neurotensin ( nts ) is a 13 amino acid peptide 
 found in the central 
 nervous system and in peripheral tissues , and plays a crucial role in a wide range of biological activities 
 such as the pathogenesis of parkinson s disease , schizophrenia , 
 antinoiception , hypothermia , and lung cancer progression . neurotensin activates neurotensin receptor 1 ( ntsr1 ) which is one 
 of the three neurotensin receptors known to date . ntsr1 is a class a , peptide - activated gpcr sharing a seven 
 transmembrane ( tm ) helical structural motif conserved in class a gpcrs . gpcrs are dynamic proteins sampling several functional 
 conformations ranging from the  inactive state  to the 
  fully active  state . analysis of 
 the crystal structures of the inactive state and the active state of the 2-adrenergic receptor 
 ( 2ar ) with the g protein bound shows that the intracellular portions of tm5 and tm6 move 
 significantly with respect to tm3 upon activation when bound to both 
 the agonist and the g protein . this outward movement of tm6 away from 
 tm3 is a characteristic conformational change observed during activation 
 in class a gpcrs . the change in the distance between tm3 and tm6 upon 
 activation is measured as the distance between 
 the c atoms of r and l. here we have used the ballesteros  weinstein amino acid numbering 
 system for class a gpcrs . the first number 
 in the superscript is the tm helix in which the amino acid is located , 
 and the second number is the position of this residue with respect 
 to the most conserved residue in that helix which is numbered 50 . this distance in various crystal structures of the active and inactive 
 states of class a gpcrs is given in table s1 of the supporting information . however , this is only one of several 
 conformational changes that occur during activation . another structural feature observed in the crystal 
 structure of 
 the fully active state of class a gpcrs is the tight packing of the 
 hydrophobic residues p and f in the 
 active state compared to their respective inactive states . this distance in various gpcr crystal structures 
 is given in table s2 of the supporting information . a third feature of the active state crystal structures is the inward 
 movement of the intracellular end of tm7 that brings this region of 
 tm7 closer to tm3 . in the active state crystal structures known so 
 far , the inter - residue distance between r and y decreases upon activation , as shown in table s3 ( supporting information ) . hildebrand and co - workers 
 recently calculated the distance between 
 the center of mass ( com ) of five residues in the intracellular region 
 of tm2 to that of tm6 to measure the outward movement of the intracellular 
 regions of tm6 . table s4 ( supporting information ) shows this distance in various active and inactive state crystal 
 structures . recently , the crystal structures of thermostable 
 mutants of ntsr1 
 with neurotensin bound have been reported . the thermostable mutant of ntsr1 , known as gw5 [ protein data bank 
 ( pdb ) code 4grv ] , contains six thermostabilizing mutations : 
 a86l , e166a , g215a , l310a , f358a , and v360a . we denote the thermostable 
 mutant as ntsr1-gw5 and the wild type ntsr1 as wt - ntsr1 . we denote 
 the crystal structure of ntsr1-gw5 with its pdb code 4grv , as ntsr1 - 4grv . this distinguishes it from ntsr1-gw5 , which is used later in the paper 
 for describing the conformations from the molecular dynamics ( md ) 
 simulations on the ntsr1-gw5 mutant . the ntsr1 - 4grv crystal 
 structure with nts bound shows outward movement 
 of tm6 with respect to tm3 that is similar in extent to that observed 
 in the crystal structure of ligand free opsin . ntsr1 - 4grv also shows an interhelical hydrogen bond between 
 r167 and n257 , a feature also observed 
 in ligand free rhodopsin ( opsin * ) and 
 the active state of rhodopsin . since 
 the outward movement of tm6 in the ntsr1 - 4grv structure is not as 
 predominant as in the fully active state of the 2ar , we call the ntsr1 - 4grv structure an  active - like  
 state , because of the many conserved features between the active states 
 of rhodopsin and 2ar and ntsr1 - 4grv . it is unlikely 
 that ntsr1 - 4grv represents a fully active structure as observed on 
 g protein binding , because it would be expected 
 that the intracellular end of tm6 should move further out when the 
 g - protein couples to ntsr1 . using the ntsr1 - 4grv crystal structure , 
 white et al . rationalized the role of 
 two thermostabilizing 
 mutations ( e166a and l310a ) , whereas 
 there was no obvious structural reason for the thermostabilizing effect 
 of the other four mutations ( a86l , g215a , f358a , and v360a ) . none of the six 
 mutant residues are located in the ligand binding site ( figure s1 , supporting information ) . the e166a mutation prevents the intrahelical salt bridge with r167 , which is a feature of the inactive state , thus facilitating the 
 movement of tm6 toward the active - like state by allowing r167 to interact with n257 . decreasing the size 
 of the side chain by the mutation of l310a promotes 
 the hydrogen bond between r167 and n257 . the ntsr1 single mutants a86l and f358a showed higher measured thermostabilities both in the presence and 
 absence of the agonist nts compared to other mutations . thus , the information gleaned from the crystal 
 structure is limited and does not explain the thermostability of all 
 the mutations . since the single snapshot of the crystal structure 
 is inadequate in providing understanding of the receptor dynamics , 
 we have used microsecond molecular dynamics ( md ) simulations to address 
 two outstanding questions : ( 1 ) how do the mutations stabilize the 
 receptor in the active - like state ? ( 2 ) what are the differences in 
