in the absence of a 
 vaccine and in lieu of a cure , antiretroviral 
 combination therapy has been the main form of treatment for individuals 
 infected with hiv .
as is the case with treatment of most rapidly evolving 
 viruses / diseases , drug resistance decreases the effectiveness of treatment . 

the high replicative capacity of hiv and the infidelity of the reverse 
 transcriptase quickly lead to a heterogeneous population of viruses 
 within patients , from which resistance has emerged to all 30 of the 
 currently used antiviral drugs .
hiv-1 protease inhibitors ( pis ) 
 have recently emerged as the most 
 effective drugs in the treatment of hiv .
pis are competitive active 
 site inhibitors that mimic the transition state of the enzyme and 
 are the most potent antiretroviral drugs for the treatment of hiv / aids .
these drugs are ideal for therapy as they target 
 the viral protease responsible for viral maturation and thus the spread 
 of the virus .
unfortunately , the rapid evolution of hiv-1 , coupled 
 with the selective pressure of therapy , results in many viable multidrug 
 resistant variants . in fact
, mutations at 45 of the 99 residues that 
 make up hiv-1 protease have been implicated in drug resistance .
while resistance due to mutations at 11 of these 
 45 residues can be explained as direct changes within the active site , 
 the resistance mechanisms for the majority of the remaining mutations 
 outside the active site of the enzyme mostly remain elusive .
drug resistance mutations in hiv-1 protease allow the enzyme to 
 become less susceptible to inhibition while retaining enzymatic activity . 

points of inhibitor  protease contact at residues within the 
 active site where the inhibitor protrudes beyond the substrate envelope 
 are sites selected for resistance , as their interactions are more 
 critical for inhibitor binding than substrate turnover . while mutations at some active site residues , 
 such as 82 and 84 , lead to resistance to all pis , other mutations 
 are signatures of specific inhibitors , such as d30n for nelfinavir 
 and i47a for lopinavir .
these mutations 
 directly impact inhibitor binding by altering or reducing contacts 
 necessary for inhibiting the enzyme , but can also simultaneously decrease 
 the catalytic efficiency or enzymatic fitness .
the mutations at the 
 remaining 34 of the 45 residues associated with drug resistance occur 
 outside the active site . these changes have often been considered 
 secondary or accessory mutations , and are thought to indirectly impact 
 inhibitor binding while assisting in enzyme fitness or stability . 
 structural studies on the effect of several hiv-1 protease secondary 
 mutations have provided insights into how inhibitor binding may be 
 affected .
however , for the most part , their specific role in protease inhibitor 
 resistance or mechanism of action has not been elucidated .
darunavir 
 ( drv ) is the most potent of the united states food and 
 drug administration ( fda ) approved hiv-1 protease inhibitors .
this 
 high potency combined with the inhibitor s fit within the substrate 
 envelope appears to account for drv s robustness against drug 
 resistance .
drug resistance to drv usually 
 occurs only in patients who have high levels of pre - existing pi resistance , 
 requiring at least seven mutations to simultaneously occur for therapeutic 
 failure .
in fact , drv is being investigated as a potential monotherapy 
 in treatment - nave patients . in drv - resistant hiv variants ,
single site mutations 
 can not confer high levels of resistance to drv , and a combination 
 of multiple mutations including those outside the active site are 
 needed to decrease potency .
however , the role of these mutations in 
 conferring resistance is not well understood : some may be enhancing 
 enzymatic activity , while others may directly confer drug resistance 
 and still others may be residual mutations from previous therapy history . 
 in this study , we examine some of the most common of these mutations , 
 v32i , l33f , l76v , and l90 m ( as a control ; not a signature of drv resistance 
 but frequent in multidrug resistance ) , 
 for their impact on drv inhibition . using a combination of static 
 and dynamic structural analyses , by determining crystal structures 
 of complexes and performing molecular dynamics simulations , we elucidate 
 the possible roles of these secondary mutations both independently 
 ( l76v , l90 m , v32i ) and in combination ( v32i / l33f ) in conferring resistance . 

