cell signaling pathways 
 are largely organized via a specific recruitment 
 of signaling effector proteins to their target membranes and a confined 
 release of calcium ions .
the quintessential example of this is the 
 action of phospholipase c ( plc ) that binds and hydrolyzes phosphatidylinositol 
 4,5-bisphosphate ( pi(4,5)p2 ) in the plasma membrane to 
 diacylglycerol ( dag ) and the water - soluble inositol 1,4,5-trisphosphate 
 ( ip3 ) , the latter inducing the release of ca from the 
 endoplasmic reticulum ( er ) into the cytosol .
another prominent example is synaptotagmin-1 , the main ca sensor of neuronal exocytosis in the presynaptic axon terminal . 

synaptotagmin-1 binding to pi(4,5)p2 directly amplifies 
 protein cooperativity and thus sensitivity to ca by a 
 factor of > 40 .
pi(4,5)p2 is 
 enriched in the inner leaflet of the plasma 
 membrane and constitutes around 1% of the total anionic 
 phospholipid content in cellular membranes . in comparison with other phospholipids
, it contains a rather bulky 
 phosphorylated inositol headgroup with a negative charge ranging from 
 3 e to 5 e , depending 
 on the ph and the presence of proteins or ions .
pi(4,5)p2 and other negatively charged lipids 
 in the cytosolic leaflet are constantly exposed to divalent cations . 
 in resting cells ,
the cytosolic concentration 
 of ca upon cell signaling has been reported to span a 
 wide range from 0.5 m to several hundred m , with a half - life 
 of 500 s to 26 ms .
ca influx primarily originates from internal stores 
 within the endoplasmic / sarcoplasmic reticulum or from specialized 
 channels within the plasma membrane providing an essentially infinite 
 supply of extracellular calcium . in all 
 cases
, ca is delivered as brief transients , forming microdomains 
 at the membrane site of influx , and thus , 
 local concentrations of ca can be expected to exceed 
 cytosolic concentrations by orders of magnitude .
meanwhile , unlike ca , the levels of free , 
 cytosolic mg are maintained within a fairly narrow concentration 
 range of 0.251 mm .
interestingly , calcium 
 but not magnesium ions have been ascribed a strong propensity to promote 
 the formation of pi(4,5)p2 clusters as demonstrated in 
 several studies , primarily by using monolayer techniques .
while the overall effects of divalent cations , including calcium , 
 on pi(4,5)p2 lateral organization have been intensely studied , 
 the mechanism of ca and pi(4,5)p2 interactions 
 at the molecular level remain unclear . experiments with
pure pi(4,5)p2 monolayers have suggested partial dehydration of both ca and pi(4,5)p2 upon interaction with each other , triggering an electron density increase in the 
 pi(4,5)p2 headgroup region as well as acyl chain region 
 thickening .
these studies , however , have typically focused on single pi(4,5)p2 molecules or used simplified 
 coarse - grained models that lack sufficient 
 details to deal with specific chemical features of phosphatidylinositides . herein , we combine protein  lipid binding assays and spectroscopic 
 experiments with atomistic molecular dynamics ( md ) simulations employing 
 refined state - of - the art force fields to unravel the functional and 
 structural consequences of the interplay between ca and 
 pi(4,5)p2 .
our data indicate a hitherto undiscovered role 
 and mechanism for ca in cellular signaling , namely the 
 direct organization of the phosphoinositide headgroup conformation 
 and the selective recognition thereof by the pleckstrin homology ( ph ) 
 domain of plc 1 , the canonical pi(4,5)p2 sensor .
to determine the 
 equilibrium dissociation constants ( kd ) for divalent cation / pi(4,5)p2 interaction , we employed 
 a simple fluorescent assay using a supported lipid bilayer platform containing 5 mol % of pi(4,5)p2 ( for details , see the supporting information ) .
significantly , the kd values differed by less than a factor of 2 , 
 with a kd of 0.6  0.2 mm for ca compared to 1.2  0.2 mm for mg ( figure s1 ) .
we therefore decided to use a cation 
 concentration of 1 mm for all follow - up experiments , matching the 
 free mg concentration in the cytosol . in order to systematically 
 study the effects of ca on pi(4,5)p2 ,
we produced 
 100 nm diameter large unilamellar vesicles ( luvs ) , facilitating the 
 control of membrane lipid composition and properties . for quality 
 control and physicochemical characterization , all preparations were 
 first subjected to thin layer chromatography ( tlc ) , dynamic light 
 scattering ( dls ) , and zeta potential measurements ( figure s2 ) . having the opposite charge of pi(4,5)p2 , it is not surprising that ca and mg equally 
 reduce the zeta potential of popc liposomes containing 5 mol % of 
 pi(4,5)p2 , the former being described previously .
in fact , the presence of either cation attenuates 
 the electrokinetic potential of the membrane down to the level of 
 popc alone ( figure s2c ) . because 
 of its extraordinary stereospecificity , the plc 1-ph domain 
 is widely used as the canonical reporter for cellular pi(4,5)p2 levels at the plasma membrane as well as with in 
 vitro assays .
size - exclusion chromatography 
 and dls confirmed that the purified plc 1-ph domain ( figure s3a , b ) was monomeric in solution , even 
 in the presence of ca and mg ( figure s3c , d ) .
next , we performed liposome flotation 
 assays to follow plc 1-ph binding efficiency to popc / pi(4,5)p2 vesicles .
interestingly , preincubation with 1 mm ca but not 1 mm mg fully inhibited liposome binding ( figure 1a , b ) . moreover , plc 
 1-ph did not bind to pure popc liposomes , highlighting its 
 specificity to pi(4,5)p2 .
( b ) plc 1-ph binding 
 to luvs with popc / pi(4,5p)2 ( 95/5 mol % ) .
( c ) guvs after 
 ecfp - plc 1-ph addition ( green ) and dil as membrane marker ( red ) . 

