when recruited from 
 the cytosol to the membrane , many signaling 
 proteins are activated in response to binding lipid activators with 
 their membrane - binding domains . as a paradigmatic class of membrane - binding 
 domains , c1 domains play vital regulatory roles in a large number 
 of signaling proteins such as protein kinase c ( pkc ) , protein kinase 
 d ( pkd ) , diacylglycerol kinase ( dagk ) , and chimerin .
each c1 domain contains about 5 short interlacing strands and a 
 c - terminal helix , which are organized around two integral zn ions ( figure 1a ) . in the sequences of c1 
 domains , a characteristic motif , hx1012cx2cx915cx2cx4hx24cx68c ,
has been identified , where h and c represent 
 the conserved histidine and cysteine residues to coordinate the zn ions , and x can be any amino acid .
it is believed that the hydrophobic half 
 of a c1 domain , comprised primarily of the 12 and 34 
 loops , penetrates into the hydrocarbon core of the membrane , while the hydrophilic half with 
 zn ions and several ionic residues on the surface is 
 exposed to the cytosol ( figure 1b  d ) . despite high sequence homology and structural similarities ,
the c1 domains of different proteins exhibit wide - ranging binding 
 affinities to anionic lipid coactivators like 1,2-sn - phosphatidyl - l - serine ( ps ) , as well as to neutral lipid 
 activators like 1,2-sn - diacylglycerol ( dag ) and phorbol-12-myristate-13-acetate 
 ( pma ) .
in particular , many typical c1 domains of pkcs bind dag or 
 pma preferentially , while others have comparable affinities to both 
 activators . there also exist atypical 
 c1 domains that bind neither dag nor pma .
these differences in binding affinities and preferences are challenging 
 to explain on the basis of the currently available , limited structural 
 data alone . at present
merely 13 distinct c1 domains have been deposited 
 to the protein data bank ( pdb ) , and thus there is no three - dimensional 
 ( 3d ) information for over 90% of the c1 domains with known sequences .
furthermore , only a single structure in complex 
 with any lipid activator has been solved with x - ray crystallography . as a result ,
key structural details of c1 domains , 
 for instance , interactions with anionic lipids and dag as well as 
 the protein orientation and degree of membrane penetration , have not 
 been elucidated fully . given the experimental challenges of studying 
 c1 domains in membrane - bound environments , computer modeling and atomistic simulations 

provide a useful means to explore the detailed interactions and conformations 
 involved in membrane binding and recognition of lipid activators , 
 which will likely enhance our understanding of the c1 domain roles 
 for the function and regulation of signaling proteins .
( a ) superimposed structures 
 of the pkc c1a domain ( a homology 
 model ; red ) , the pkc c1b domain ( pdb code : 2eli ; green ) , and the 
 ligand - bound pkc c1b domain ( pdb code : 1ptr ; gray ) .
the c rmsd is 0.1  between the pkc c1a model 
 and the pkc c1b structure , and 0.7  between the pkc 
 and pkc c1b structures .
surface representations of ( b ) the 
 pkc c1a domain , ( c ) the pkc c1a domain , and ( d ) the 
 ligand - bound pkc c1b domain are also shown . in panels
b , c , 
 and d , basic , acidic , and neutral polar residues are shown in blue , 
 red , and green , respectively .
a combined computational and experimental study is presented 
 in 
 this work with the aim of investigating the interactions between the 
 c1 domains in the pkc alpha isoform ( pkc ) and their lipid partners 
 ( ps , dag , and pma ) . implicated in a large number of human diseases including cancers , cardiovascular diseases , diabetes and complications , as well as bipolar 
 disorder , pkc is widely studied as a model 
 for conventional pkcs ( cpkcs :  , i , ii , and  
 isoforms ) for understanding how c1 domains regulate signaling proteins . similar to other cpkcs , pkc possesses 
 two tandem c1 domains , namely , c1a and 
 c1b , in addition to a c2 targeting domain and a c - terminal kinase 
 domain .
the evidence shows that a mature 
 pkc in its compact inactive state is activated via sequential 
 binding of individual domains to the plasma membrane surface . at first , calcium triggers c2 domain binding to anionic lipids and 
 the entire pkc protein is directed to the membrane .
dissociating 
 from the kinase domain , the inhibitory c1a and c1b domains are then 
 recruited to the membrane to bind lipid coactivators ( such as ps ) 
 and activators ( such as dag and pma ) .
this process results in an activated 
 pkc , which catalyzes the phosphorylation of substrate proteins .
significant recent progress highlights the importance 
 of the two c1 domains in the activation mechanism , and the need for 
 further computational and experimental efforts are required to better 
 understand the membrane interactions of these domains .
a growing body 
 of experimental evidence suggests that the two c1 domains in cpkcs 
 are not equivalent , but the molecular basis for this nonequivalence 
 is only partly understood .
for example , 
 although both c1 domains are thought to interact with membranes , some 
 evidence suggests that only one of the two cpkc c1 domains can bind 
 to a lipid activator .
other results suggest 
 that , in the case of pkc , both domains bind to activators yet with opposite affinities . it is generally believed 
 that the pkc c1a domain has a higher affinity for dag than 
 the c1b domain , while the latter has a higher affinity for pma . regarding mutations of equivalent 
 or ionic residues , more pronounced impacts appeared in the c1a domain 
 than in the c1b domain on pkc membrane binding and activation .
lastly , in the cpkc activation mechanism , recent work indicate that 
 c1a stabilizes the predominant activation intermediate by binding 
 first to the membrane , even in the absence of activating lipid . in this model ,
recruitment of c1b to the membrane 
 by activating lipid is the key step in kinase activation . to further elucidate the roles of the c1a and c1b domains in pkc 
 activation , it is essential to understand their detailed molecular 
 interactions with membranes . therefore , we have combined computational 
 and experimental approaches in this work to characterize the interactions 
 of the pkc c1a and c1b domains with various lipids in different 
 membrane environments . because of the shortage of experimentally 
 determined structures 
 and membrane - docking geometries of the cpkc c1 domains , it is a challenge 
 to employ computer simulations to compare the two c1 domains from 
 the same cpkc in membrane - bound environments .
while most prior docking 
 and simulation studies focused on the c1b domains of pkc and 
 pkc in solution , only the pkc c1b domain has been simulated in a lipid bilayer . to the best of our knowledge ,
a comparative 
 study of both c1a and c1b domains in membranes has never been reported . 