we find how mutations at residues with no direct contact with the 
 inhibitor can alter the structure and dynamics of the protease to 
 affect inhibitor binding through common mechanisms , which we define 
 through a  network hypothesis  .
to determine 
 how mutations remote from the active site contribute 
 to drv resistance in hiv-1 protease , the impact of four mutations 
 ( l76v , l90 m , v32i , and v32i / l33f ; figure 1 ) 
 in a subtype b background was investigated in terms of enzyme inhibition , 
 inhibitor - bound crystal structures , and molecular dynamics simulations .
crystal structures 
 of mutant protease variants superimposed with the wt protease complex 
 structure in blue .
the enzyme inhibition 
 constant for 
 drv was measured against each of the protease mutants , in addition 
 to wt subtypes b and c for comparison ( table 1 ) .
drv is highly potent against wt subtype b protease with a ki of 2 pm , as we previously reported .
the level of inhibition for the mutants varied 
 from 2 pm to 45 pm , with the l90 m mutant being inhibited as potently 
 as the wt protease and the v32i / l33f double mutant exhibiting the 
 greatest decrease in susceptibility to drv with a fold - change greater 
 than 20 .
hence , single mutations are not enough to confer high levels 
 of drv resistance , as expected , and the mutations had varying degrees 
 of effects on drv susceptibility .
drv inhibition constants ( ki ) of hiv-1 
 protease variants , with fold - changes 
 relative to subtype b wt protease in parentheses .
the overall vdw 
 interaction energy between the inhibitor and protease was determined 
 from crystal structures . to structurally characterize the 
 effects of the mutations on drv binding , we determined the crystal 
 structures of variants l76v , l90 m , v32i , and v32i / l33f , which diffracted 
 to resolutions of 1.51.9  in the p212121 space group ( table 2 ) .
alignment of the four complex structures on our previously 
 determined structure of the wt protease  drv complex ( 1t3r ) showed that the variants had only minor backbone variations , 
 mainly in the 20s loop likely due to crystal packing differences ( figure 1 ) .
therefore , the mutations had very little impact 
 on the overall backbone structure of the protease . from surleraux 

the high - resolution cocrystal structures enabled detailed analysis 
 of protease  drv contacts in each of the five complexes .
the 
 wt complex had the most extensive van der waals ( vdw ) contacts with 
 the inhibitor with a favorable energy of 44.5 kcal / mol , similar 
 to v32i and l90 m variants ( table 1 ) .
the l76v 
 variant and v32i / l33f double mutant lost more than 1 kcal / mol in vdw 
 contact energy with drv relative to the wt complex .
thus , despite 
 no large - scale changes in the protease backbone , subtle changes in 
 repacking occurred around drv in these two complexes to weaken protease 
 interactions with the inhibitor .
however , the extent of contacts lost 
 with drv in the mutant crystal structures with respect to wt protease 
 does not correlate completely with the fold - change loses in ki values ( table 1 ) .
contacts involving specific drv moieties ( figure 2 ) and protease active site residues ( figure 3 ) were analyzed in detail . in general , the impact of mutations 
 on drv contacts are larger at the p2 and p2 than the central 
 p1 and p1 moieties .
the bis - thf group of drv p2 moiety forms 
 the most extensive contacts in all of the complexes ( figure 2 ) , but also loses considerable contacts due to the 
 mutations , except in the v32i structure . in the case of v32i ,
drv 
 contacts are retained as in the wt complex , consistent with no significant 
 change in total vdw or ki values ( table 1 ) .
when this mutation occurs together with l33f 
 in the double mutant though , contacts are lost in all three of p2 , 
 p1 , and p2 moieties . in the l90 m variant , although interactions 
 get weaker at the p2 position , gain of contacts at p1 compensate for 
 this loss yielding comparable total vdw contacts and susceptibility 
 to drv as wt protease .
( a ) chemical 
 structure of drv ( tmc114 ) with the inhibitor moieties p2p2 
 indicated .
( b ) vdw interaction energy ( kcal / mol ) of drv moieties for 
 contacts with the protease active site in the crystal structures , 
 and changes in vdw interaction energy in mutant structures relative 
 to the wt complex .
( a ) the two monomers of wt protease in surface representation 
 with the bound drv displayed as sticks .
active site residues are colored 
 from blue to red for increasing vdw contacts with the inhibitor .
the 
 monomer that interacts mostly with the p2p1 moieties of drv 
 is on the left , and the primed - side monomer is on the right .
( b ) the 
 vdw interaction energy of active site residues in crystal structures 
 ( top ) , and changes in mutant complexes relative to the wt structure 
 ( bottom ) .
only the residues displaying considerable changes relative 
 to wt are included for both monomers .
see figures s1 and s2 in the supporting information for all active site residues . 