 the dynamics of the active - like state of ntsr1-gw5 compared to the 
 wt - ntsr1 ? we have performed a total of 4 s of md simulations 
 using 
 the anton specialized computers for the 
 wt - ntsr1 and ntsr1-gw5 , starting from the ntsr1 - 4grv crystal structure 
 of the thermostabilized rat ntsr1 bound to the carboxy terminal portion 
 ( nts813 ) of the endogenous peptide agonist nts . finally , the ntsr1 - 4grv structure does not show the amphipathic helix 
 8 at the carboxy terminus of the receptor . instead , the intracellular 
 region of tm7 was elongated by three helical turns beyond the well - conserved 
 npxxy motif . subsequent crystal structures of ntsr1 thermostable mutants 
 showed the presence of helix 8 . to examine the stability of helix 
 8 in the ntsr1 - 4grv structure , we used an additional 2 s of 
 md simulations to examine the dynamic behavior and stability of helix 
 8 by homology modeling . the md simulations provide insights into the 
 dynamics of ntsr1-gw5 and how the mutations specifically stabilize 
 the active - like state compared to the wild type receptor . the md simulations 
 for the agonist nts bound mutant ntsr1-gw5 were 
 started from its crystal structure ( pdb code 4grv ) . the initial conformation of the wild type receptor was 
 generated by mutating the residues in the crystal structure of ntsr1 - 4grv 
 back to the wild type sequence using maestro v9.2 ( schrdinger , 
 inc . ) . we omitted t4 lysozyme from all the simulations , and did not 
 model the unresolved 31-residue stretch in the intracellular loop 
 3 ( icl3 ) between tm5 and tm6 in the crystal structure ( residues h269r299 ) , 
 since the loop addition programs are not reliable for this long stretch 
 of loop residues . the residues in the amino and carboxy termini that 
 were either deleted from the crystallized construct or not resolved 
 in the crystal structure were also omitted from the simulations . the 
 four disordered residues in the intracellular loop 1 ( k93q96 ) 
 were added by homology modeling ( modeler v9.7 ) . all ntsr1 systems were embedded in a hydrated palmitoyl - oleoyl - phosphatidyl - choline 
 ( popc ) bilayer in all the md simulations . all the atoms ( including 
 those in lipids and water ) were represented explicitly . hydrogen atoms 
 were added to the crystal structures using maestro ( schrdinger , 
 llc ) , and n and c termini were capped with neutral groups ( acetyl 
 and methylamide , respectively ) . the protein structures were inserted 
 into an equilibrated popc bilayer solvated with 0.15 m nacl . all four 
 systems based on the ntsr1 crystal structure initially measured about 
 80   80   85  ( 0.54  10  ) and contained 173 lipid molecules , 2800 water 
 molecules , 40 chloride ( cl ) ions , and 28 sodium 
 ions ( na ) , with a total of approximately 58  900 
 atoms . for all the md simulations , we used the charmm27 parameter set with cmap terms and the tip3p water model . a modified 
 charmm22 lipid force field was used . 
 after energy minimization and heating the system to 310 k , all simulations 
 consisted of a 50 ns equilibration run followed by a 2 s production 
 run . all the systems were equilibrated in the npt ensemble ( 310 k 
 at 1 bar of pressure ) , with initial velocities sampled from the boltzmann 
 distribution and with 5 kcal / mol /  harmonic position 
 restraints applied to all non - hydrogen atoms of the protein and peptide 
 ligand . both equilibration and production 
 runs were performed on anton , the special - purpose supercomputer that 
 significantly accelerates standard md simulations . the shake algorithm was used to constrain 
 all bond lengths involving hydrogen atoms , allowing 2 fs time steps 
 to be used . for the analysis , the cutoff distance of 13.5  was used for nonbonded 
 interactions , and the k - space gaussian split ewald method was used for long - range van der waals interactions . 
 the simulation details are summarized in table s5 ( supporting information ) . rms 
 fluctuations for ntsr1-gw5 and wt - ntsr1 were calculated using the g_rmsf program in the gromacs v4.6 molecular dynamics package . these c rmsfs have been directly 
 compared to the crystallographic temperature factor ( b ) using the conversion factor rmsf = ( 3b/8 ) . protein structures 
 from anton trajectories for ntsr1-gw5 and wt - ntsr1 were prepared using 
 the protein preparation wizard protocol available in the schrdinger 
 suite ( maestro9.2 software package from schrdinger , llc ) . the nts813 peptide , water molecules , and popc bilayer 
 lipids were deleted for protein enthalpy calculation . each single - point 
 enthalpy was calculated by the charmm27 force field with bond orders 
 of protein reassigned . during the course 
 of this work , more 
 crystal structures of other thermostable ntsr1 mutants bound to nts 
 were published . unlike the ntsr1 - 4grv 
 structure , these crystal structures showed helix 8 ( h8 ) . to examine 
 the stability of helix 8 for the gw5 mutant , we added the helix 8 
 structure from the crystal structures of egloff et al . , on to ntsr1 - 4grv and to the corresponding wild 
 type derived from the ntsr1 - 4grv structure . the homology models were 
 generated using the structure of ntsr1 - 4grv ( pdb code 4grv ) until p366 , followed by the crystal structure of 4buo(17 ) to the end of the carboxy terminus of amphipathic helix 
 8 . modeler v9.7 was used to build a homology 
 model of helix 8 with the 4buo as the template structure for helix 8 . the resulting 
 homology models of the ntsr1-gw5 and wt - ntsr1 with helix 8 ( denoted 
 as ntsr1-gw5-h8 and wt - ntsr1-h8 , respectively ) were optimized to reduce 
 the side chain steric clashes . later on , the potential energy of the 
 entire receptor was minimized and equilibrated using the gromacs v4.6 