given our recent computational and experimental progress in pkc 
 research , it has now 
 become viable to employ a combined approach to reveal and compare 
 details of the two pkc c1 domains in membranes .
as presented 
 in this work , we have built the atomistic pkc c1a and c1b models 
 in different membrane - bound environments and performed a systematic 
 investigation combining findings from simulations and experiments . 

synergy between simulations and experiments enabled us to access structural 
 and dynamic details of the c1 domain - lipid complexes at various spatial 
 and temporal scales .
our combined study aimed to help understand the 
 atomistic details of the pkc c1 domain - lipid interactions that 
 have not been fully described before , and to provide valuable new 
 insight about the roles of the c1 domains in pkc regulation 
 and activation .
since ps 
 lipid is an essential coactivator for cpkc binding to membranes , we first performed atomistic molecular dynamics 
 ( md ) simulations with an individual c1a or c1b domain in the 3:1 pc : ps 
 membrane , to examine the interactions between the proteins and ps . 
 in both cases , the c rmsds rise over the first 2026 
 ns , and then stabilize between 1.5 and 2.0  for the rest of 
 the simulations , which demonstrates structural stability of our membrane - bound 
 protein models . the major deviation from the reference , the pkc 
 c1b structure in solution ( pdb code : 2eli ) , is the increased separation between 
 the 12 and 34 loop tips , defined as the c distance between q46 and f60 in the c1a domain or s111 and l125 
 in the c1b domain .
the increased loop 
 separation leads to opening of the activator - binding groove , albeit 
 to different extents in these two domains ( see supporting information ( si ) , figure s1a ) .
the c1b domain shows 
 a slight increase in the loop - tip distance from 11.5  to an 
 average value of 12.2  , with a broad distribution from 9 to 
 16  .
the conformations sampled in our simulations agree well 
 with the open - loop conformations ( 12.5  ) that were observed 
 in an earlier simulation study with the same c1b domain . compared to the c1b domain
, the c1a domain 
 appears to possess a wider activator - binding groove , presumably due 
 to the presence of more flexible 12 and 34 loops .
the 
 loop - tip distance is generally 23  longer , compared 
 to the corresponding distance of the c1b domain , with a distribution 
 centered at 15.3  ( figure s1a ( si ) ) .
overall , in the absence of activators , the increased separation 
 of the loop tips as well as the resulting open activator - binding groove 
 in the pkc c1 domains , suggests that interactions with pc : ps 
 membrane stabilizes the open - loop conformations and may facilitate 
 activator interactions .
ps headgroup density maps of the pkc 
 c1a domain ( left panel ) 
 and the c1b domain ( right panel ) .
the blue contours show the surface 
 density of ps lipid head groups in the lower membrane leaflet based 
 on the 300 ns simulations . with a viewpoint from the bottom of the 
 simulation box ,
the protein backbones are shown as ribbons and the 
 c atoms of ionic residues as beads . while association with the pc :
ps membrane induces 
 conformational 
 changes of the c1 domains , the proteins also shape the local ps distribution . 

the lateral distributions of the ps head groups are shown in figure 2 , which reveals the similarities and differences 
 between the c1a and c1b domains in terms of interactions with the 
 ps lipids .
on one hand , given no detectable density in the protein - occupied 
 regions , the ps head groups only associate at the periphery of the 
 c1 domains , remaining excluded from the activator - binding grooves 
 in both cases .
it is likely that , in their roles as coactivators , 
 anionic lipids such as ps hardly interfere with activator binding . 
 on the other hand , as shown by the distinct high - density regions , 
 the ps lipids interact with the two c1 domains at very different sites . 