while the apo form of the protease is a symmetric homodimer , 
 drv 
 induces asymmetry to the complex and thus despite identical residues 
 mutating in both monomers , the effect of these mutations on protease 
specifically , l76v and l90 m mutations cause considerable 
 loss of contacts at i47 , but to a lesser extent at i47. other 
 active site residues whose contacts are altered in mutant structures 
 include i50 at the tip of the flaps , and 818284 at 
 the 80s loop .
contrary to previous reports , we do not see any major enhancement of drv contacts with the catalytic 
 d25 in the l90 m mutant , or any of the other 3 variants .
residue 
 32 is at the periphery of the active site , and v32i mutation 
 causes a unique pattern of rearrangement of inhibitor contacts than 
 the other variants studied . unlike l76v and l90 m , contacts with 47 
 are retained in v32i . although the backbone is not shifted significantly , 
 the proximity of residue 32 to the 80s loop causes subtle rearrangements 
 to result in repositioning of the drv away from i84 s and more 
 toward i50s at the tip of the flaps . as a result ,
drv contacts with 
 residues 84 , 81 , and 84 are lost but those with 50 
 and 50 are enhanced .
the larger isoleucine also forms additional 
 contacts with the inhibitor in the unprimed - side monomer . in the v32i / l33f 
 variant , which loses an additional 7-fold in binding affinity relative 
 to v32i ( table 1 ) ,
in contrast with v32i alone , additional loss of interactions 
 at residues 47 and 50 are observed .
these losses of contacts are similar 
 to the alterations observed in the l76v and l90 m variants .
thus , the 
 double mutant v32i / l33f variant alters the active site in a synergistic 
 manner , leveraging both alterations similar to l76v and l90 m , and 
 some changes from v32i .
the change in variants affinity is 
 not simply due to a loss of van der waals contacts , but an interdependent 
 change in optimal contacts . in the wt complex ,
drv forms a network 
 of hydrogen bonds within 
 the active site involving both backbone and side chains .
most of these 
 bonds , including the two water - mediated ones with i50 , are conserved 
 in the variant complexes .
two exceptions occurred in the l76v and 
 v32i complexes : consistent with the loss of vdw contacts , in the l76v 
 complex a hydrogen bond to the backbone of d30 is lengthened from 
 2.0 to 3.0  . in the v32i variant , an additional water - mediated 
 hydrogen bond with the side chain of d30 is formed .
nevertheless , 
 overall the hydrogen bonds with drv within the various complexes are 
 conserved .
analysis of crystal 
 structures above revealed that the mutations away from the active 
 site are able to influence interactions of drv - contacting residues 
 at the active site .
the alterations in vdw contacts or hydrogen bonds 
 lost , however , only partly correlate with the experimentally determined 
 enzyme inhibition constants .
another possible mechanism by which these 
 secondary mutations could alter inhibitor binding is by influencing 
 the dynamic ensemble of the enzyme .
starting from the crystal 
 structures of the drv complexes , three replicates of fully hydrated 
 10 ns md simulations of each drv complex were performed and analyzed . 