 package with the charmm22 lipid force field with tip3p water molecules . these two homology models were equilibrated by performing 5 ns of 
 md at 310 k using the npt ensemble with harmonic restraint followed 
 by 1000 ns of npt production run . to understand the similarities 
 and differences in the protein global motion in ntsr1-gw5 and wt - ntsr1 the g_anaeig and g_covar programs in gromacs v4.6 were employed to calculate covariance matrix 
 elements in pca . in our analysis , only the main chain atoms of six 
 tm helices ( tm2tm7 ) were considered and tm1 was omitted for 
 the pca due to the missing amino terminus that would control its movement . for understanding 
 the effect of point mutations on the effects of lipid packing ( interaction 
 of the protein and the popc lipid bilayer ) , we have calculated the 
 sasa for those residues that are within 5  of the popc molecules 
 in wt - ntsr1 and the ntsr1-gw5 mutant . we used the vmd tcl command 
  measure sasa 1.4 $ all restrict $ selection  , where $ all is an atom selection for the 
 whole protein and $ selection is the protein atoms 
 that are within 5  of the lipid molecules . we have 
 compared the flexibility of the wt - ntsr1 to the thermostable 
 mutant ntsr1-gw5 . figure 1 shows the comparison 
 of the root - mean - square fluctuations ( rmsfs ) in coordinates from the 
 average structure from md simulations for each residue for the two 
 systems ( wt - ntsr1 and ntsr1-gw5 ) in addition to that computed from 
 the x - ray b - factor . lower flexibility 
 was observed for the transmembrane regions , and these were in agreement 
 with the b - factors of the crystal structure . the 
 ntsr1-gw5 shows less receptor flexibility compared to the wt - ntsr1 , 
 especially in the extra- and intracellular loops . rms fluctuation for the 
 c atoms over the entire 
 2 s simulation for ntsr1-gw5 mutant ( black ) and its wild type 
 ( red ) , and then compared with the rmsf of the 4grv crystal structure 
 ( blue ) ( conversion from b - factor to rmsf ) . we have analyzed four different inter - residue distances that show 
 a change upon activation in class a gpcrs , described in the introduction section , to understand the differences 
 in the dynamics of the wt - ntsr1 compared to the mutant ntsr1-gw5 ( figure 2 ) . our md simulations show that the distance between 
 the c atoms of the residues r167 and 
 l303 ( cc ) in ntsr1-gw5 stays between the corresponding 
 distance in the inactive state of 2ar and in the 
 active - like state of the ntsr1 - 4grv crystal structure . the r167 and l303 distance in the wt - ntsr1 is between 
 the crystal structure of ntsr1 - 4grv and the fully active state of 
 2ar , as shown in figure 2a . we also calculated the most populated ( observed in many conformations 
 in the md ensemble ) distance between the center of mass of the last 
 three helical turns of tm3 and tm6 that supports the same observation 
 ( shown in figure s2 , supporting information ) . figure 2b shows the receptor population 
 density variation with the inter - residue distance between p249 and f317 in both the wt - ntsr1 and ntsr1-gw5 . there is little difference in 
 the dynamics between the wt - ntsr1 and ntsr1-gw5 in this inter - residue 
 distance . figure 2c shows the population density 
 variation with the inter - residue distance r and y. the wt - ntsr1 shows a lower spread in this distance , keeping 
 it close to the crystal structure ( 11.9  ) , a more active - state - like 
 behavior compared to the ntsr1-gw5 . the increase in the distance between 
 r and y comes from rotation of the side 
 chain of y in the dynamics of ntsr1-gw5 and infiltration 
 of three water molecules ( with 40% of the snapshots having these waters ) . such a rotation is not seen in the wt - ntsr1 , which keeps the tm3 and 
 tm7 distance intact . figure 2d shows the distance 
 between the center of mass of five residues in the intracellular part 
 of tm2 and tm6 . the wt - ntsr1 opens up more like g protein bound 2ar , while ntsr1-gw5 does not . summarizing these inter - residue 
 distance analyses shows that the dynamics of wt - ntsr1 are closer to 
 the fully active state of the 2ar conformation compared 
 to the dynamics of ntsr1-gw5 . the wt - ntsr1 shows more of the active - state - like 
 structural features than the ntsr1-gw5 . variation of inter - residue 
 distances of active - like wt - ntsr1 and 
 ntsr1-gw5 . ( a ) the population distribution density of the cc distance of r167l303 for the ntsr1-gw5 ( black ) and wt - ntsr1 ( red ) . ( b ) the 
 receptor population density variation with the inter - residue side 
 chain distance between p249 and f317 in the wt - ntsr1 and ntsr1-gw5 . ( c ) the population density variation 
 with the inter - residue distance r167y369 observed in the md simulation . ( d ) the observed population 
 density of the com ( center - of - mass ) distance using five residues in 
 the intracellular tm2 and tm6 ( dtm2-tm6 ) for ntsr1-gw5 ( black ) and wt - ntsr1 ( red ) . the inter - residue distance 
 in the crystal structure , ntsr1 - 4grv , is shown as a blue dashed line . the same inter - residue distances in the crystal structures of the 
 fully active ( pdb code 3sn6 ) and inactive ( pdb code 2rh1 ) states of 2ar are 
 shown in bold and thin magenta dashed lines , respectively . we used principal component 
 analysis ( pca ) to analyze the global motion of the wt - ntsr1 and ntsr1-gw5 
 receptors during the md simulations . we performed pca of the md trajectories 
 of the main chain atoms in the tm helices of ntsr1-gw5 and wt - ntsr1 , 
 and visualized the movement of the first two principal components 
 that show the direction and magnitude of major movements in the tm 
 region of the receptors ( shown in figure 3 ) . two principal 
 components represented by the double arrows showing 
 the dominant motion in the ntsr1-gw5 ( a ) and wt - ntsr1 ( b ) simulations . 