( d ) the distribution of 
 median ecfp - plc 1-ph intensity per pixel of individual guvs 
 and different sizes of control ( blue ) and after preincubation with 
 1 mm mg ( green ) or ca ( red ) ( data from two 
 additional independent experiments are provided in figure s5 ) .
the median intensity 
 values with mean and standard deviation are depicted in the inset . 
 the mann 
whitney test was used as significance test ( p value < 0.0001 for all cases ) .
circular dichroism ( cd ) spectroscopy excluded a direct effect 
 for 
 cations on the secondary structure of the protein ( figure s3e , f ) .
as such , although ca and mg bind to pi(4,5)p2 with comparable kd values and reduce electrokinetic membrane properties 
 in an equal manner , only ca was capable of inhibiting 
 plc 1-ph binding .
this indicates that pi(4,5)p2 recognition 
 by proteins can not be solely based on electrostatic interactions . because a concentration of 1 mm ca corresponds to 
 twice its kd for pi(4,5p)2 interaction , 
 we performed additional flotation assays with lower ca concentrations . here , a significant reduction in protein binding 

could be observed already at a concentration of 0.6 mm ca ( figure s4 ) . in this context , recent 
 data by milovanovic and colleagues show that ca but not 
 mg promotes syntaxin-1/pi(4,5)p2 domain formation 
 by an underlying mechanism in which ca clusters pi(4,5)p2 and syntaxin-1 independently from each other
. moreover , ca acts as a charge bridge that merges multiple syntaxin-1/pi(4,5)p2 clusters into larger domains . also here , ca was 
 found to be effective at a concentration of 0.5 mm while even 1 mm 
 mg had no effect .
we therefore 
 additionally followed the binding of monomeric ecfp - plc 1-ph 
 fusion protein to giant unilamellar vesicles ( guvs ) ( figure 1c ) . despite limited control 
 over membrane lipid composition at the individual guv level , guvs
provide the most appropriate synthetic 
 approach for flat and freestanding bilayer systems . in this system , 
 the presence of 1 mm ca drastically reduced ecfp - plc 
 1-ph binding ( figure 1d and figure s5 ) , demonstrating 
 the robustness of the observed effect , irrespective of membrane curvature . 

magnesium , however , also reduced ecfp - plc 1-ph domain binding , 
 halfway toward the ca effect . to understand this result , 