the pkc c1a domain has a number of basic residues ( i.e. , r42 , 
 k45 , k62 , k76 and r77 ) on one face , corresponding to a semiannular 
 region with a high ps density .
the other face adjacent to the low - density 
 region is made up mainly of several aromatic residues ( i.e. , f49 , 
 f56 , w58 , and f72 ) and an acidic residue d55 .
in contrast , the pkc 
 c1b domain has high ps density only near two basic residues , k105 
 and k141 . most of the membrane region around the c1b domain shows 
 a much lower ps density than observed around the c1a domain periphery . 

this phenomenon can not be attributed simply to fewer basic and more 
 acidic residues in the pkc c1b domain than in the c1a domain . 

our further analysis of ionic residues reveals a stable salt - bridge 
 network in the c1b domain , which leads to the following explanation : 
 two sets of charged residues that are consistently interacting , k103-d133-k105 
 and k131-d136 , form a salt - bridge network in the c1b domain ( figure 3 ) .
such a salt - bridge network ( i ) reduces the number 
 of free basic residues and ( ii ) weakens the protein - ps contacts , so 
 that the c1b domain is less able to attract ps lipids .
by contrast , 
 a similar network is not observed in the case of the c1a domain : there 
 are varying electrostatic contacts between residues k76-e80-r77 , but 
 the basic residues are still allowed to bind ps lipids ( figures 3 and 4 ) .
therefore , our simulations 
 suggest that distinct electrostatic detailed interactions are responsible 
 for the observed different association of the pkc c1a and c1b 
 domains with ps lipids . ( a ) time evolution of intramolecular salt bridges 
 in the 300 ns 
 simulations of the pkc c1a ( top panel ) and c1b domains ( bottom 
 panel ) in the pc : ps membrane .
the raw and smoothed data are illustrated 
 as thin and thick lines , respectively .
( b ) structural representations 
 of the salt - bridge network at 290 ns of the apo c1a simulation ( pink 
 backbone ) and at 293 ns of the apo c1b simulation ( green backbone ) . 
 zn ions are shown as silver spheres . to illustrate the 
 membrane insertion ,
a shadow box is used to indicate the membrane 
 as a guide for the eyes .
the ligand binding sites are open in both 
 conformations . even though transiently 
 in our simulations the pkc c1a domain 
 can bind as many as 45 ps lipids while the pkc c1b 
 domain can bind as many as 23 ps lipids ( figure 4 ) , the ps binding stoichiometry ( time averaged ) is only 1.2 
  1.0 and 0.3  0.6 , respectively .
the large uncertainties 
 of those values highlight the dynamic nature of the c1 domain - ps association .
each line in the eventplot represents the occurrence of the 
 basic residue with a bound ps lipid .
subsequent to these md studies , further evidence 
 consistent with 
 the higher positive charge and ps stoichiometry of the c1a domain 
 relative to the c1b domain has been obtained experimentally by single 
 molecule tirf microscopy ( sm - tirfm ) studies .
illustrated in figure 5 , these experiments have revealed that the membrane 
 binding affinity of the c1a domain is much more sensitive to the ps 
 density than the c1b domain . as expected
, both proteins bind bilayers 
 more efficiently ( relative to pure pc bilayers ) when ps lipids are 
 present .
interestingly , our sm - tirfm experiments found that the c1a 
 domain has significantly higher affinity for ps - containing membranes 
 than the c1b domain , a finding that follows the trend of their relative 
 ps binding stoichiometry suggested by our md simulations .
dependence 
 of c1a and c1b bilayer binding on phosphatidylserine 
 ( ps ) density .
c1a or c1b concentration 
 was fixed at 5 pm and added to supported phosphatidylcholine ( pc ) 
 bilayers containing ( a ) increasing amounts of ps lipid or ( b ) increasing 
 amounts of ps lipid where total anionic lipid was kept at 40% using 
 phosphatidylglycerol ( pg ) lipid . after a brief incubation to ensure 
 steady state binding , the density of fluorescent , membrane bound proteins 
 was measured for 5 temporally isolated frames from three separate 
 movie streams in three separate titration experiments . in order to 
 remove purely electrostatic binding due to pg lipid , the additional 
 binding signal measured from 0 to 40% ps lipid was removed . the resulting 
 binding is thus due to specific protein interactions with ps since 
 nonspecific electrostatic recruitment has been removed .
closer examination of the ps binding events in 
 our simulations 
 shows that none of the identified interacting sites were persistently 
 bound to ps ( figure 4 ) .
for example , each one 
 of the residues r42 , k45 , k62 , and k76 in the pkc c1a domain 
 was found associated with ps lipids for 2630% of the time 
 and with r77 for 17% of the time , while k105 and k141 in the c1b domain 
 showed association with ps lipids for only about 10 and 18% of the 
 time , respectively .
these results indicate that the c1 domain - ps association 
 is not saturated , and thus is not high affinity .
however , as depicted 
 by the snapshots from our simulations ( figure 4 ) , at least some of the ps binding sites provide multiple headgroup 
 contacts .
given the basic residues in the pkc c1 domains , the 
 ps enantiomer ( 1,2-sn - phosphatidyl - l - serine ) 
 appears to be highly suitable to bind more than one of the basic residues , 
 presumably due to the two separated negatively charged centers as 
 well as the stereochemistry .
we estimate that 8090% of the 
 c1-ps multivalent contacts in our simulations are better enabled by 
 the physiological enantiomer 1,2-sn - phosphatidyl - l - serine than by the nonphysiological enantiomer 2,3-sn - phosphatidyl - d - serine .
this is consistent with 
 the findings of newton and co - workers , which show that the nonphysiological 
 enantiomer 2,3-sn - phosphatidyl - d - serine 
 exhibits significantly lower affinity for pkcs .
in addition , our simulations also provide structural evidence 
 to support earlier experiments which discovered that 
 cpkc has higher affinity for ps than for other lipids ( like pc , pa , 
 and pe ) .
thus , although there is no high affinity ps - binding site 
 observed in the pkc c1 domains , the multivalent c1-ps interactions 
 can explain the observed stereospecificity of c1 for the ps enantiomer .
taken together , the findings of our md simulations and sm - tirfm 
 experiments suggest a simple mechanism for the positive cooperativity 
 observed in experimental c1 domain binding measurements when the bilayer 
 ps density is varied and a fixed bilayer negative charge is maintained 
 ( figure 5 ) . in this model ,
the c1 domain first 
 associates with the bilayer by interacting with a single ps molecule , 
 then subsequently interacts with one or more additional ps molecules . 