root - mean - square deviations ( rmsd ) of c atoms during the simulation 
 and the average root - mean - square fluctuations ( rmsf ) about their mean 
 positions readily reveal that the secondary mutations alter the overall 
 enzyme dynamics ( figure 4a ) .
the l90 m and v32i / l33f 
 variants display larger fluctuations throughout the enzyme compared 
 to the other variants , although the catalytic d25 stays relatively 
 rigid in both monomers .
these altered fluctuations are not restricted 
 to the sites of mutation , but propagate throughout the enzyme .
( a ) rmsd of c atoms from the initial positions , and rms fluctuations 
 of residues averaged over three 10 ns trajectories .
( b ) significantly 
 altered change in distance between residue pairs around the active 
 site relative to wt complex , sampled during the md simulations ; increased 
 and decreased distances are indicated by blue and red , respectively . 

see figure s3 for the distance distributions . to further analyze the impact 
 of mutations on the dynamic ensemble 
 sampled by the protease , the distance distributions were calculated 
 across the active site at a variety of positions ( figure 4b and figure s3 ) . in 
 all the variant complexes , the dynamic ensemble sampled by the protease 

many of the distances displaying a significant 
 change are longer than the wt distance , indicating a widening of the 
 active site . in the l76v complex , the changes are highly asymmetric , 
 with one side of the active site constricting and the other widening 
 ( figure 4b ) . in all variant complexes ,
the 80s loops in both monomers 
 form the  side walls  of the active site .
relative to 
 the wt complex , the distance between residues 81 in the two monomers 
 are shorter , and that between 8484 are longer in all 
 variants .
part of the side walls 
 are closer and the  lower  part is farther away in the 
 mutant complexes compared to the wt . in addition to the 80s loop
, 
 certain distances involving residue 50 at the tip of the flaps , and 
 even the catalytic d25 are altered in the variant complexes .
the catalytic 
 site is the most invariant and dynamically restricted region of the 
 protease , both when different crystal structures are compared and 
 dynamics analyzed by simulations and nmr experiments .
therefore , widening of the d25d25 distance in the 
 v32i / l33f double mutant is an unusually profound impact of remote 
 changes on the catalytic region .
the md simulations also permitted 
 a detailed analysis of the interaction 
 network both for direct interactions within the active site to drv 
 and the internal hydrogen - bonding network throughout the enzyme ( figure 5 , figures s4s6 ) . throughout the md simulations the wt complex maintains a network 
 of stable hydrogen bonds .
starting from the bottom of the enzyme the 
 c - terminal -helix forms a network of hydrogen bonds that links 
 the termini of the protein to the flap regions .
the backbone of residue 
 95 links to residue 90 which in turn contacts residue 86 , residue 
 88 bridges to residues 29 , 31 , and 74 , and residue 76 bonds to both 
 residues 31 and 33 which is bonded to residue 78 .
the hydrogen 
 bonds linking residues 47 and 54 within the flaps stay tightly hydrogen 
 bonded throughout the simulation .
thus , as we previously observed , the hydrogen bonding network is stably retained 
 within the wt md simulation .
( a ) crystal structure 
 of drv bound to the active site , and only one monomer of the protease 
 is shown for clarity .
the sites of mutation ( l76 , l90 , v32 , and l33 ) 
 have colored side chains .
( b ) histograms of the changes of the percentage 
 time hydrogen bonds are formed relative to the wt simulation for each 
 of the complexes .
( c ) schematic hydrogen bond network of the hiv-1 
 protease dimer with the percentage time hydrogen bonds are formed 
 during the wt simulation ( figure s4 ) .
( d ) 
 schematic representation of the v32i_l33f complex simulation with 
 the change in hydrogen bonding relative to the wt simulations .
the 
 remaining variants schematic are shown in figure 
 s5 . in comparing the simulations of 
 the variant drv complexes with 
 the wt ,
subtle changes are seen in the vdw contacts within the active 
 site ( figure s6 ) , similar to what was observed 
 in the crystal structures .
however , in each of the four variants , 
 with the notable exception of the 4754 linkages , the hydrogen 
 bond network is disrupted to a greater or lesser extent asymmetrically , 
 including the direct hydrogen bonds with drv ( figure 5b , s4 , s5 ) .
the v32i / l33f variant 
 is the most disrupted with 12 hydrogen bonds changing by greater than 
 20% relative to the wt complex throughout the dimer , with 11 being 
 weakened ( figure 5d ) including most dramatically 
 the interactions of the side chain of asn 88 .
eight of these changes 
 are within the monomer that coordinates the highly rigid bis - thf moiety 
 including weakened interactions at points of contact between the protein 
 and drv .
thus , mutations distal to the active site often weaken the 
 strength of the hydrogen bonds in the network , which is propagated 
 through to the active site including altering vdw packing , pushing 
 the flaps , and thereby the contact of i47 with drv .
taken together , 
 one can decipher how contacts between protein and inhibitor are affected 
 by these changes even for residues that are packed through vdw contacts 
 or covalently linked along the backbone and are not directly involved 
 in the hydrogen bonding network ( figure 5a 
 and c ) .
hiv-1 protease evolves 
 in complex combinations to evade inhibition , 
 but still maintains biological function .
however , in highly resistant variants , active 
 site mutations often coexist with mutations outside the active site . 