it is important to note that liposome flotation experiments with proteins 
 are nonequilibrium assays because much of the protein stays in the 
 bottom of the tube . at the same time ,
by contrast , 
 protein binding in the guv experiment is at equilibrium and binding 
 events are quantified at the individual guv level . to analyze the 
 molecular basis for the cation specificity , vibrational sum frequency 
 spectroscopy ( vsfs )
was employed to study the effects of ca and mg on pure pi(4,5)p2 monolayers at the 
 air / water interface .
the spectra were recorded over frequency ranges 
 corresponding to the headgroup and acyl - chain portions of the lipid 
 molecules and included the adjacent interfacial water structure .
we present vsfs spectra from the inositol ring and phosphate regions 
 of pi(4,5)p2 in the absence and presence of 1 mm ca and mg ( figure 2a , detailed peak assignments in figure s6 and table s1 ) . in the absence of cations in the 
 subphase ,
both the inositol ring vibrations and the phosphate stretches 
 were rather weak ( black data points ) .
this is because of a relatively 
 disordered arrangement of the pi(4,5)p2 headgroups adopted 
 in a pure buffer with a wide range of tilt angles relative to the 
 surface normal . with 1 mm ca , however , the inositol ring 
 signal ( 961 cm and 1012 cm peaks from the c  c and c  o coupled vibrations , respectively ) increased substantially ( red data points ) .
in 
 fact , the resonances showed 2.7- and 3.6-fold increases , respectively , 
 in oscillator strength ( table s1 ) .
these 
 changes reflect both reorientation of the inositol rings and a narrowing 
 of their orientational distribution upon cation binding .
significantly , 
 the changes were not nearly as strong upon the addition of 1 mm mg ( blue data points ) . in that case ,
the oscillator strength 
 of the inositol ring vibrations was increased by only a factor of 
 1.5 and 2.1 , respectively .
such results indicated that ca rigidified the configuration of the pi(4,5)p2 headgroups 
 much more effectively than mg .
vsfs spectra of ( a ) the 
 inositol ring and phosphate regions and 
 ( b ) the carbonyl c = o symmetric stretch region of pi(4,5)p2 on a buffer subphase ( black spectra ) containing 1 mm mgcl2 ( blue spectra ) or 1 mm cacl2 ( red spectrum ) at 
 a surface pressure of 17 mn / m .
the open circles represent vsfs data 
 points , and the solid lines are fits to the data .
spectra of the same data 
 offset along the y - axis are provided in figure s7 .
in addition to the inositol ring 
 modes , the phosphate peaks ( e.g. , sympo3 at 982 cm , sympo2 at 1086 cm , asypo3 at 1115 cm , asypo2 at 1154 cm , detailed assignments 
 in figure s6 and table s1 ) also showed a substantial intensity increase upon the introduction 
 of ca to the subphase .
this indicates a strong net orientation 
 and/or ordering of the headgroup phosphates upon ca binding . 

it should be noted that ca binding may help to deprotonate 
 the second monoesterified phosphate , which 
 would prompt additional changes in the spectra beyond those related 
 to ordering and tilt angle .
moreover , upon the addition of ca , the symmetric po3 stretch 
 exhibited a relatively large 20 cm blue shift , 
 while the asymmetric po3 and po2 stretches shifted by 6 cm and 8 cm , respectively ( table s1 ) .
the shifts of both po3 peaks are consistent with phosphate dehydration upon cation binding 
 and/or a symmetry change of the c3v point group .
the shift of the asymmetric po2 peak also suggests headgroup phosphate 
 dehydration upon ca binding .
the spectral 
 change brought about by 1 mm mg in the 
 phosphate region was much less pronounced overall compared to that 
 with 1 mm ca .
the difference in the interactions of ca and mg with phosphate could be explained at 
 least in part by different dehydration penalties for these two cations . 