 shown in this figure the 
 orange - colored structures are the average structures for each system 
 generated from covariance matrix and from filtering the trajectories , 
 and the cyan - colored structures are the representative structures 
 of the most populated cluster in the ensembles . the principal component 1 ( pc1 ) is dominated by the outward 
 movement 
 of the intracellular part of tm5 and tm6 . as shown in figure 3 , the ntsr1-gw5 shows less movement in the intracellular 
 regions of tm5 and tm6 compared to wt - ntsr1 . thus , the wt - ntsr1 , which , 
 in contrast to ntsr1-gw5 , activates the g protein , shows an outward 
 movement of tm5 and tm6 with respect to tm3 . such a movement also 
 leads to activation , and we had observed this type of movement in 
 our previous study of the dynamics of ligand free 2ar . pca shows that the flexibility of 
 the wt - ntsr1 receptor is higher , while the ntsr1-gw5 remains rather 
 inflexible . taking the results of the pca combined with the analysis 
 on the changes in inter - residue distances that characterize activation 
 processes , we summarize that the dynamic behavior of wild type ntsr1 
 shows more active - like characteristics compared to ntsr1-gw5 . this 
 observation may explain why the ntsr1-gw5 mutant does not catalyze 
 the gdp  gtp exchange at gq , as efficiently 
 as the wt - ntsr1 , an experimentally observed behavior of the two receptors . to understand 
 the energetic contribution of the mutations in the thermostabilization 
 of ntsr1 , we compared the enthalpy of the wild type receptor and the 
 ntsr1-gw5 mutant . figure 4 shows the population 
 density distribution of the enthalpy of various conformations in the 
 md ensemble calculated over the md trajectories for the thermostable 
 mutant ntsr1-gw5 ( black ) and the wt - ntsr1 ( red ) receptors . the enthalpy 
 is the total potential energy of the system ( etot ) , and provides information on the stability of the nts813ntsr1 complex and interaction energy between 
 protein / popc and protein / waters . the difference in the 
 energies between the most populated conformations of ntsr1-gw5 and 
 wt - ntsr1 shows that the thermostable mutant is more stable by 515 
 kcal / mol compared to the wt - ntsr1 . the components of the potential 
 energy such as the nonbonded energy of the receptor , receptor  ligand 
 interaction energy , receptor  lipid ( popc ) interaction energy , 
 and receptor  water interaction energy are all shown in figure 
 s3 ( supporting information ) . these results 
 show that the significant contribution to the stability of ntsr1-gw5 
 comes from the receptor energy and the receptor  popc interaction 
 energy ( figure s3a and b , supporting information ) . the protein energy of ntsr1-gw5 is lower due to the improvement 
 in the interhelical packing interactions , as discussed in the section 
 below . the annular lipids around the ntsr1-gw5 show better packing 
 than the lipids around wt - ntsr1 ( figure s3c and d , supporting information ) . this is reflected by the more compact 
 radius of gyration calculated for the ntsr1-gw5 compared to the wt - ntsr1 
 ( figure s3c , supporting information ) . popc 
 interaction energy in the mutant ntsr1-gw5 compared to the wt - ntsr1 . figure s4a ( supporting information ) shows 
 the nonbonded interaction energy between the lipid bilayer and the 
 residues that are within 7  of the mutation positions in the 
 receptor . as seen in this figure , 80% of the favorable interaction 
 energy between the receptor and popc in ntsr1-gw5 comes from the lipid 
 interaction with the residues that are in the vicinity of the mutations . figure s4b ( supporting information ) shows 
 the breakdown of this interaction energy for each point mutation in 
 ntsr1-gw5 ( wt - ntsr1 shown in red and ntsr1-gw5 in black ) . figure s4c 
 ( supporting information ) shows the difference 
 in the coulombic and van der waals components of the lipid protein 
 interaction energies between the ntsr1-gw5 and the wt - ntsr1 , for the 
 residues in the vicinity of each mutation position . it is seen that 
 the mutations a86l , e166a , and l310a that are located in the intracellular regions of transmembrane 
 helices contribute significantly to the stronger protein we 
 also calculated the solvent accessible surface area of the protein 
 regions that are exposed to the lipid bilayer ( calculated as described 
 in the methods section ) in the ntsr1-gw5 and 
 wt - ntsr1 , shown in figure s4d ( supporting information ) . the protein surface area exposed to the lipid bilayer is higher 
 in the ntsr1-gw5 than in the wild type ntsr1 that leads to better 
 interaction energy between the ntsr1-gw5 and the lipid bilayer . population 
 density distribution of the conformations in the md 
 ensemble with the total potential energy for the ntsr1-gw5 ( black ) 
 and wt - ntsr1 receptors ( red ) . to understand the structural basis of the enthalpic stabilization 
 of the ntsr1-gw5 mutant , we calculated the number of interhelical 
 hydrogen bonds and van der waals ( vdw ) packing interactions averaged 
 over the trajectories for each system . changes in the interhelical 
 interactions play an important role in the packing of the tm core in the membrane protein structures . figure 5 shows the analysis of the differences in the number 
