many human diseases , such as type ii diabetes mellitus , alzheimer 's disease , parkinson 's disease , and huntingdon 's disease , are associated with protein aggregation and amyloid formation [ 14 ] . in type
ii diabetes mellitus , the cytotoxicity is most likely related to membrane damage , which leads to attrition of insulin - producing -cells [ 57 ] .
the primary component of islet amyloid and actual fibril - forming molecule is human islet amyloid polypeptide ( hiapp or amylin ) , a 37-residue peptide which is synthesized in pancreatic islet -cells and cosecreted with insulin .
the normal physiological role of hiapp is still unclear , but it is believed to have correlations with gastric emptying , suppression of food intake , and glucose homeostasis [ 810 ] . like other amyloidogenic peptides , it is believed that hiapp forms amyloid deposits via a nucleation - dependent aggregation pathway characterized by a lag phase associated with the formation of a nucleus .
the early intermediates were reported to play important nucleating roles in hiapp fibrillation and nmr experimental studies have shown that these intermediates are large in size [ 1216 ] .
increasing evidence suggests that the prefibrillar intermediates , such as oligomers and protofibrils , are the primary toxic species to trigger pathological processes [ 1214 ] , while mature amyloid fibrils themselves exert only a minimal cytotoxic effect on pancreatic -cells [ 1719 ] .
the intermediate oligomers as well as the fibrillization process can disrupt membrane integrity and thereby cause toxicity [ 2022 ] .
experimental studies reported that monomeric hiapp exhibits predominantly a random coil conformation in aqueous solution , and residues 8~19 of the peptide transiently adopt an -helical structure [ 2325 ] . in the presence of lipid membranes , hiapp initially binds to the membrane in a helical state
electron paramagnetic resonance ( epr ) spectroscopy study showed that the -helical region of hiapp at neutral ph spans residues 9~22 and is oriented parallel to the surface of large unilamellar vesicles containing negatively charged lipids .
an earlier nuclear magnetic resonance ( nmr ) study demonstrated that residues 7~17 and 21~28 adopt helical structure in sodium dodecyl sulfate ( sds ) detergent micelles .
as the concentration of membrane - bound peptides rises , hiapps cooperatively convert from -helical intermediates to -sheet aggregates [ 24 , 2931 ] .
it was reported that the n - terminal 1~19 fragment of hiapp is primarily responsible for membrane interaction , while the amyloidogenic 20~29 fragment is mainly responsible for fibrillar aggregates [ 26 , 32 , 33 ] .
these hiapp aggregates may reconstitute membranes and form amyloid ion channels , which would mediate ion transport and destabilize the cell ionic homeostasis [ 5 , 3436 ] .
nonselective ion channel activity of polymorphic hiapp double channels was reported recently by experimental and md simulation studies [ 37 , 38 ] .
in addition , previous experiments demonstrated that the toxic hiapp and its variants primarily interact with the curved regions of the membrane , and lipids of phosphatidylethanolamine ( pe ) type exhibit intrinsic curvature strain , indicating that membrane curvature may play very important roles in the polymerization of hiapp .
pore - like structures and channel activities are also reported in the studies of cytotoxicity induced by a and prp [ 35 , 41 ] .
lipid composition was suggested to be one of the major factors that influence hiapp aggregation , and the presence of membranes that contain negatively charged lipids , such as phosphatidylglycerol ( pg ) or phosphatidylserine ( ps ) , can significantly accelerate the aggregation process [ 13 , 27 , 29 , 42 , 43 ] . however , the effect of lipid composition on the structures and orientations of hiapp on membrane surface at atomic level are not well understood . on the computational side ,
several studies have investigated the structures of monomeric / oligomeric species of full - length and the fragments of hiapp in aqueous solution [ 4451 ] and in membrane environment [ 5255 ] .
our previous study demonstrated that monomeric hiapp has a preferential orientation on anionic palmitoyloleoyl - phosphatidylglycerol ( popg ) bilayers .
