66.1 ± 9.7 (-3.8)
54.0 ± 7.4* (-21.4)
52.8 ± 7.1* (-23.2)
4:6
36.6 ± 5.5 (0)
41.3 ± 7.5 (12.8)
48.0 ± 6* (31.1)
28.5 ± 6.6 (-22.2)
2:8
22.9 ± 3.2 (0)
23.8 ± 3.6 (3.9)
32.1 ± 3.9* (41.5)
20.1 ± 6.7 (-12.3)
G
PC:PE
Con ol
PA
MA
GG
10:0
100.0± 1.9 (0)
86.0 ± 12.7 (-14.0)
104.5 ± 16.6 (4.5)
125.0 ± 12.2* (25.0)
8:2
134.9 ± 15.8 (0)
164.5 ± 5.5* (21.9)
170.1 ± 12.3* (26.1)
169.6 ± 5.4* (25.7)
6:4
207.9 ± 16.7 (0)
250.3 ± 12.2* (20.4)
210.3 ± 24.5 (1.1)
266.6 ± 5.9** (28.2)
4:6
289.3 ± 24.8 (0)
287.5 ± 47.2 (-0.6)
309.7 ± 51.0 (7.5)
298.4 ± 29.6 (9.1)
2:8
334.7 ± 9.8 (0)
277.3 ± 33.9 (-17.2)
374.5 ± 13.5 (11.9)
339.5 ± 40.3 (1.4)
Gi1
PC:PE
Con ol
PA
MA
GG
10:0
100.0± 3.4 (0)
118.1 ± 18.5 (18.1)
95.2 ± 18.2 (-4.8)
108.8 ± 8.8 (8.8)
8:2
140.1 ± 5.4 (0)
175.3 ± 20.9* (25.1)
141.3 ± 26.8 (0.8)
179.0 ± 6.7* (27.8)
6:4
166.3 ± 14.5 (0)
228.8 ± 28.7** (37.6)
172.2 ± 23.9 (3.5)
288.8 ± 23.0** (73.7)
4:6
225.7 ± 17.9 (0)
233.2 ± 35.7 (3.3)
224.3 ± 30.9 (-0.6)
331.2 ± 32.1* (46.7)
2:8
272.5 ± 13.0 (0)
285.7 ± 30.1 (4.8)
267.7 ± 36.8 (-1.8)
371.1 ± 51.3* (36.2)
23
Table 2 Binding o G p o eins o GUVs assessed by con ocal mic oscopy.
Gi, G and Gi we e luo escen ly labeled wi h Alexa Fluo 488 and he binding o GUVs
composed o PC:PE was de e mined om he amoun o luo escence in he memb ane esicle
(a.u., a bi a y uni s), using he luo escence o su ounding a eas as backg ound. Da a a e
exp essed as a e age alues ± S.D o a leas 5 esicles pe condi ion. - es s we e used o
de e mine s a is ical signi icance * p < 0.05, ** p< 0.01, *** p< 0.001.
PC:PE
Gi1
G
Gi1
10:0
11.18 ± 1.23
0.53 ± 0.23
1.07 ± 0.76
8:2
3.84 ± 0.88***
1.18 ± 0.18
2.94 ± 0.98
6:4
0.41 ± 0.3***
3.6 ± 0.56*
3.35 ± 1.11*
4:6
No binding
6.04 ± 0.66**
7.47 ± 0.37***
2:8
No binding
14.82 ± 1.08***
10.51 ± 0.59***
24
FIGURE LEGENDS
Figu e 1 The e ec o G p o ein lipids on memb ane lipid s uc u e.
(A) DSC he mog ams o bo ine li e PE memb anes in he p esence o absence o 5 mol% PA,
MA o GG. The peaks co espond o he lamella - o-hexagonal phase ansi ion. (B) 31P-NMR o
bo ine li e PE memb anes in he p esence o absence o 5 mol% PA, MA o GG. NMR scans
we e eco ded a he empe a u es indica ed on he le o he panel.
Figu e 2 Binding ene gies o GG, MA and PA o model memb anes.
Binding ene gies o he G p o ein lipids o POPC ( illed ba s) and POPC:POPE (6:4, mol:mol)
(g ey ba s) memb anes. The ba s co espond o he mean binding ene gy alues ± S.D. o h ee
simula ion expe imen s.
Figu e 3 E ec s o GG, MA and PA on he bilaye la e al p essu e.
The igu e shows he la e al p essu e in (A) POPC and (B) POPC:POPE (6:4, mol:mol)
memb anes in he absence (black line) o p esence o GG ( ed line), MA (g een line) and PA
(blue line). The X-axis indica es he dis ance om he cen e o he memb ane.
Figu e 4 E ec s o G p o ein lipids on he binding o G p o eins o lipid bilaye s.
Model memb anes composed o PC:PE (10:0) o PC:PE (6:4; mola a io), in he p esence (g ay
ba s) o absence ( illed ba s) o 5 mol% PA, MA o GG. Ba s co espond o he mean ± SEM o
i e independen expe imen s o he binding o Gαi1, G and G o lipid bilaye s.
Rep esen a i e immunoblo s o each g aph a e shown abo e each his og am. An i-Gi1 was used
o de ec Gi1 and G, o an i-G o de ec G. * indica es s a is ical signi icance (p < 0.05)
compa ed o he same memb ane composi ion in he absence o PA, MA o GG.
Figu e 5 Con ocal images o G p o ein binding o GUVs.
Gαi1, Gβγ and Gαβγ we e luo escen ly labeled wi h Alexa Fluo 488 and incuba ed wi h GUVs
composed o di e en mol a ios o PC:PE (10:0, 8:2, 6:4, 4:6, 2:8). Lipid memb anes we e
s ained wi h DiI and a e shown in yellow, while he luo escen ly labeled G p o eins appea in
ed. Binding o G p o ein subuni s o lipid esicles was assessed by quan i ica ion o he Alexa
488 luo escence signal associa ed o memb anes. The luo escence su ounding he esicle was
used as backg ound. Ba =10 µm.
25
Figu e 1
32
G p o ein-memb ane in e ac ions II: E ec o G P o ein-linked Lipids on Memb ane S uc u e
and G P o ein-memb ane In e ac ions
Jesús Casasa, Mai ane Iba gu ena,b,*, Ra ael Ál a eza, Da id J. Lópeza,b, Sil ia Te ésa,b, Vic o ia
Lladóa, S e ano P. Pio oc, Simona Conciliod, Xa ie Busque sa,b and Pablo V. Esc ibáa,b,*
Highligh s
My is oyl, palmi oyl and isop enyl g oups in luence biophysical p ope ies o memb anes.
My is oyl, palmi oyl and isop enyl moie ies o G p o eins modula e hei binding o
memb anes.
The isop enyl moie ies a o he accumula ion o he e o ime ic G p o eins in
phospha idyle hanolamine-en iched domains.
Changes in he lipid composi ion o G p o eins migh modula e cell signaling.