 of interhelical hydrogen bonds and vdw interactions in the 2 s 
 of md simulations on ntsr1-gw5 and wt - ntsr1 . details of the specific 
 interhelical hydrogen bond and vdw interactions observed are listed 
 for these systems in tables s6 and s7 ( supporting 
 information ) . total number of interhelical interactions between various 
 pairs 
 of helices , calculated for the two ntsr1 systems . parts a and b show 
 the interhelical hydrogen bonds between each transmembrane helix in 
 the wt - ntsr1 and ntsr1-gw5 , while parts c and d show the interhelical 
 vdw interactions . the number on each line is the total number of each 
 type of interhelical interactions between pairs of tm helices . as seen in figure 5 , ntsr1-gw5 has a higher 
 number of pairwise interhelical hydrogen bonds and vdw interactions 
 than those of the wt - ntsr1 . the difference in the number of interhelical 
 vdw interactions is much higher than that in the interhelical hydrogen 
 bond interactions , since the six point mutations in ntsr1-gw5 lead 
 to repacking of the side chains in the neighborhood of the mutations 
 as explained in the next section . tm3 shows stronger vdw interactions 
 with tm2 , tm4 , tm5 , tm6 , and tm7 in ntsr1-gw5 compared to the wild 
 type receptor . the five mutations in ntsr1-gw5 , a86l , e166a , l310a , f358a , and v360a , are located in tm1 , tm3 , tm6 , and tm7 , 
 respectively , whereas the sixth mutation g215a is located 
 on the extracellular loop 2 . the mutations a86l , e166a , and l310a are located in the intracellular 
 parts of tm1 , tm3 , and tm6 , respectively , while the mutations f358a and v360a are positioned in the middle 
 of tm7 . out of these six mutations , the single mutants a86l and f358a show higher thermostability than any other 
 single mutation ; however , the structural basis 
 of this thermostability is not obvious from the crystal structure . 
 to rationalize the stability of each mutation , we calculated the difference 
 in the interhelical hydrogen bonds as well as vdw interactions between 
 ntsr1-gw5 and the wild type receptor in the neighborhood of the mutation 
 positions 86 , 166 , 310 , 358 , and 360 . shibata et al . measured the experimental 
 stability of the a86l single mutation to be 656% of 
 the wild type in the presence of full length [ h]nts . we calculated the difference in the interhelical hydrogen bonds 
 as well as vdw interactions between ntsr1-gw5 and the wild type receptor 
 in the neighborhood of the mutation position a86l . figure 6 shows interhelical hydrogen bonds 
 that are within 5  of the mutation a86l . compared 
 to wt - ntsr1 , ntsr1-gw5 shows an increased number of interhelical hydrogen 
 bond interactions between tm1 , tm2 , and tm7 in the neighborhood of 
 the a86l mutation . ( a ) the direct and indirect contacts of n82 with a110 and s362 in the 
 vicinity of the a86l mutation in ntsr1-gw5 ( b ) and 
 the wild type receptor . two waters that have important roles in indirect 
 contacts between n82 and s362 were observed 
 in 53 and 83% of the snapshots from md simulation trajectories , respectively . ( c and d ) hydrophobic interaction pattern near the a86l mutation in ntsr1-gw5 and wild type receptor . the t79l114 and a86l  l114 interactions in ntsr1-gw5 and t85a110 hydrophobic interaction in the wild type receptor are 
 highlighted by orange double arrows . the van der waals surfaces of 
 the atoms are shown as mesh , and the distances shown in orange are 
 in  . the residue n82 makes a water mediated hydrogen 
 bond with the backbone carbonyl of s362 and another 
 water mediated hydrogen bond with the side chain of s362 . these two water molecules are present in more than 40% of the conformations 
 in the md ensemble . the side chain ( nd2 ) of n82 makes 
 a hydrogen bond with the backbone carbonyl ( o ) of a110 on tm2 . in contrast , wt - ntsr1 has no direct interactions between 
 tm1 , tm2 , and tm7 . in addition to strong interhelical hydrogen bond 
 interactions near the a86l mutation in ntsr1-gw5 , the 
 effect of a86l mutation from a short methyl side chain 
 to a long hydrophobic side chain also resulted in improved interhelical 
 vdw interactions between a86l with a110 and l114 , as shown in figure 6c , that is absent in the wild type receptor ( shown in figure 6d ) . the wild type ntsr1 shows interaction between 
 t85 and a110 that is absent in ntsr1-gw5 
 dynamics . the variations of the interhelical hydrogen bond and hydrophobic 
 interaction distances near the a86l mutation with time , in ntsr1-gw5 
 and wt - ntsr1 , are shown in figures s5 and s6 ( supporting information ) , respectively . similar to the effect of a86l , these 
 mutations lead to a strong interhelical hydrogen bond network between 
 n82 , d113 , and s362 and 
 a water mediated hydrogen bond between n82 and n365 , as shown in figure 7a . these hydrogen 
 bond networks are not observed in the md simulation of the wt - ntsr1 
 ( figure 7b ) , except for the hydrogen bond interaction 
 between d113s362 . strong interhelical 
 hydrogen bonds between tm1 , tm2 , and tm7 in ntsr1-gw5 compared to 
 wt - ntsr1 are shown in figure s7 ( supporting information ) . ntsr1-gw5 also shows favorable interhelical -stacking interaction 
 between the sulfur atom of m121 and the aromatic side 
 chain of y359 in the extracellular side of tm2 and 
 tm7 ( figure s8 , supporting information ) . 