in such a system , the initial ps association will increase the affinities 
 of subsequent ps binding events , leading to positive cooperativity . to explore the detailed c1 domain - activator interactions , we simulated 
 the activator - bound c1 complexes in the pc : ps membrane with additional 
 unbound activators . in the absence of structural information for ligand - bound 
 pkc c1 domains , we tested different activator - bound poses and 
 obtained only one pose for each complex that is stable for the entire 
 300 ns membrane - bound simulation , with the protein c rmsd mainly 
 fluctuating between 1.0 to 2.6  .
compared to the activator - free 
 cases , dag binding induced 1.8 and 1.6  decreases on average 
 in the loop - tip distance for c1a and c1b respectively , while pma binding 
 induced 3.0 and 0.5  decreases , respectively , with all ligand - bound 
 states yielding much narrower distributions ( see figure s1 ( si ) ) .
these observations suggest that activator 
 binding leads to closing of the groove and a higher degree of protein 
 rigidity .
( a1 ) structural representations of superimposed dag - bound c1a and 
 c1b conformations .
cartoon illustrations of ( a2,3 ) 
 hydrogen bonds between dag and the c1a domain , and ( a4,5 ) hydrogen 
 bonds between dag and the c1b domain .
( b1 ) structural representations 
 of superimposed pma - bound c1a and c1b conformations .
( c1,2 ) illustration 
 of the hydrogen bonds between the activators and the proteins .
the association of ps with the pkc c1 domains is not 
 altered 
 significantly in our simulations by the presence of lipid activators . 

it is estimated that the average number of bound ps lipids for the 
 c1a and c1b domains are 1.0  1.0 and 0.2  0.4 with dag 
 and 0.9  1.0 and 0.4  0.6 with pma , suggesting that the 
 interaction of the c1 domains with ps is still highly dynamic .
major 
 sites of ps interaction are identical to the activator - free cases : 
 r42 , k45 , k62 , k76 and r77 in the c1a domain and k105 and k141 in 
 the c1b domain . these results again support the notion that binding 
 of the c1 domains to lipid activators has little direct impact on 
 their association with ps lipids . in order to further examine 
 why the pkc c1a and c1b domains 
 might have opposite affinities to bind dag and pma ,
surprisingly , a highly conserved hydrogen - bonding 
 pattern is observed , although this pattern does not appear sufficient 
 to account for the different activating lipid preferences of the two 
 domains . the backbone of conserved residues , including t48 , i57 , and 
 g59 of the c1a domain and t113 , l122 , and g124 of the c1b domain , 
 form consistent hydrogen bonds with dag and pma ( figure 6 ) .
the 1-hydroxyl of dag or the 20-hydroxyl of pma , serving 
 as both a donor and an acceptor of hydrogen bonds , connects backbone 
 atoms of i57 and t48 in the c1a domain or l122 and t113 in the c1b 
 domain ( figure 6c1,c2 ) . the i57/l22 backbone 
 nh donates a hydrogen bond to the t48/t113 backbone carbonyl , so that 
 the 12 and 34 loops are bridged ( figure 6c1,c2 ) .
this hydrogen - bonding triangle likely determines the 
 activator orientation in the binding groove .
additionally dag and 
 pma also form hydrogen bonds with backbone atoms of g59 and g124 to 
 further anchor the complex conformations . in the absence of water
, 
 this hydrogen - bonding network plays a key role for activators to access 
 the binding groove , as well as to stabilize the c1 domains in their 
 membrane - bound states .
it is noteworthy that the raf-1 c1 domain , an atypical c1 domain lacking binding to dag , 
 is incapable of forming these hydrogen bonds due to a much shorter 
 34 loop ( see figure s2 ( si ) ) , which 
 highlights the importance of these specific hydrogen bonds . while the hydrogen - bonding network involving the protein backbone 
 seems to be conserved among the pkc c1 and the pkc c1b 
 domains , the side - chain polar contacts 
 appear as an obvious difference between dag and pma . in our simulations , 