it has been suggested that ca binding to phosphate groups 
 is favored because ca is more easily dehydrated than 
 mg .
this difference in the 
 hydration shell chemistry may , in turn , act to disfavor the bridging 
 of the inositol rings of pi(4,5)p2 , which would weaken 
 the ordering effect of mg .
in addition to phosphate 
 and inositol resonances , vsfs spectra 
 were also obtained in the carbonyl c = o symmetric stretch ( 1730 
 cm ) region before 
 and after addition of 1 mm cacl2 or mgcl2 ( figure 2b ) .
in fact , a 1.6-fold increase in the oscillator 
 strength of this peak was observed upon binding of ca , while only a 1.3-fold increase was found for mg ( table s2 ) .
this oscillator strength increase 
 should correspond to a backbone ordering effect , thus helping to reinforce 
 a more rigid configuration of the headgroup inositol rings . ordering 
 of the lipid acyl chains was also observed ( figure s8 and table s3 ) .
taken 
 together , the changes in the vsfs spectra provide strong 
 experimental evidence for distinct conformational changes within the 
 lipid headgroup region in the presence of ca , but less 
 with mg .
such results should be important for the plc 
 1-ph domain selectivity of pi(4,5)p2 found above 
 with liposomes and guvs . with the aim 
 of obtaining mechanistic insights into the effects of ca and
mg on pi(4,5)p2 molecules at a molecular 
 level , we employed atomistic md simulations . in order to reduce methodological 
 bias , we used two all - atom force fields ( opls - aa and charmm36 ) as 
 well as the united - atom force field from berger ( table s4 ) .
importantly , to further account for electronic polarization 
 effects of charged groups in a mean field manner , for ca interacting with pi(4,5)p2 phosphates we also employed 
 the recently developed electronic continuum correction with rescaling 
 ( eccr ) method .
this , to a large extent , 
 dampens the unrealistically high ion pairing found when employing 
 nonpolarizable force fields .
it is particularly 
 useful in the present case where strong electronic polarization can 
 be expected in the vicinity of multiple - charged moieties .
we 
 generated multiple sets of 1 s long trajectories for different 
 initial pi(4,5)p2 distributions prior to and after the 
 addition of ca or mg .
for all simulations , 
 consistently with all force fields used , we find that ca interacts with pi(4,5)p2 and has a pronounced effect 
 on the lipid headgroup orientation ( figure 3 and figures s9 and s13 ) .
moreover , control simulations with mg showed that 
 the effects induced by magnesium are much weaker than those induced 
 by calcium for all simulations ( figure 3c , d and si ) , in full agreement 
 with experiments .
snapshots from md simulations of the lipid bilayer taken 
 at 1 s 
 ( a ) without and ( b ) with ca .
( c ) tilt angle distribution 
 of the pi(4,5)p2 headgroup and ( d ) density profiles of 
 lipid headgroups without ( blue ) and with mg ( green ) or 
 ca ( red ) . numbers in ( c ) represent mean tilt angles for 
 each system . here , only the results of the berger force field simulations 
 are presented .
additional force field simulations with similar outcomes 
 can be found in the supporting information .
the addition of ca or mg immediately 
 leads to a significant reduction of the area per lipid ( figure s10 and table s5 ) .
this macroscopic effect 
 is in agreement with lateral condensation of the pi(4,5)p2-containing monolayers by ca   and our vsfs analysis of the ch stretches ( figure s8 ) . at the microscopic level
, we found 
 that each pi(4,5)p2 molecule binds on average 1.63.1 
 ca molecules , depending on the force field that is employed 
 ( table s5 ) .
this is consistent with the 
 water peak spectral changes , which show that each lipid molecule binds 
 more than two ca ions ( figure s8 ) .
ca binds mostly to the phosphate groups at positions 
 4 and 5 , but it also penetrates deeper into the lipid bilayer to interact 
 with the carbonyl groups ( figure s11 ) . 

ca binding to the lipid carbonyl group is consistent 
 with the vsfs data in the carbonyl stretch region , as documented herein 
 ( figure 2b ) and elsewhere . in agreement with
previously published computational and experimental 
 results , we observed that ca increases 
 the order parameters of the pi(4,5)p2 acyl chains ( figure s12 ) .
the acyl chain ordering is also 
 fully in line with the effects observed in the vsfs spectra ( figure s8 ) .
the most prominent feature 
 observed by simulations is a pronounced 
 headgroup reorientation , primarily caused by the ability of ca to bridge two pi(4,5)p2 headgroups ( figure 3a , b ) .
to quantitatively 
 analyze the headgroup reorientation , we monitored the tilt angle between 
 the c1c4 atoms of the pi(4,5)p2 inositol ring and 
 the bilayer normal .
the average tilt angle in the control simulation 
 without ca was in the range of 3541 , depending 
 on the employed force field .
this result is in agreement with previously 
 published md studies . in the presence of ca ions , however , 
 the average tilt angle significantly increased for all of the force 
 fields up to 65 ( figure 3c and figures s9 and s13 ) .
simulations 
 thus consistently showed bending of the pi(4,5)p2 headgroup 
 toward the plane of the bilayer and away from bulk water ( table s5 ) .
moreover , consistent with a narrowing 
 of the inositol ring s distribution as indicated by vsfs results 
 above ( figure 2a ) , 
 ca slowed pi(4,5)p2 headgroup rotational diffusion 
 as revealed by the rotational correlation function ( figure s9e ) .
the ca effect was also manifested 
 in the density profiles ( figure 3d ) , where the location of the pi(4,5)p2 headgroups 
 shifted in the presence of calcium toward the bilayer center . moreover
, 
 ca significantly decreased the solvent accessible surface 
 area of pi(4,5)p2 , which correlated with a reduced average 
 number of hydrogen bonds between the pi(4,5)p2 headgroups 
 and water molecules ( table s5 ) .
these data 
 also match the experimentally observed partial dehydration of pi(4,5)p2 in the presence of ca as measured here by vsfs 
 and elsewhere .
the charge state 
 of pi(4,5)p2 in lipid membranes is 
 highly sensitive to the cellular ph and the presence of proteins and 
 ions . by using
not only the default parametrization 
 ( charmm36 and opls - aa ) but also the eccr corrected charges for the 
 ions and pi(4,5)p2 phosphate groups ( berger , opls - aa ) , 
 we were able to assess the potential effects of the lipid charge state . 