 in summary , the mutations a86l , f358a , and v360a improve the interhelical interactions 
 in the mid region of tm1 , tm2 , and tm7 of ntsr1-gw5 compared to the 
 wt - ntsr1 . changes in the interhelical interaction in the neighborhood of 
 the mutations f358a / v360a . ( a ) the direct 
 and indirect hydrogen bond interactions centered on the f358a / v360a region of ntsr1-gw5 . the water molecule 
 located between n82 and n365 has been 
 observed in 59% of the snapshots in the md simulation ensemble . the e166a is a charged residue 
 to neutral residue mutation that would abolish the intrahelical salt 
 bridge with r167 that has been observed in the inactive 
 state crystal structures of several class a gpcrs , thus freeing up 
 this arginine residue . the side chain of r167 turns 
 around and makes a direct hydrogen bond with n257 in 
 the crystal structure ntsr1 - 4grv . however , 
 this hydrogen bond becomes water mediated during the md simulations 
 of ntsr1-gw5 and facilitates the formation of another interhelical 
 hydrogen bond between r167 and s373 ( figure 
 s9 , supporting information ) . thus , r167 serves as a bridge between tm5 and tm7 , making a two - pronged 
 water mediated hydrogen bond network between n257 and 
 s373 . the side chain dihedral angle of n257 rotates by about 60 ( figure s10 , supporting 
 information ) , facilitating the formation of a hydrogen bond 
 with s164 with 98% of the conformations in the md ensemble 
 of ntsr1-gw5 showing this hydrogen bond . however , the same interaction 
 in the wt - ntsr1md simulation was observed in less than 50% of the 
 conformation ensemble . mutating e166 to ala abolishes this hydrogen bond that is however compensated 
 by a direct hydrogen bond between s162h105 , and a water mediated interaction between the backbone 
 atoms of a166 and h105 ( figure s9a , supporting information ) . thus , the e166a mutation strengthens the interactions in the intracellular 
 part of the tm3 with tm5 and tm7 . like e166a , 
 the l310a mutation also modulates the interhelical 
 interactions in the intracellular region of tm3 , tm5 , tm6 , and tm7 
 ( figure s11 , supporting information ) . in 
 the wt - ntsr1 , the side chain of l310 affects the rotamer 
 of n257 , such that the interaction between s164 and n257 is mediated by a bridging water 
 molecule ( see figure s11b , supporting information ) . mutating this residue to ala in ntsr1-gw5 shortens the side chain 
 and allows a rotamer change of n257 that can then form 
 a hydrogen bond directly with the side chain of s164 ( figure s11a , supporting information ) , 
 thus strengthening the interaction between the two helices . in addition , 
 residue r311 ( a neighboring residue of l310a ) has a water mediated hydrogen bond with the backbone of n257 in the ntsr1-gw5 in 73% of the snapshots in the md trajectories . there is a water molecule present at this position in the crystal 
 structure ntsr1 - 4grv . in the wt - ntsr1 , this interaction is not sustained 
 and observed in less than 50% of the snapshots of the md simulations . 
 in summary , the l310a mutation again facilitates the 
 strong interaction in the intracellular region of tm3 with tm5 and 
 tm6 . these interactions could hold the tm3 , tm5 , and tm6 stable in 
 the active - like conformation and possibly restrict the dynamics of 
 the receptor in this region . the 
 number of interhelical water mediated hydrogen bonds is higher in 
 ntsr1-gw5 compared to wt - ntsr1 . therefore , to examine the difference 
 in the extent of hydration between wt - ntsr1 and ntsr1-gw5 , we calculated 
 the radial distribution function of water molecules near the receptors 
 for the 2 s of md simulation trajectories ( figure 8a ) . this function gives a quantitative measure of 
 the density of water molecules within 3  of the protein residues 
 in the tm region . the density of water molecules in the interior of 
 the ntsr1-gw5 mutant is higher compared to wt - ntsr1 , as seen in figure 8a . we calculated the water map near the ligand binding 
 site and the presumed na ion binding region as well as 
 near other well - conserved and structurally important motifs such as 
 the e(/d)ry motif in 
 tm3 and npxxy in tm7 , shown in figure 8b and 
 c. as seen in figure 8b , the water population 
 near the highly conserved structural motifs , namely , ndn , e(/d)ry , and npxxy , in the ntsr1-gw5 system is higher than that in the wild type receptor . most of the waters that have persistent density in the interior 
 of the tm region in both the ntsr1-gw5 and wt - ntsr1 are involved in 
 mediating interhelical contacts . figure 8c 
 shows the van der waals surface of the water molecules that are within 
 3  of the residues in the tm region , both in ntsr1-gw5 and in 
 wt - ntsr1 . however , the waters within 3  of the residues in the 
 tm region of ntsr1-gw5 form larger hydrogen bonded water clusters 
 that improve the interhelical packing compared to the wt - ntsr1 . the 
 water clusters form tight hydrogen bond networks between transmembrane 
 helices leading to extra stability , as shown in figure s12 ( supporting information ) . most of the crystal structures 
 of the class a gpcrs show an interhelical hydrogen bond network ( either 
 direct or water mediated ) between n , d , and n residues . this 
 region is also the na ion binding region , as seen in the 
 inactive state crystal structures of adenosine receptor a2a , 1-adrenergic receptor , protease - activated receptor ( par1 ) , and an opioid receptor ( -or ) . upon agonist binding to the a2a receptor , 