this is particularly necessary when resistance is achieved to the 
 highly potent inhibitor drv , which fits well within the substrate 
 envelope .
however , the role of these changes outside the active site 
 has long been thought to be only in recovering viral fitness , or protease 
 stability . in the current study , we have primarily chosen enzyme 
 variants 
 that are associated with drv resistance : l76v , v32i , and l33f . although l76v causes only a 1.5-fold decrease 
 in drv binding affinity ( table 1 ) , this mutation 

is often observed in highly mutated drv - resistant variants , as well as variants with hyper - susceptibility to other pis .
l33f 
 is a highly networked mutation co - occurring with many others in highly 
 drug - resistant patient isolates , often together with v32i .
therefore , comparison of v32i and the v32i / l33f 
 double mutant permits the context - dependence of mutational effects 
 in drug resistance .
while not directly associated with drv resistance , 
 l90 m is a canonical highly networked mutation that typically arises 
 in multidrug resistant proteases .
the large and rigid p1/p1 
 moieties in nfv and sqv have been implicated in susceptibility to 
 l90 m , a feature lacking in drv .
l90 m 
 has been found in more than half of patient isolates with at least 
 one pi resistance substitution , and hence is often present in patients 
 needing drv - based salvage therapy .
thus , 
 elucidating the physical impact of these secondary mutations on drv 
 binding provides a detailed perspective on how the enzyme accommodates 
 such frequently observed changes .
specifically , we find that 
 mutations outside the active site impact 
 inhibitor binding thereby playing a direct role in conferring drug 
 resistance .
compared to the wt complex , the overall structure and 
 backbone conformations are very similar in the cocrystal structures 
 of the variant complexes .
however , the mutations cause subtle but 
 significant rearrangements in the structure to cause altered interactions 
 with the bound inhibitor , as well as impacting the dynamic ensembles 
 of these complexes . we had previously hypothesized and tested that alterations 
 in the hydrophobic core of the enzyme could alter the conformational 
 dynamic ensemble through changes in the hydrophobic sliding of internal 
 residues potentially impacting drug resistance .
this impact on dynamics 
 is not localized to the points of mutation but would propagate throughout 
 the enzyme .
in the present study we hypothesize these mutations outside 
 the active site share a common pathway of altering the overall enzyme 
 dynamics and propagating their effects to the active site .
although 
 the resistance - associated mutations are located at a variety 
 of positions in the protease and away from the active site , they all 
 may utilize a common mechanism or pathway of altering the protease 
the mutations cause subtle changes through the repacking 
 of the active site ; in particular , these are observed in the crystal 
 structures at residues 47 , 50 , and 84 in both monomers , and also observed 
 in the md analysis ( figure s6 ) . within 
 the crystal structures of both the l76v and l90 m complexes
residue 
 i47 , which is located in the flap , loses contact with drv .
in contrast , 
 in the v32i complex i47 contacts are retained , while this loss is 
 restored when l33f occurs in v32i / l33f ( these changes are also observed 
 in subtle differences in the md simulation figure 
 s6 ) .
interestingly , v32i and i47v are the second most frequent 
 pair of residues often found to coevolve , thus compensating for each 
 other .
mutations at i47 , together with 
 i54 , which its backbone is hydrogen bonded with , is a major drv resistance 
 site . among about 30 total active site residues that contact drv
, 
 i47 is consistently the residue whose contacts are affected the most 
 in l76v , l90 m , and v32i / l33f variants ( figure 
 s3 ) .
these results suggest that the interactions of residue 
 47 with inhibitors within the active site may represent a pivotal 
 site in conferring drug resistance to pis , and these interactions 
 can be altered by changes propagated through the enzyme from remote 
 sites .
in addition to repacking around the inhibitor in the 
 crystal structures , 
 the secondary mutations share a common pattern of altering the dynamic 
 ensemble sampled by the protease , and the shape of the active site . 

overall in the dynamic ensemble of the v32i and the v32i / l33f variants 
 the active site is expanded , with the double mutant expanding the 
 active site more , while l76v active site contracts and the l90 m active 
 site displays asymmetric changes .
hence , even though not located at 
 the active site , mutations at all these remote sites affect the shape 
 of the active site in the dynamic ensemble .
how are single mutations 
 at a remote site able to alter interactions 
 and dynamics of the active site with highly common molecular mechanisms ? 

we propose a  network hypothesis  where the perturbation 
 introduced by mutation of a distal residue is propagated to the active 
 site through a network of interactions within the protease structure 
 ( figure 5 ) .
the distal mutation sites we studied 
 are all part of a hydrogen - bonded network connecting to the active 
 site where the inhibitor binds .
our network hypothesis postulates 
 that the mutations have similar effects and common mechanisms as they 
 all cause a rearrangement of this same network .
this hypothesis is 
 supported by the alterations observed during the md simulations in 
 the stability of the hydrogen bonding networks ( figure 5 ) , where changes propagate from residues 7478 and 
 8790 , through 2833 , to 8485 and 25 .
this altered 
 interaction network includes repacking of the vdw contacts with residues 
 47 and 54 , which are pivotal in linking the networked residues to 
 the rearrangement of the flaps , residues 29 and 30 that directly hydrogen 
 bond to drv , and 82 and 84 that are key sites within the active site 
 cavity .
we hypothesize that all these are the active site residues 
 where the impact of distal mutations is propagated as a common mechanism 
 of resistance in all variants and their subtle rearrangements can 
 cause inhibitor specific resistant changes .
these common mechanisms 
 provide an explanation for why some mutations 
 are redundant and thus are not observed together in patient sequences , 
 while others are synergistic and occur together to confer higher levels 
 of drug resistance as they impact one another at pivotal sites that 
 confer resistance often through expanding the active site
. this hypothesis 
 does not exclude the possibility that some changes may still provide 
 additional stability , increasing the combined fitness of the variants . 

most significantly , our findings show that all of the mutations we 
 have studied , although outside the active site , still directly alter 
 the shape and flexibility of the active site , thus likely play a direct 
 role in conferring resistance .
each of the four protease 
 mutants was constructed using a standard site - directed mutagenesis 
 protocol on a wt - hxb2 protease gene with a codon sequence optimized 
 for e. coli expression . the wt pr gene contained 
 the amino acid substitution q7k to minimize the enzyme s autoproteolytic 
 activity .
briefly , the mutant hiv-1 protease gene was cloned into 
 the pxc-35 plasmid , which was then transformed into the tap56 strain 
 of escherichia coli .
transformed cells were grown 
 in 6  1 l cultures from which cell pellets were harvested 3 
 h after induction .
the cell pellets were lysed and the protease was 
 retrieved from inclusion bodies with 100% glacial acetic acid .
the 
 protease was separated from higher molecular weight proteins by size - exclusion 
 chromatography on a sephadex g-75 column .
the purified protein was 
 refolded by rapid dilution into a 10-fold volume of 0.05 m sodium 
 acetate buffer at ph 5.5 , containing 10% glycerol , 5% ethylene glycol , 
 and 5 mm dithiothreitol ( refolding buffer ) .
the protease solution 
 was concentrated , followed by dialysis to remove any remaining acetic 
 acid .
protease used for crystallization was further purified with 
 a pharmacia superdex 75 fast - performance liquid chromatography column 
 equilibrated with refolding buffer .
crystals were set up with 
 a 5-fold molar excess of inhibitor to protease , which ensures ubiquitous 
 binding .
the final protein concentration ranged from 0.8 to 1.6 mg / ml 
 in refolding buffer .
the hanging - drop method was used for crystallization 
 as previously described . for the l76v , 
 l90 m , and v32i mutants ,
the reservoir solution consisted of 126 mm 
 phosphate buffer at ph 6.2 , 63 mm sodium citrate , and ammonium sulfate 
 at a range of 2429% . for the v32i / l33f double mutant ,
the 
 reservoir solution consisted of 0.1 m citrate - phosphate buffer , 7% 
 dmso , and 2530% ammonium sulfate .
a substrate 
 peptide mimicking the ma - ca ( p17-p24 ) cleavage site labeled with k - e(edans)-s - q - n - y - p - i - v - q - k(dabcyl)-r 
 ( 0.5 m , final concentration ) was added just prior to the reading 
 to each well containing 50 nm of pr and varying concentrations of 
 inhibitor . the fret pair ( edans , the donor and dabcyl , the quencher ) 
 was attached to the indicated amino acids of the peptide ( molecular 
 probes ) .
fluorescence intensity increase upon hydrolysis of the fluorogenic 
 substrate was monitored at 490 nm ( emission of edans ) from the highest 
 inhibitor concentration to the lowest , as well as the no inhibitor 
 control well .
initial velocities were obtained from the progress 
 curves and plotted against inhibitor concentration to get inhibition 
 curves .
resulting curves were globally fitted to morrison s 
 equation to obtain the ki value , as described 
 previously .
the maestro component 
 of the schredinger software suite 
 was used to analyze the hydrogen bonds between the inhibitor and the 
 protease residues and neighboring waters after optimization of the 
 complex structure .
briefly , a hydrogen bond was defined by a distance 
 between donor and acceptor of < 3.5  and a donor - hydrogen 
 acceptor angle of > 120. the vdw contacts between the inhibitor 
 and protease were calculated using a simplified lennard 
the md simulations 
 were performed using 
 the program sander in the amber 8 package , as previously described .
a set of three simulations was run for each 
 of the four mutants and the wt - pr yielding a total of 15 trajectories 
 for analysis .
each simulation was assigned initial velocities according 
 to the maxwellian distribution and random seeds were assigned with 
 five different values for each pr .
an in - house script was used to 
 determine the intra and intermonomeric c distances between 
 various residues using the trajectories . to calculate the hydrogen 
 bond duration between various residues within the network from the 
 simulations , the visual molecular dynamics ( vmd ) version 1.9.1
vmd was used to write out the trajectory 
 in a pdb format using the coordinates and trajectory files generated 
 by ptraj from the amber simulation software .
vmd was also used to 
 generate the trajectory pdb files to determine the vdw contact energies 
 over the simulations .
the in - house vdw script was then modified to 
 assess vdw contacts from the simulations .
once the robustness of 
 the system was assessed , the three trajectories for each system were 
 concatenated into one file containing 1500 frames and the total vdw 
 contacts were analyzed .
 hiv-1 protease inhibitors are part 
 of the highly active antiretroviral 
 therapy effectively used in the treatment of hiv infection and aids . 
 
 darunavir ( drv ) is the most potent of these inhibitors , soliciting 
 drug resistance only when a complex combination of mutations occur 
 both inside and outside the protease active site . with few exceptions , 
 
 the role of mutations outside the active site in conferring resistance 
 remains largely elusive . through a series of drv  protease complex 
 crystal structures , inhibition assays , and molecular dynamics simulations 
 , 
 we find that single and double site mutations outside the active site 
 often associated with drv resistance alter the structure and dynamic 
 ensemble of hiv-1 protease active site . 
 these alterations correlate 
 with the observed inhibitor binding affinities for the mutants , and 
 suggest a network hypothesis on how the effect of 
 distal mutations are propagated to pivotal residues at the active 
 site and may contribute to conferring drug resistance . 
Introduction
Results
Discussion
Methods