as a first step to understand how lipid composition modulates the aggregation of full - length hiapp , in this study , we investigate the binding orientation and membrane interaction of hiapp at zwitterionic palmitoyloleoyl - phosphatidylcholine ( popc ) bilayer by conducting multiple atomistic md simulations and then compare the results with those obtained at anionic popg bilayers . through the comparison of binding behaviors and lipid interactions of monomeric hiapp at popc and popg bilayers , we try to understand at atomic level the membrane - modulated hiapp aggregation in the membrane environment .
the amino acid sequence of hiapp is kcntatcatqrlanflvhssnnfga ilsstnvgsnty , with the cys2 and cys7 forming a disulfide bond that constrains the first four residues in a disordered hairpin loop . to mimic experimental conditions ,
the n - terminus was charged and the c - terminus was amidated . at neural ph ,
we constructed the zwitterionic membrane using popc lipids because phosphatidylcholine ( pc ) is the most abundant phospholipids in pancreatic islets .
this model membrane consists of 2  64 popc lipids and the initial atomic coordinates were obtained from a previous computational study of a neat popc lipid bilayer by tieleman and bentz .
na and cl ions were added to neutralize the system and provide an additional 0.1  m salt concentration .
numerous experimental studies reported that hiapp adopt predominantly -helical structure when initially bound to the membrane [ 21 , 2327 ] .
consistently , a recent spectroscopic study reported that hiapp transiently sample an -helical structure in solution that becomes fully stabilized when bound to the surface of a membrane containing negatively charged lipids .
as the time scale for protein folding at water / membrane interface is on the order of milliseconds to seconds , it is still out of reach to sample conformational transition from a random coil to a helical structure at physiological temperature .
therefore , we took one of the nmr - derived conformations ( pdb i d : 2kb8 ) solved in sds micelles   as the starting point of our md simulations , as done in previous studies [ 54 , 6163 ] .
it consists of a helix running from residue 5 to 28 and disordered structures for the n - terminal residues 14 and the c - terminal residues 2937 .
the choice of an -helical conformation as a starting structure in our study is an md strategy to speed up the simulation outcome as the time for coil - to - helix transition at water / membrane interface is on the order of milliseconds to seconds .
although the interfacial folding of shorter peptides could be probed by replica - exchange molecular dynamic ( remd ) simulations as we did for hiapp(119 ) in our recent study , it would be too expensive for a peptide of 37 residue with current computational resources .
the helical region of hiapp monomer was initially orientated parallel to the membrane surface with a minimum distance 1.4  nm between the peptide and the popc bilayer .
we chose four different starting orientations of hiapp with respect to popc bilayer surface ( see figure 1 ) so that the peptide was allowed to adjust itself before adsorption to the bilayer surface . in the initial state of s(0 ) , the side chain of residue k1 points toward the membrane surface .
the initial state s(90 ) , s(180 ) , and s(270 ) were generated by rotating the hiapp peptide in s(0 ) by 90 , 180 , and 270 around the axis of the helix , respectively .
all md simulations have been performed in the isothermal - isobaric ( npt ) ensemble using the gromacs 3.3.3 software package .
currently , several force fields are available for protein - lipid system , such as gromos87/berger , opls - aa / berger , amber99sb - ildn / slipids , gromos54a7 , and charmm36 [ 6773 ] . the gromos force field and berger force field have been widely used for proteins and lipids , respectively .
berger force field borrowed the standard parameters of the gromos force field for bonds , valence angles , improper dihedrals , and the dihedral angles in the headgroup region of lipids .
thus , the combination of gromos force field with berger force field is appropriate for peptide - membrane system .
the lipid is described with the berger force field , and the peptide is described with gromos87 force field .
the popc parameters used in this study have the correction on the double bond suggested by bachar et al . .
peptide bonds are constrained by the lincs algorithm   and water geometries are constrained by settle .
the pressure is maintained at 1  bar using a semi - isotropic scheme in which the lateral and perpendicular pressures are coupled separately with a coupling constant of 1.0  ps and a compressibility of 4.5  10  bar .