 the volume of the na ion binding pocket in the receptor 
 shrinks and is no longer able to accommodate the na ion . the number of water molecules around the na ion binding 
 site is less in wt - ntsr1 compared to ntsr1-gw5 ( see figure 8b ) , implying that the wild type receptor shows greater 
 shrinkage of the na ion pocket compared to ntsr1-gw5 . ( a ) radial 
 distribution function of water from the center of mass 
 ( com ) of ntsr1-gw5 ( black ) and wt - ntsr1 ( red ) . ( b ) number 
 of water molecules observed in the md simulations near conserved motifs . ndn motif , ndn ; e(/d)ry 
 motif , e(/d)ry ; npxxy 
 motif , npxxy . ( c ) water 
 density representation within the transmembrane domain of ntsr1-gw5 
 ( left ) and wt - ntsr1 ( right ) . the water densities near the ligand binding 
 site and near the e(/d)ry motif are shown by black and blue boxes , 
 while the na ion binding region including the ndn and 
 npxxy motifs is represented by a red box . volumetric density maps 
 were isosurface contoured ( probe radius of 1.4  ) using vmd software . although most of all high 
 resolution class a gpcr structures possess 
 an amphipathic helix 8 , surprisingly the structures of par1 ( proteinase 
 activated receptor 1 ) , cxcr4 ( chemokine 
 receptor 4 ) , and ntsr1 - 4grv do not show helix 8 in their structures . however , 
 helix 8 is present in a more recent crystal structure of a different 
 thermostabilized mutant of ntsr1 , although the helix 8 region was 
 found to be less stable than that of other gpcrs . the reason for the absence of helix 8 in ntsr1 - 4grv is 
 unclear and may be the result of a crystallization artifact , but it 
 may also be caused by its intrinsic instability . thus , we explored 
 the dynamic behavior of helix 8 in md simulations of ntsr1-gw5 and 
 of wild type receptor , by modeling helix 8 , using the homology modeling 
 technique described in the methods section . we generated homology models of both ntsr1-gw5 and wt - ntsr1 , starting 
 from the crystal structure ntsr1 - 4grv ( pdb code 4grv ) until p366 of tm7 ( npxxy region ) ( figure 
 s13a , supporting information ) . for the 
 rest of the carboxy terminus sequence that includes helix 8 , we used 
 the helix 8 conformation of the crystal structure of ntsr1-tm86v ( pdb 
 code 4buo ) . we followed the same procedure to generate the 
 structure of the wt - ntsr1 with helix 8 . the initial structures of 
 ntsr1-gw5 with helix 8 , denoted as ntsr1-gw5-h8 , and of ntsr1 - 4grv 
 are shown in figure s13a ( supporting information ) . it is seen that the residues at the turn of helix 8 show a van 
 der waals clash with the residues in the intracellular edge of tm7 
 in the initial ntsr1-gw5-h8 structure . starting from these initial 
 structures , we performed 1 s of md simulations on both of these 
 ntsr1 systems with helix 8 . we observed that the helix 8 unravels 
 spontaneously during these simulations in both ntsr1-gw5-h8 and wt - ntsr1-h8 , 
 as shown in figure s13b ( supporting information ) . this could mean that helix 8 is not stable in ntsr1-gw5 , due to 
 steric hindrance between the hydrophobic residues of p366 and f380 and n370 and f380 . details of the specific electrostatic and van der waals interaction 
 in the initial ntsr1-h8 homology models , that lead to the clashes 
 of tm7 and helix 8 , are listed in table s8 ( supporting 
 information ) . we performed a detailed analysis of the 
 helix 8 unraveling in other class a gpcrs for which we have done at 
 least 1 s of md simulations in explicit lipid bilayer and water . table s9 of the supporting information shows that , while helix 8 remains largely intact during the md simulations 
 of the inactive state gpcrs , they are rather flexible and unravel 
 in the active state or the active - like conformations . finally , 
 we have analyzed the r3.50 and y7.53 inter - residue distance 
 in the ntsr1-gw5 and wt - ntsr1 in the presence of helix 8 ( figure s14 , supporting information ) . we observed that the 
 distance of tm3 and tm7 does increase in the ntsr1-gw5 more than wt - ntsr1 
 with and without helix 8 present ( see figure 2c also ) . we also observed a strong and persistent hydrogen bond between 
 r167 and s373 and van der waals interaction 
 between y369 and v160 in the wild type 
 that is absent in gw5 , which could lead to the inward movement of 
 tm7 that is absent in the gw5 . our 
 study is , to our knowledge , the first md simulation study on 
 the neurotensin peptide receptor ntsr1 with nts bound , with the aim 
 of understanding the structural basis of thermostabilization and activation . we performed 2 s of md simulation on the two systems , namely , 
 the thermostable mutant ntsr1-gw5 and its wild type counterpart ( wt - ntsr1 ) . the ntsr1-gw5 is stabilized by enthalpic contributions from increased 
 interhelical hydrogen bonds and van der waals interactions within 
 the receptor , as well as receptor  lipid ( popc ) interactions . the layer of annular lipids ( popc ) packs tighter around the ntsr1-gw5 
 compared to the wt - ntsr1 , and this could lead to increased stability 
 and reduced flexibility in the dynamics of the thermostable mutants . this observation is consistent with our previous studies on the dynamics 
 of thermostable mutants of the 1-adrenergic receptor , adenosine a2a receptor . experimentally , the thermostability of adenosine 
 a2a receptor mutants reconstituted in surfactants was found 
 to be dependent on the amount of lipid in the surfactant micelles . adding lipids to the surfactant micelles led 
 to improved thermostability in some of the mutant receptors . the tight 
 packing of lipids around the gw5 mutant observed in our simulations 
 could be a possible mechanism of thermostabilization in other gpcrs . the reduced flexibility and dynamics of the thermostable mutant receptors 
 could result in reduced efficacy of these receptors to couple to g - proteins . in ntsr1-gw5 , all the thermostabilizing mutations ( with the exception 
 of a86l ) are alanine mutations . alanine mutations are 
 presumed to lead to  loss of effect  where the effect 
 of the mutation is usually detrimental to the receptor stability compared 
 to the wild type , due to the loss of side chain packing interactions . however , in ntsr1 ( and other gpcrs such as 1ar and 
 a2ar ) , alanine mutations lead to increased thermostability 
 by improving the nonbonded contacts among neighboring residues . the 
 point mutations show increased water mediated interhelical hydrogen 
 bonds in the neighborhood of the respective mutation positions . each 
 of the six mutations in ntsr1-gw5 lead to tighter interhelical packing 
 through water mediated inter helical hydrogen bonds in the middle 
 of tm1 , tm2 , and tm7 , as well as at the intracellular end of tm3 , 
 tm5 , and tm6 . water molecules form hydrogen bonded water clusters 
 similar to  ice - like waters  in the ntsr1-gw5 md simulations . the md simulations also showed 
 improved interhelical van der waals packing in the neighborhood of 
 the point mutations due to rearrangement of side chain conformations . this is consistent with our observations in the previous work on 2ar and a2ar thermostable mutants . analysis of four different types of inter - residue 
 distances during 
 the md simulations that characterize receptor activation in gpcrs 
 ( obtained from analyzing crystal structures of active and inactive 
 states ) shows that ntsr1-gw5 , in contrast to wt - ntsr1 , is an active - like 
 state that shows reduced propensity to go to the fully active r * conformation , 
 as defined by the structure of 2ar bound to g protein . the global motion analysis obtained from pca shows that ntsr1-gw5 
 is less dynamic and less flexible than wt - ntsr1 . the extent of the 
 movement of tm6 away from tm3 is higher in the wt - ntsr1 compared to 
 the ntsr1-gw5 mutant . the wt - ntsr1 has a lesser number of water molecules 
 near the putative na ion binding site in the tm1tm2tm7 
 region and near the well conserved motif npxxy in tm7 , showing that 
 the wt - ntsr1 in the active - like state shows more potential to be activated 
 than the ntsr1-gw5 . these analyses that signify activation are consistent 
 with the experimental observation that the wild type of ntsr1 activates 
 the g - protein but ntsr1-gw5 does not . in the gw5 mutant , the thermostabilizing mutations stabilized an 
 active - like receptor conformation , and in the process reduced the 
 flexibility of the wild type receptor that is essential for gpcr activation 
 and coupling to the g - protein . this is possibly due to the strong 
 interhelical hydrogen bonds and van der waals contacts that are formed 
 in the thermostable mutant due to the single point mutations leading 
 to the rearrangement of side chains in the neighboring residues . the 
 single point mutations also lead to improved lipid interactions with 
 residues within 7  of the mutation positions . there is formation 
 of a tight annular lipid bilayer around the ntsr1-gw5 compared to 
 wt - ntsr1 . thus , it is could be a challenge to design a gpcr thermostable 
 mutant that preserves the full signaling efficacy of the wild type 
 receptor , since it is difficult to decouple the thermostabilizing 
 effect from the conformational flexibility . our simulations showed 
 that the presence of helix 8 in the active - like ntsr1-gw5 conformation 
 leads to van der waals clash and instability .
the neurotensin receptor ntsr1 binds 
 the peptide agonist neurotensin 
 ( nts ) and signals preferentially via the gq protein . 
 recently , 
 grisshammer and co - workers reported the crystal structure of a thermostable 
 mutant ntsr1-gw5 with nts bound . understanding how the mutations thermostabilize 
 the structure would allow efficient design of thermostable mutant 
 gpcrs for protein purification , and subsequent biophysical studies . 
 using microsecond scale molecular dynamics simulations ( 4 s ) 
 of the thermostable mutant ntsr1-gw5 and wild type ntsr1 , we have 
 elucidated the structural and energetic factors that affect the thermostability 
 and dynamics of ntsr1 . 
 the thermostable mutant ntsr1-gw5 is found 
 to be less flexible and less dynamic than the wild type ntsr1 . 
 the 
 point mutations confer thermostability by improving the interhelical 
 hydrogen bonds , hydrophobic packing , and receptor interactions with 
 the lipid bilayer , especially in the intracellular regions . during 
 md , 
 ntsr1-gw5 becomes more hydrated compared to wild type ntsr1 , with 
 tight hydrogen bonded water clusters within the transmembrane core 
 of the receptor , thus providing evidence that water plays an important 
 role in improving helical packing in the thermostable mutant . 
 our 
 studies provide valuable insights into the stability and functioning 
 of ntsr1 that will be useful in future design of thermostable mutants 
 of other peptide gpcrs .