the side chains of q63 of the c1a domain and q128 of the c1b domains 
 are hydrogen bonded to not only the backbone of both 12 and 
 34 loops but also with dag ( figure 6a3,a5 ) .
no such contact is observed , however , between q63/q128 and 
 pma , since in this case the glutamine side chain is less stretched 
 and can not reach to provide the contacts .
our results show further , 
 general evidence to support an earlier finding regarding q128 of the 
 pkc c1b domain , which suggests 
 that this conserved glutamine residue plays a key role to modulate 
 the shape and the contacts of the activator - binding groove . while the conserved hydrogen - bonding network still does not explain 
 the opposite activator lipid preference of the pkc c1a and 
 c1b domains , our analysis of nonpolar contacts does provide a plausible 
 explanation of this disparity . in figure 6d , 
 the number of nonpolar contacts between the activators and the c1 
 domains are plotted against increasing cutoffs in the definition of 
 the contact distances .
dag always has more nonpolar contacts with 
 the c1a domain than with the c1b domain , regardless of the cutoffs 
 employed .
the opposite behavior is observed for pma , which always 
 has more nonpolar contacts with the c1b domain .
when changing from 
 dag to pma , the number of nonpolar contacts decreases for c1a but 
 increases for c1b .
estimates of the pisa interfacial energies show that the c1a - dag complex is more stable 
 than the c1a - pma complex by 2.1 kcal / mol , while the c1b - pma complex 
 more stable than the c1b - dag complex only by 0.8 kcal / mol .
these results 
 therefore provide semiqualitative evidence for the binding preferences 
 of c1 domains . moreover , our simulations 
 are also able to identify the most relevant residues ( with over 2 
 activator contacts on average ) , which include f43 , p47 , w58 , f60 , 
 and l63 in the c1a domain when bound to dag , and p112 , y123 , l125 , 
 q128 in the c1b domain when bound to pma .
these findings are in agreement 
 with prior mutagenesis experiments , which 
 showed that some of these residues ( in particular w58 and f60 ) are 
 essential for dag activator binding .
in addition to dag / pma 
 interactions , we also monitor the unbound 
 ligands in each simulation ( see figure s3 ( si ) ) .
even though the unbound dag or pma molecules are close enough 
 to the c1 domain in our starting conformations , they do not dwell 
 near the proteins . at the end of the 300 ns simulations , all unbound 
 dag and pma ligands move away from the protein , exhibiting a separation 
 of over 10  from the closest protein heavy atom . in line with 
 previous experimental observations , our 
 simulations
does not show a secondary site in the c1 domains for dag 
 or pma binding , suggesting a binding stoichiometry of 1:1 for activators 
 binding to each pkc c1 domain .
we 
 did not observe obvious tilting of the membrane - bound pkc c1 
 domains in simulations , as demonstrated by the small values and narrow 
 distributions of the angles between the longest protein principal 
 axis and the z - axis .
these angles are 17  8 
 for the c1a domain and 16  8 degrees for the c1b domain in the 
 pc : ps membrane , while they reach values of 16  8 for the c1a 
 domain and 12  7 degrees for the c1b domain in pc : ps+dag membrane 
 as well as 14  8 ( c1a ) and 13  9 ( c1b ) degrees in pc : ps+pma . 