the temperature of the system is maintained at 310  k with a coupling constant of 0.1  ps , above the gel - liquid crystal phase transition temperature ~270  k of popc and popg lipid bilayers [ 78 , 79 ] .
long - range electrostatic interaction is calculated using the particle mesh ewald ( pme ) method   with a real space cutoff of 1.2  nm , as recommended for membrane simulations , especially for those involving charged lipids .
three independent 120 ns md runs were carried out for each system starting from the four initial states , using different initial velocity distributions .
we analyze the md trajectories using our in - house - developed codes and the gromacs facilities .
the z - position of each amino acid residue is described by the z - component of the main chain or side chain centroid with respect to the average z - position of the phosphorus atoms .
a residue is considered to be the one closest to the bilayer surface if the z - position of its centroid is the smallest among all the residues .
the number of hydrogen bonds ( h - bonds ) is calculated using gromacs tool g_hbond .
a h - bond is considered to be formed if the distance between n ( h ) and o is less than 0.35 ( 0.25 )  nm and the angle of n  h  o is greater than 150. this geometrical criterion for hydrogen - bond formation is widely used in many previous studies [ 55 , 63 , 8287 ] .
the interaction energy u between peptide and lipid is computed using the gromacs tools g_ener and mdrun - rerun ( using the formula uinter = u(peptide + lipid )  u(peptide )  u(lipid ) ) . to examine the effect of hiapp on the ordering of bilayer surface
, we calculate the thickness of lipid bilayer and the order parameter scd of the lipid acyl chain ( sn-1 ) .
the thickness of lipid bilayer is estimated by the average of the phosphorus - to - phosphorus distance .
trajectory data of hiapp monomer at popg bilayer membrane are obtained from our previous study .
the initial relative orientations of the peptide with respect to the membrane surface are the same for popc and popg lipid bilayers .
we have calculated the z - position of the centroid of each residue with respect to the popc bilayer surface and present the closest residue index as a function of time in figure 2 .
we observe that in 11 out of 12 md runs , the c - terminal residues are observed to adsorb to the popc bilayer surface prior to the n - terminal residues , namely , the adsorption of hiapp monomer is initiated from the c - terminal residues .
this membrane adsorption behavior of hiapp may be attributed to the dipole - dipole interaction between the polar residues ( such as ser34 , asn35 , and thr36 ) and the zwitterionic popc lipids . in our previous study
, we observed that the positively charged residues k1 and r11 in the n - terminal region have a preference to adsorb to the anionic popg lipid bilayer .
previous experimental studies reported that the n - terminal residues are involved in the membrane entry of hiapp peptide , and result in membrane damage at high peptide concentration [ 19 , 89 ] .
our results show that the adsorption process of hiapp monomer to popc and popg bilayer membranes is distinct , which may lead to different binding behaviors and may influence the aggregation of membrane - bound hiapps . to give the detailed adsorption process
, we show in figure 3 the snapshots at different time points and the time evolution of the contact number and hydrogen bond number between residue 1~19/20~37 and popc headgroups in a representative md run started from the initial state s(0 ) .
it can be seen from figure 3 that in the initial state , the hiapp monomer is placed in water parallel to the popc bilayer with the side chain of residue k1 pointing toward membrane surface .
the contact numbers between the c - terminal residues 20~37 and popc lipids increase with simulation time . at t
= 12  ns , the c - terminal residues 20~37 adsorb to the membrane surface prior to the n - terminal residues .
then , it takes tens of nanoseconds for residues 20~37 to adjust their side chains . at t
= 50  ns , hiapp monomer is mostly adsorbed to membrane surface and stays on the bilayer surface in the remaining 70  ns of md simulation .
the larger contact number of c - terminal residues 20~37 with popc lipids with respect to the n - terminal residues
1~19 indicates that the c - terminal residues 20~37 interact with the membrane more strongly than the n - terminal residues 1~19 . as seen from figure 3(b ) , the adsorption process is accompanied by the formation of h - bonds between hiapp and the headgroups of popc lipids .  
figure 3(c ) gives the time evolution of the z - position of the positively charged residues ( k1 and r11 ) and their interaction energy with popc bilayer within the first 50  ns of md simulation .