namely , the charge used for pi(4,5)p2 varied from 3.75 
 to 5 , depending on the particular force field ( for more details , 
 see the si ) .
reassuringly , we found semiquantitatively 
 the same effect of ca on the pi(4,5)p2 tilt 
 angle with ca in all the systems which were tested .
this 
 indicates that under the conditions of these investigations the protonation 
 state of pi(4,5)p2 was not particularly critical for the 
 observed effects .
by means of protein  lipid 
 binding assays and spectroscopic 
 experiments , together with atomistic md simulations , we have unraveled 
 and characterized in molecular detail the pronounced effect of ca on pi(4,5)p2 headgroup presentation .
first , we 
 confirmed the previously observed increase of the pi(4,5)p2 acyl chain order and pi(4,5)p2 cluster formation , as evidenced here by vsfg spectroscopy and md simulations .
second , 
 we characterized at the molecular level the interactions of ca with pi(4,5)p2 headgroup phosphates , as well 
 as the more deeply seated carbonyl groups .
we observed the hitherto 
 unrecognized consequences of ca binding for pi(4,5)p2 at the molecular level .
namely , we observed a dramatic change 
 in the pi(4,5)p2 headgroup tilt angle . by means of liposome 

flotation and guv binding assays , we show that ca has 
 a strong propensity to render the pi(4,5)p2 headgroup invisible 
 to the plc-1 ph domain .
our data lead to the plausible 
 conjecture that the calcium - induced 
 switching of phosphoinositide conformational states may serve as a 
 potential cellular mechanism for lipid recognition and thus play a 
 decisive role in cell signaling and membrane trafficking
. a systematic 
 correlation of kinetics and curvature sensitivities at the nanoscale in vitro(58 ) will be key to understanding 
 the general applicability of our data to other proteins and to different 
 endomembranes .
 the 
 orchestrated recognition of phosphoinositides and concomitant 
 intracellular release of ca2 + is pivotal to almost every 
 aspect of cellular processes , including membrane homeostasis , cell 
 division and growth , vesicle trafficking , as well as secretion . 
 although 
 ca2 + is known to directly impact phosphoinositide clustering , 
 little is known about the molecular basis for this or its significance 
 in cellular signaling . here , we study the direct interaction of ca2 + with phosphatidylinositol 4,5-bisphosphate ( pi(4,5)p2 ) , the main lipid marker of the plasma membrane . 
 electrokinetic 
 potential measurements of pi(4,5)p2 containing liposomes 
 reveal that ca2 + as well as mg2 + reduce the 
 zeta potential of liposomes to nearly background levels of pure phosphatidylcholine 
 membranes . 
 strikingly , lipid recognition by the default pi(4,5)p2 lipid sensor , phospholipase c delta 1 pleckstrin homology 
 domain ( plc 1-ph ) , is completely inhibited in the presence 
 of ca2 + , while mg2 + has no effect with 100 nm 
 liposomes and modest effect with giant unilamellar vesicles . 
 consistent 
 with biochemical data , vibrational sum frequency spectroscopy and 
 atomistic molecular dynamics simulations reveal how ca2 + binding to the pi(4,5)p2 headgroup and carbonyl regions 
 leads to confined lipid headgroup tilting and conformational rearrangements . 
 
 we rationalize these findings by the ability of calcium to block a 
 highly specific interaction between plc 1-ph and pi(4,5)p2 , encoded within the conformational properties of the lipid 
 itself . 
 our studies demonstrate the possibility that switchable phosphoinositide 
 conformational states can serve as lipid recognition and controlled 
 cell signaling mechanisms . 
Introduction
Results
and Discussion
Conclusion