these results show a consistent orientation of the pkc c1 domains 
 inserted in membranes and confirm once more the overall stability 
 of the models employed in our simulations .
furthermore , we examined 
 the membrane penetration of the pkc c1 domains . our simulations 
 show that these two domains are not fixed in the membranes . defined 
 as the distance from the domain center of mass to the n - plane of dopc , 
 the membrane insertion depth of the c1a and c1b domains can vary continuously 
 from the deep state ( 2  ) to the shallow state 
 ( 10  ) , with the distributions shown in figure s4 ( si ) . in the deep state the hydrophobic half of 
 the c1 domains
is embedded in the membrane , while in the shallow state 
 the tips of the loops barely touch the membrane .
these results confirm , 
 at the molecular level , the two states of the membrane - bound pkc 
 c1 domains identified in our earlier experiments , although the md simulations may not be long enough to fully 
 define the ratio of time spent in the deep and shallow states .
the 
 average membrane insertion depth is 3.94.0  for all 
 our constructs except for the pma - bound c1b domain , which has a mean 
 value of 4.4  ( see figure s4 ( si ) ) .
it is suggested that dag or pma binding does not generally modify 
 membrane penetration for the c1 domains except that pma induces the 
 c1b domain to move to more shallow positions .
in addition to 
 the orientation and membrane penetration , we have 
 also measured protein diffusion coefficients via simulations as well 
 as by experiments . while such calculations in membranes are very difficult 
 to converge and their results should be viewed with caution , we note 
 that our atomistic simulations compare reasonably well to earlier 
 coarse - grained ones of membrane proteins in free - standing lipid bilayers . although the absolute diffusion coefficients from our atomistic 
 simulations are about 1 order of magnitude larger than the experimental 
 values , we achieve qualitative agreement between simulations and experiments 
 ( table 1 ) . in spite of the different membrane 
 constructs ( simulations with free - standing bilayers versus experiments 
 with supported bilayers ) , both simulations and experiments agree that 
 the pkc c1 domains have very similar diffusion coefficients 
 ( dl ) in membranes with and without activators , 
 except that the pma - bound c1b domain has a much higher dl than other assemblies . as suggested in prior work
it is likely that the shallow insertion depths 
 of the pma - bound c1b domain observed in our simulations is related 
 to the increased diffusion coefficient measured in both simulations 
 and experiments .
a unique finding from simulations for the pma - c1b 
 complex is that its membrane insertion depth becomes shallower by 
 about 2.8  from 150 to 300 ns .
in fact , the fraction of the 
 conformations with insertion depth shallower than 4.4  increases 
 for 20% every 50 ns only for the pma - bound c1b domain , while 
 no obvious increase is observed for other constructs . additionally , 
 unique to the pma - bound c1b domain , the activator tail - to - tail contacts 
 to the pc or ps lipids decrease for 50% from 200 to 300 ns 
 in our long simulation .
we therefore hypothesize that the joint effect 
 of weak interactions with the ps head groups and reduced contacts 
 to the membrane hydrophobic core results in a higher occurrence of 
 the shallow state , which could be responsible for the comparatively 
 fast diffusion of the pma - bound c1b domain .
the relatively larger 
 effect of pma on the experimental diffusion coefficient of c1b may 
 arise from the longer time scale and spatial dimension of the diffusion 
 measurement .
error bars of 
 simulation data 
 were calculated from 2 trajectories of the same construct .
error bars 
 of experimental data were calculated from 5 experiments of the same 
 construct , each containing at least 300 trajectories .
connecting the evidence from our systematic combined 
 study , we propose the possible detailed membrane - binding mechanism 
 of the pkc c1 domains .
compared to the pkc c2 domain , 
 which has been studied in our previous work , we found that the interactions of ps with c1 are weaker than with 
 c2 . in line with the previously proposed activation mechanism , the c2 domain rather than
for the subsequent 
 activation steps , although the solvent exposure of these two c1 domains 
 in the full - length pkc might differ , our study supports the notion 
 that the c1a domain is recruited to the membrane before the c1b domain 
 due to its much stronger interactions with anionic ps lipids .
once 
 it is bound to the membrane , the c1a or c1b domain likely undergoes 
 conformational changes to open the activator - binding groove , while 
 at the same time the entire domain fluctuates between the shallow 
 and deep states of membrane insertion .
deep membrane insertion enhances 
 the stability of the open groove conformations , which may relate to 
 the searching mechanism for activators .
when a pkc c1 domain 
 binds an activator , the activator - binding groove likely becomes closed , 
 and the entire domain turns more rigid .
the conserved hydrogen bonds 
 are important for activator recognition and binding orientation in 
 the membrane - bound environment .
however , it is the nonpolar contacts 
 between the c1 domains and the activators that lead to the opposite 
 activator - binding preference .
finally , pma binding appears to favor 
 the shallow binding state of the c1b domain , as observed in the md 
 simulations and suggested by experimental diffusion coefficients . 

the findings of strong nonpolar contacts between pkc c1b and 
 pma , reduced contacts between the membrane and the c1b - pma complex , 
 and abnormally fast diffusion of the pma - bound c1b domain may be relevant 
 to the molecular mechanism of tumor promotion induced by pma and other 
 phorbol esters .
we have combined modeling , 
 simulations , and experiments to study 
 the c1a and c1b domains of pkc in membranes , a difficult task 
 with a single approach alone .
our findings of previous and current work suggest the 
 following detailed mechanism involving the c1a and c1b domains during 
 pkc activation : after the entire pkc is associated with 
 the membrane , both c1 domains can bind to the membrane during activation . 