it is observed that k1 and r11 approach to the membrane surface at ~50  ns ( solid line in figure 3(c ) ) , while the c - terminal residues reach to the bilayer surface within 15  ns ( see figure 2 ) .
in addition , the interaction energy between the popc bilayer and residue k1/r11 is positive during the first 40/30  ns of simulation , reflecting the existence of repulsive interaction between the positively charged residues and the popc lipids in the beginning of the simulations .
it is known that a popc lipid molecule is composed of a positively charged choline , a negatively charged phosphate group and hydrophobic fatty acids .
although the popc lipid has no net charge , the positively charged choline is located closer to the membrane - water interface than the negatively charged phosphate group ( see section 3.3 for more details about the location of choline and phosphate groups ) , which leads to net repulsive interactions during the adsorption process .
this net repulsive interaction disfavors the n - terminal residues to adsorb first to the membrane surface , which explains the observed c - terminal - initiated adsorption behavior ( see figure 2 ) .
interestingly , both insertion and some helical folding were observed in our recent remd study on hiapp(119 ) peptide .
based on the results of our remd study , we deduce that the next step of hiapp - bilayer interaction might proceeds through insertion of partially ordered structures followed by helical folding within the interface [ 90 , 91 ] .
however , the exact mechanism remains clearly to be determined . to investigate the peptide orientation at the membrane - water interface , we plot in figure 4 the z - positions of c-atom and side chain centroid of each residue .
as experimental results have shown that the membrane - bound hiapp monomer adopts an -helix spanning residues 8~19 [ 2325 ] , we classify the membrane binding orientation of hiapp into four different of orientations ( labeled as fa , fb , fc , and fd ) according to z - positions of the residues in the core helix region ( residues 8~19 ) .
our recent md study showed that the side chains of residues r11 , f15 , and s19 insert more deeply into the anionic popg bilayer than their neighboring residues .
this binding resembles the binding orientation fd of hiapp at the zwitterionic popc bilayer ( figure 4 ) .
however , four different membrane binding orientations are observed for hiapp at zwitterionic popc bilayers , with almost equal probability .
we give in table 1 the initial states and the final hiapp orientations in each md run .
as seen from table 1 , hiapp with the same initial orientation can lead to different final binding orientations , and those with different initial orientations can lead to the same final binding orientation .
these results suggest that hiapp monomer adopts multiple binding orientations at popc membrane surface independent of its initial orientation . to identify the important interactions that stabilize each binding orientation of hiapp at popc bilayer , we plot in figure 5 the interaction energy between a peptide and a lipid bilayer ( per lipid ) .
the interaction energy is decomposed into electrostatic and van der waals ( vdw ) components .
as seen from figure 5 that hiapps with four binding orientations have nearly the same vdw interaction energy with lipids , and the electrostatic interaction energy is also similar .
however , the electrostatic interaction is much stronger than vdw interaction , indicating that the former plays a dominant role in stabilizing the binding of hiapp monomer to the popc lipid bilayer although the net charge of a popc lipid is zero .
different from our result , a recent md study by zhao et al . reported that the electrostatic and vdw interaction energy between hiapp ion - channel and a dopc bilayer are quite similar .
it is of particular interest to note that for the four different binding orientations , the peptide - lipid electrostatic interaction energy overlaps with each other ( see the error bar of the average value ) .
the small differences in total peptide - lipid interaction energy ( the same vdw + similar electrostatic interaction energy ) allow multiple binding orientations of hiapp at the popc lipid bilayer ; that is to say , hiapp has no preferred binding orientation to the membrane surface . in order to compare the binding orientations of hiapp at popc bilayer with those at popg bilayers , we plot in figure 6 the z - position of each amino acid residue of hiapp relative to the average z - position ( z = 0 ) of phosphorus atoms , averaged over the last 20  ns of twelve independent 120 ns md runs .
it is seen that , at the popc membrane surface , the z - position of each residues is very close to the position of lipid phosphorus atoms ( green dotted line ) .
the similar z - position of all residues reflects the uncertainty of peptide orientations . at the popg bilayer surface , the positively charged residues k1 and r11 anchor to the bilayer surface by electrostatic interactions , and
the z - position gradually increases from the n - terminal to c - terminal residues .
the z - position of residues in figure 6 shows that the -helix region ( residues 5~19 ) of hiapp is parallel to the popg bilayer surface , with residues r11 , f15 , and s19 pointing to the lipid bilayer while the hydrophobic residues l12 , a13 , l16 , and v17 exposed to the water , revealing a preferred binding orientation at anionic popg bilayers .
previous experimental studies reported that the n - terminal residues of hiapp are mainly responsible for membrane insertion and the c - terminal residues for fibrillar aggregation [ 26 , 32 ] .
figure 6 shows that the whole hiapp peptide binds tightly to the lipid headgroups of popc , with the c - terminal residues buried below the phosphorus atoms , thus restraining the flexibility of the c - terminal residues .
when hiapp monomer binds to the popg membrane , the c - terminal residues 20~37 are immersed in the water solution .
this allows the amyloidogenic 20~29 region to have more freedom than the residues inside lipid bilayer , which facilitate peptide - peptide interaction . on the other hand ,
the helix - helix association of hiapp is believed to proceed before the -sheet formation of the disordered c - terminal region [ 29 , 92 ] .
our md simulations show that hiapp monomer exposes the hydrophobic face of the amphipathic helical region to the solvent when binding to popg lipid bilayer surface .
the preferential binding orientation of hiapp may be attributed to the strong electrostatic interaction between the n - terminal positively charged residues k1 and r11 and the negatively charged popg lipid headgroups .
when hiapp monomers interact with popc lipid bilayers , their multiple binding orientations would reduce the solvent exposure probability of hydrophobic residues , thus disfavoring the peptide - peptide association .
these results are consistent with experimental reports that negatively charged membranes can promote hiapp aggregation [ 29 , 43 ] . through detailed structural analysis
, we find that although the chemical components of popc and popg lipids are similar , the locations of these components in the membrane are different .
we plot in figure 7 the electron density of popc and popg along the membrane normal ( i.e. , z - axis ) , as done in a previous study of lipid bilayers .
it shows that the popc ester , phosphate , and choline groups are located in turn from the membrane center ( z = 0 ) to the water solution , while the popg phosphate and glycerol groups are nearly at the same depth in the membrane with ester groups buried deeper .
in addition , the peak value of each popc headgroup component is smaller than that of popg , and the average area per lipid of popc membrane is higher than that of popg ( 61.6  0.7   versus 54.6  0.6   ) , consistent with previous computational and experimental studies [ 82 , 93 , 94 ] .
overall , the headgroup region of the popc bilayer is less compact than that of the popg bilayer , which is helpful for the insertion of hiapp monomer into popc membrane .
the symmetric distributions of popc / popg lipid atoms in the upper and lower leaflets of the bilayer reveal that hiapp monomer does not cause membrane disruption , in agreement with experimental observations [ 29 , 95 ] .
we also calculate the number of hydrogen bonds formed between hiapp monomer and different groups of the popc and popg membrane .  
figure 7(c ) shows that hiapp monomer forms hydrogen bonds most with the phosphate groups and less with the ester groups and least with the glycerol groups .
the formation of hydrogen bonds between the popc ester groups and residues n31 , s34 , n35 , and y37 of hiapp ( see figure 7(c ) ) allow hiapp to interact with the hydrophobic lipid tails , which may result in the c - terminal residues insertion deep into popc bilayer .
it is noted that the popc choline groups can not form hydrogen bonds with hiapp monomer , while the popg glycerol groups can .
the formation of h - bonds between hiapp and the glycerol groups , together with the formation of h - bonds between hiapp and the phosphate groups , would stabilize the specific binding orientation of the peptide at popg bilayer .
on the other hand , the formation of these h - bonds would constrain hiapp monomer at the popg membrane surface and thus hinders the peptide inserting into the bilayer , which might be helpful for the peptide - peptide association through the water - exposed hydrophobic residues .