the c1a domain is recruited first with strong interactions to lipid 
 coactivator ps and activator dag , and the c1b domain is recruited 
 later with a preference to bind activator pma .
the two pkc 
 c1 domains are encoded in their sequences to play different roles , 
 via the distinct surface electrostatic contacts with coactivors as 
 well as nonpolar contacts with activators .
our study has provided 
 evidence to support the notion that c1b binding to the membrane by 
 activating lipids could likely be the key step in the pkc activation 
 model . in addition
, corroborating evidence is obtained from 
 simulations 
 and experiments in terms of lipid binding and protein diffusion .
simulations 
 and experiments complement each other and enable us to connect evidence 
 in multiple spatial and temporal scales of the c1 domains interacting 
 with membranes
. our combined approach will be useful in exploring 
 the roles of the c1 domains in many signaling proteins , even in the 
 absence of detailed structural information , and help to further understand 
 their molecular mechanisms in normal cellular function and disease 
 development . given the approach of atomistic md simulations used here , 
 it is important to be aware of the difference between our simulations 
 and experiments in time and length scales .
future efforts will be 
 to develop accurate coarse - grained lipid and protein models to better 
 explain and predict protein dynamics on experimental time scales . 
 in order to gain further information on pkc activation , we 
 are also simulating a full - length model with the knowledge gained 
 from the individual domains in our pkc studies as well as from this work .
we have modeled and 
 simulated the individual c1a and c1b domains in membranes and buffers 
 that mimic the experimental conditions . in many experiments ,
an engineered 
 c1 domain is fused with a 300-residue maltose - binding protein 
 ( mbp ) , which serves to enhance the solubility of the c1 domains . as shown in the supporting 
 information
, we confirmed that both the mbp and the peptide 
 linkers have negligible impact on the interactions between the c1 
 domains and membranes ( see figure s5 ( si ) ) .
it is , therefore , reasonable to only model individual c1 domains 
 in order to understand the detailed protein  lipid interactions . at the beginning ,
the 3:1 pc : ps symmetric bilayer model , containing 120 
 dopc and 40 dops lipids , was set up with the charmm - gui membrane builder and pre - equilibrated in a water box ( containing 
 150 mm nacl solution ) for 20 ns using the charmm36 force field and 
 the desmond 3.0 simulation package .
the 
 membrane model was then aligned to the x  y plane , perpendicular to the z - axis . the 
 pkc c1b model ( residue 102151 )
was based on the coordinates 
 of the solution structure from residue 1867 ( pdb code : 2eli ) .
the pkc 
 c1a model ( residue 3786 , figure 1 ) 
 was generated by the homology - modeling server swiss - model , with 42% sequence identity to the template ( pdb 
 code : 1ptr ) . 
 to examine
the protonation states of the ionic residues , the protein 
 preparation wizard implemented in maestro ( version 9.3 , schrdinger , 
 llc , 2012 ) was used .
each zn ion was ligated by three 
 cysteine residues in the thiolate form and one histidine residue singly 
 protonated on the -nitrogen atom in our starting protein conformations .
the pkc c1a and c1b models were then combined with the pc : ps 
 membrane .
each c1a or c1b structure was aligned , rotated , and moved 
 below the membrane model , so that the long axes of the protein was 
 almost parallel to the z - axis and the tip of the 
 12 and 34 loops could point toward the lower leaflets 
 of the membranes . as suggested by preliminary experimental data , the protein center was set 6  below the n - plane of dopc in 
 the lower leaflet to insert the protein model into the membrane model . 

almost half of the c1a / c1b domain was in the lower leaflet , but did 
 not reach the upper leaflet . with system builder in maestro , a water 
 box containing 140 mm nacl and 10 mm kcl
was created in each construct , 
 whose boundary was at least 15  from the closest protein or 
 lipid atoms in the z direction . since trapping water between a c1 
 domain and the membrane is generally unfavorable , water molecules were excluded near the protein  membrane 
 interface .
dag and pma molecules were inserted to the membrane , 
 in order to 
 model the c1 domains in the presence of activators .
the pma - bound 
 models were built according to alignment of the ligand - bound pkc 
 c1b structure ( pdb code : 1ptr ) to the protein structure in the above - mentioned models , 
 followed by modification of the phorbol ester ligand to pma . since 
 dag has been found to compete with pma for the same binding site , we aligned selected oxygen atoms of dag to the 
 ones of the phorbol compound in the crystal structure , and thus several 
 dag - bound c1a and c1b constructs were built and tested in our search 
 for stable complexes .
in addition to the bound activator , two dag 
 or pma molecules were inserted adjacent to the proteins to examine 
 any secondary binding or interacting sites . in short , three 
 membrane compositions ( pc : ps , pc : ps+dag , and
pc : ps+pma ) 
 for the pkc c1a and c1b domains were used to build 6 constructs . 

each construct contains about 52  000 atoms in a 85 
   85   85  box with periodic boundary 
 conditions .
we applied 
 the tool viparr 
 in desmond to assign all - atom force field parameters .
the protein 
 parameters were obtained from the charmm27 cmap force field , except 
 that the thiolate parameters were adopted from a prior report .
the charmm36 parameters were used for dopc and 
 dops , and the charmm general force field for dag and pma .
after parameter assignment , the starting models were minimized 
 to remove steric clashes and relaxed with the standard protocol in 
 the maestro - desmond package .
we used a script in the package with 
 the m - shake algorithm to constrain the bond length of all bonds involving 
 hydrogen atoms , as well as the angle in all water molecules .
our production 
 simulations were conducted with desmond 3.0 with a 2 fs time step . 

the bonded and near interactions were updated every step , while the 
 far interactions were updated every three steps .
these semi - isotopic 
 simulations were performed at constant temperature ( 296 k ) and constant 
 pressure ( 1 bar ) . the nos 
the long - range coulombic interactions 
 were treated with the smooth particle mesh ewald method .
two replica 
 simulations were run for each construct : a short one for 150 ns and 
 a long one for 300 ns .
consistence of our short and long simulations 
 suggests that our simulation time scale is sufficient to remove starting 
 conformation bias .
conformational 
 analyses were 
 performed with vmd and pymol ( schrdinger , 
 llc ) . in this work , root - mean - square deviations ( rmsds ) of protein 
 conformations
were computed on c atom pairs with 
 alignment to the reference pkc c1b structure ( residue 1867 , 
 pdb code : 2eli ) .
a salt bridge or a charged contact is defined when two polar atoms 
 with opposite charges are within 4.0  .
bound lipids are defined 
 as those which have heavy atoms within 4.0  of the closest protein 
 heavy atom with the opposite charge .
a hydrophobic contact is defined 
 when two nonpolar atoms ( partial charge < 0.3 unit ) are within the 
 cutoff distance .
the protein orientation in the membrane is measured 
 as the angle between the protein s longest principal axis and 
 the z - axis .
the peripheral diffusion coefficient 
 ( dl ) of the pkc c1 domains was 
 calculated from the mean - square displacement ( msd ) over time according 
 to eq 1:1where r is the center of mass 
 vector of the c1a or c1b domain .
the overall drift of the lower 
 leaflet was removed from the protein diffusion , and only the second half of each trajectory was used during 
 our msd calculation .
the angle between the longest 
 protein principal axis and the z - axis was measured 
 to examine the protein orientation in 
 membranes . to quantify membrane penetration , the membrane insertion 
 depth of the pkc c1 domains
was defined as the distance from 
 the protein s center of mass to the closest nitrogen plane 
 of the dopc lipids .
reagents and experimental 
 protocols are 
 consistent with previously described work and will be described briefly .
bacterial 
 expression constructs of human pkc c1a and c1b regulatory domains 
 were constructed by inserting dna sequences encoding c1a domain ( residues 
 26100 ) and c1b domain ( 90165 ) into a pmal - c2 g expression 
 vector . for each protein ,
primers were designed to incorporate an 
 n - terminal 11-amino acid recognition sequence for sfp phospho - pantethienyl - transferase 
 to enable sequence - specific enzymatic labeling with a coa - linked fluorophore .
the c1a and c1b domains were expressed in escherichia coli rosetta 2(de3 ) cells ( novagen ) .
overnight expression at 20 c 
 was followed by purification on amylose resin ( neb ) and eluted with 
 excess maltose .
the n - terminal sfp labeling tag was covalently modified with 
 an alexa fluor 555-coa by the sfp enzyme , and excess fluorophore was 
 removed using vivaspin concentrators ( sartorius stedim , gttingen , 
 germany ) . to generate supported bilayers , sonicated unilammelar 
 vesicles 
 ( suvs ) comprised of synthetic dioleolyl phospholipids pc ( phosphatidylcholine ; 
 1,2-dioleoyl - sn - glycero-3-phosphocholine ) , ps ( phosphatidylserine ; 
 1,2-dioleoyl - sn - glycero-3-phospho - l - serine ) , 
 dag ( diacylglycerol ; 1,2-dioleoyl - sn - glycerol ) , pg 
 ( 1,2-dioleoyl - sn - glycero-3-phospho-(1-rac - glycerol ) ) [ all from avanti polar lipids ( alabaster , 
 al ) ] and pma ( phorbol-12-myristate-13-acetate ) [ from sigma - aldrich 
 ( st .
tirf microscopy measurements were carried out at 22 c  
 0.5 c on a home - built , objective - based instrument as previously 
 described .
supported bilayers were imaged before and after the addition of 
 physiological buffer ( 140 mm kcl , 15 mm nacl , 0.5 mm mgcl , 26 m 
 cacl2 , 20 m egta , 5 mm reduced l - glutathione , 
 25 mm hepes , ph 7.4 ) and a blocking step with bsa in order to account 
 for fluorescent contaminants , which typically were few . after a 5 
 min incubation with protein , samples were bleached at high laser powers 
 to minimize contributions from immobile fluorescent particles followed 
 by a 60 s recovery . for each sample
, multiple movie streams were acquired 
 at a frame rate of 20 frames / s and a spatial resolution of 4.2 pixels/m . 

particle tracking analysis and fitting were carried out using imagej , 
 graphpad prism 5 and mathematica .
 protein kinase c- ( pkc ) 
 has been studied widely as 
 a paradigm for conventional pkcs , with two c1 domains ( c1a and c1b ) 
 being important for the regulation and function of the kinase . 
 however , 
 it is challenging to explore these domains in membrane - bound environments 
 with either simulations or experiments alone . in this work , 
 we have 
 combined modeling , simulations , and experiments to understand the 
 molecular basis of the pkc c1a and c1b domain interactions 
 with membranes . 
 our atomistic simulations of the pkc c1 domains 
 reveal the dynamic interactions of the proteins with anionic lipids , 
 as well as the conserved hydrogen bonds and the distinct nonpolar 
 contacts formed with lipid activators . 
 corroborating evidence is obtained 
 from additional simulations and experiments in terms of lipid binding 
 and protein diffusion . 
 overall , our study , for the first time , explains 
 with atomistic detail how the pkc c1a and c1b domains interact 
 differently with various lipids . on the molecular level , the information 
 provided by our study helps to shed light on pkc regulation 
 and activation mechanism . 
 the combined computational / experimental 
 approach demonstrated in this work is anticipated to enable further 
 studies to explore the roles of c1 domains in many signaling proteins 
 and to better understand their molecular mechanisms in normal cellular 
 function and disease development . 
Introduction
Results and Discussion
Conclusions
Materials and Methods