this result is resembling the results reported for an antimicrobial peptide msi-78 by nmr and fluorescence experiments where the peptide insertion was measured with the variation of the pc  :  pg ratio , showing that the peptide inserts more deeply in zwitterionic lipid bilayers than that in anionic lipid bilayers .
the toxicity of hiapp and membrane disruption are suggested to be associated with hiapp - membrane interactions [ 29 , 95 ] . to examine the effect of membrane - bound hiapp monomer on the popc membrane , we calculate the lipid tail order parameter scd of acyl chain 1 ( sn-1 ) and the local membrane thickness ( see figure 8) .
the scd value is calculated by the formula scd = 0.53cos  1 , where  represents the angle of the c  h bond vector ( in the simulation ) or the c  d bond vector ( in the experiment ) with the bilayer normal .
as seen from figure 8 , the averaged scd value is within the error bar of the neat popc lipid bilayer , implying that lipid interaction of hiapp monomer does not disturb the membrane integrity .
the calculated local thickness of lipid bilayers in figure 8(b ) using different cutoff demonstrates that hiapp - lipid interaction influences the local thickness of popc bilayer .
the influence can be neglected when the cutoff is larger than 3  nm . these results suggest that the binding of hiapp monomer at the popc membrane surface has negligible disturbance on the integrity of the popc bilayer , which provides atomic - level evidence that membrane - bound hiapp monomer does not cause membrane disruption [ 29 , 95 ] .
however , it is expected that when the concentration of membrane - bound peptide reaches a critical value , the hiapp - lipid interaction may cause membrane disruption .
in this study , we have investigated the binding orientation and lipid interaction of hiapp monomer at the zwitterionic popc bilayer by carrying out multiple md simulations .
we have also examined the influence of lipid composition on lipid binding by comparing results of hiapp at anionic popg bilayer .
we have found that hiapp monomer adopts multiple orientations at popc bilayers while it has a preferential orientation at popg bilayers .
the specific binding orientation of hiapp at popg bilayer allows the hydrophobic residues exposed to water , thus facilitating peptide - peptide association by hydrophobic interactions .
our results also show that the hiapp monomer forms more hydrogen bonds with the headgroups of popg lipids , which constrains hiapp monomer to the popg bilayer surfaces , while the formation of less h - bonds allows hiapp ( especially the amyloidogenic c - terminal residues ) inserts deep into popc bilayer , thus reducing the probability of peptide - peptide interaction via solvent - exposed hydrophobic residues .
our studies provide atomic - level information of the binding behavior of hiapp and the effect of lipid composition on hiapp - membrane interactions , which may improve our understanding of membrane - mediated hiapp aggregation .
 increasing evidence suggests that the interaction of human islet amyloid polypeptide ( hiapp ) with lipids may facilitate hiapp aggregation and cause the death of pancreatic islet -cells . 
 however , the detailed hiapp - membrane interactions and the influences of lipid compositions are unclear . in this study , as a first step to understand the mechanism of membrane - mediated hiapp aggregation , we investigate the binding behaviors of hiapp monomer at zwitterionic palmitoyloleoyl - phosphatidylcholine ( popc ) bilayer by performing atomistic molecular dynamics simulations . the results are compared with those of hiapp at anionic palmitoyloleoyl - phosphatidylglycerol ( popg ) bilayers . 
 we find that the adsorption of hiapp to popc bilayer is mainly initiated from the c - terminal region and the peptide adopts a helical structure with multiple binding orientations , while the adsorption to popg bilayer is mostly initiated from the n - terminal region and hiapp displays one preferential binding orientation , with its hydrophobic residues exposed to water . 
 hiapp monomer inserts into popc lipid bilayers more readily than into popg bilayers . 
 peptide - lipid interaction analyses show that the different binding features of hiapp at popc and popg bilayers are attributed to different magnitudes of electrostatic and hydrogen - bonding interactions with lipids . 
 this study provides mechanistic insights into the different interaction behaviors of hiapp with zwitterionic and anionic lipid bilayers . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions