eukaryotic topoisomerase i is a monomeric enzyme that catalyzes the relaxation of supercoiled dna during important processes including dna replication , transcription , recombination and chromosome condensation ( 13 ) .
human topoisomerase i ( htop1 ) is composed of 765 amino acids , and proteolytic experiments have shown that the enzyme is composed of four different domains : n - terminal domain ( residues 1214 ) , core domain ( residues 215635 ) , linker domain ( residues 636712 ) , and c - terminal domain ( residues 713765 ) ( 4 ) .
the 3d structure of the enzyme without the n - terminal domain ( topo70 ) , complexed with a linear double - helix dna substrate , has permitted a further classification of the core domain in subdomain i ( residues 215232 and 320433 ) , ii ( residues 233319 ) and iii ( residues 434635 ) , and has shown that topoisomerase i is a bi - lobed protein that clamps completely around duplex dna through protein 
one lobe is constituted by the c - terminal catalytic domain and core subdomain iii , the other one ( called  cap ) by core subdomains i and ii .
the protein forms a substantially closed clamp between loops , designated lip1 and lip2 , belonging to core subdomains i and iii , respectively .
the two lips interact directly through a non - covalent interaction between the carboxylic lateral group of glu497 and the side - chain amino group of lys369 ( 5 ) .
the catalytic cycle of the enzyme is composed of five steps : dna association , cleavage , strand rotation , ligation and dissociation ( 6 ) . a complete understanding of the dna recognition event and the conformational changes that follow dna binding would require determination of the 3d structure of the enzyme in the free and dna - bound states .
large domain movements are required for the protein to bind the supercoiled dna substrate , likely through the flexible hinge that has been hypothesized to be located between the  cap and subdomain iii ( helices 8 and 9 ) ( 5 ) .
indeed a molecular dynamics ( md ) simulation study suggested the occurrence of a movement of 1014   between the lips , during the dna rotation step ( 7 ) .
two articles have demonstrated that introduction of two cysteines on the two lips , by site - directed mutagenesis , traps htop1 in the closed conformation , through a disulfide bridge upon dna addition , confirming the existence of ample conformational changes ( 8,9 ) . until now
, the structure of the protein has been solved only in the closed form , when it is fully embracing the dna , and no information is available concerning the preferred conformation in the absence of the substrate .
a paper describing the open state of the topoisomerase ib from deinococcus radiodurans has recently appeared ( 10 ) .
the bacterial enzyme is much smaller than the human one , being constituted by a c - terminal domain of about 250 residues , comparable to the core subdomain iii and c - terminal domain of the human enzyme , and by an n - terminal domain of about 90 residues that has no counterpart in the human enzyme .
the two domains are splayed apart in an open conformation in which the surface of the catalytic domain containing the active site is exposed for dna binding . in the case of the human enzyme ,
the internal flexibility of the protein in the absence of dna , and the consequent conformational heterogeneity , makes its crystallization and analysis through x - ray diffraction very difficult .
md simulation is a powerful technique that is widely used to probe the flexibility of proteins , dna , and protein  dna complexes , and also has been used to examine the properties of dna - topoisomerase i complexes ( 1114 ) . starting from the htop1
closed crystallographic structure , the flexibility of the different domains have been monitored , but it has not been possible to monitor the so - called open state conformation , because of the strict non - covalent interactions occurring between the protein domains and dna ( 11 ) .
md can also be applied to sample large conformational changes after introducing a perturbation or a force that imposes the system to sample events occurring on a timescale not accessible by the current computational resources .
this approach has been used to study the conformational changes of several proteins ( 15,16 ) , and to propose a model for the relaxation of positive and negative dna supercoils in the htop1dna complex ( 7 ) . in this work ,
we have carried out a simulation of the human topoisomerase enzyme , starting from the closed configuration coordinates of the topo70-dna non - covalent complex after elimination of the 22-bp dna duplex oligonucleotide coordinates .
a repulsion force between the two lips has been introduced for a short time to induce the destabilization of the local minimum , and then an unperturbed simulation was carried out for 10  ns .
analysis indicates the occurrence of a large reorientation of the different domains that however , fully maintain their secondary and tertiary structures .
beside such a large conformational change , which induces movements as large as 8090   between the domains , the active site residues remain preassembled , and the open structure of the protein displays a vast positive region with the active site located nearly at its center , perfectly suited to interact with the negatively charged dna substrate .
the initial configuration of htop1 was modeled obtaining the starting position for residues 215633 and 641765 from the crystal structure 1a36 ( 6 ) , and those for residues 203214 from the crystal structure 1ej9 ( 17 ) .
the seven residues constituting the loop region that connects the linker to the core domain ( residues 634640 , which are lacking in the 1a36 pdb structure because of thermal fluctuation ) were added to the system by molecular modeling , as already described ( 14 ) , using the sybyl program ( st louis , mo ) .
the same procedure has been followed to reconstruct the partially missing side chains of the residues not fully detected in the x - ray diffraction structure .
the spatial environment of each new residue was checked for close contact or overlap with neighboring residues , and stereochemical regularization of the structures was obtained by the powell minimization method implemented in the sybyl program .
the system was modeled with the amber95 all - atom force field ( 18 ) , and placed in a rectangular box ( 111    152    133 
the dimension of the box was sufficiently large to accommodate the protein also in the open state , without any interactions with its periodic images .
the system was , in fact , simulated in periodic boundary conditions , using a cutoff radius of 9   for the non - bonded interactions , and updating the neighbor pair list every 10 steps .
the shake algorithm ( 22 ) was used to constrain all bond lengths involving hydrogen atoms .
optimization and relaxation of solvent and ions were initially performed , keeping the solute atoms constrained to their initial position with decreasing force constants of 500 , 25 , 15 , and 5  kcal/(mol  ) .
the system has been simulated at a constant temperature of 300  k using berendsen 's method ( 23 ) and at a constant pressure of 1  bar with a 2-fs time step .
simulation a refers to the protein in the closed state without the dna double strand and in the presence of 21 cl counterions to neutralize the system ( a total of 219  655 atoms , with 9415 protein atoms and 70  073 water molecules ) ; in simulation b , the ionic strength has been increased introducing 21 atoms of na and 21 more cl atoms ( a total of 219  487 atoms , with 9415 protein atoms and 70  003 water molecules ) ; in c , a positive charge value has been given to the lateral chain of the glu 497 residue to introduce a repulsion with the close lys369 in the opposite lip ; in d , the repulsion between the two lips has been increased , introducing a positive charge value also to the main - chain carbonyl group of glu497 and to the lateral chain carbonyl group of asn366 ; in the final part of the simulation ( simulation e ) , the charge perturbation was eliminated and an unperturbed simulation has been carried out for 10  ns . 

table 1.conditions and lengths of the different simulations carried out for htop1simulationabcdecharge perturbationnoneglu497glu497  and asn 366nonetime ( ps)350050020020010  000main - chain perturbation.side-chain perturbation .
conditions and lengths of the different simulations carried out for htop1 main - chain perturbation .
the atomic rmsf have been computed by using the following definition : 

 where the averages have been computed over the equilibrated md trajectory of simulation a , i.e. from 500 to 3500  ps .
the per - residue root mean square deviations ( rmsds ) between two aligned structures x and y have been computed by using the following definition : 

 where the distances between all atoms in a certain residue are averaged .
the average per - residue rmsd for simulation e has been calculated by least - square fitting a group of main chain atoms to their initial position , calculating the per - residue rmsd with the previous equation , and averaging the resulting value on the entire 10  ns simulation time .
all the analyses have been carried out using the gromacs md package version 3.3.1 ( 24 ) .
figures 2 , 4 and 5 , and the movies of md dynamics , shown as supplementary data at nar online , were created using the vmd visualization package ( 25 ) .
in the first 3500  ps of simulation in the absence of dna , defined here as simulation a , the protein is maintaining a conformation very close to the crystallographic state , being the largest rmsd calculated on the main chain atoms of 3.5   .
this value is very close to that found for the protein simulated in complex with the dna substrate for an identical simulation time length ( 13 ) .
this result indicates that the closed configuration of the protein is very stable , independently of the presence of dna , and that the intra - domain and inter - domain protein interactions are sufficient to maintain the closed conformation .
also , the overall flexibility of the protein with or without dna is quite similar , as can be observed in figure 1 , where the per - residue rmsf in the absence of dna , evaluated for the last 3000  ps of simulation a , is compared to the rmsf evaluated on the same time window for the simulation previously carried out on the protein  dna complex ( 13 ) .
the maxima and minima of the rmsf are identical in the two simulations , being very similar to the protein regions characterized by the largest flexibility .
in fact , in the absence of dna , the linker and the loop connecting -strands 12 and 13 centered on gly520 are the regions characterized by the largest fluctuations , as already observed for the enzyme in the closed conformation in the presence of dna ( 12 ) .
differences are observed in the absolute values of the rmsf of definite regions , such as the  nose - cone helices ( helices 5 and 6 in core subdomains ii and i , respectively ) and the small loop at the n - terminal of the linker domain ( residues pro636-met645 ) , which show larger rmsfs in the simulation without dna than those observed in the simulation of topo70 in complex with dna ( figure 1 ) . residues 634640 are known to be highly flexible , and are not detected in several topoisomerase 
dna complex crystals because of their thermal fluctuations , while they are seen in the structure of the topo70dna  topotecan ternary complex ( 26 ) .
this region seems to display a scale of disorder , being more ordered in the ternary topo70dna  topotecan complex , less ordered in the binary topo70dna complex and even more disordered in the absence of dna . on the contrary ,
the maximum fluctuations , located in the loop of the linker domain between helices 18 and 19 , are less ample in the simulation without the dna . 

figure 1.average per - residue rmsf represented as a function of the residue number of htop1 in the absence of dna for the initial unperturbed simulation a ( full line ) in comparison with the ones previously calculated for the protein in complex with dna ( dashed line ) . 

figure 2.four snapshots of the final unperturbed simulation e taken at different times , showing the opening of the protein .
core subdomains i , ii and iii are rendered in yellow , blue and red , respectively . linker and c - terminal domains are rendered in green and cyan , respectively .
the hinge residue glu445 is indicated by an arrow in panel d. average per - residue rmsf represented as a function of the residue number of htop1 in the absence of dna for the initial unperturbed simulation a ( full line ) in comparison with the ones previously calculated for the protein in complex with dna ( dashed line ) .
four snapshots of the final unperturbed simulation e taken at different times , showing the opening of the protein .
core subdomains i , ii and iii are rendered in yellow , blue and red , respectively . linker and c - terminal domains are rendered in green and cyan , respectively .
the hinge residue glu445 is indicated by an arrow in panel d. per - residue - rmsds ( data not shown ) give higher values in the simulation in the absence of dna , for the linker domain and the nose - cone helices , which move away from the space previously occupied by dna .
both these regions have been shown to form several direct and water - mediated contacts with the dna phosphate groups in the strands downstream of the scissile base ( 5,11,13 ) .
it is worth noting , therefore , that the greater changes of dynamics and structure , in the absence of dna but in the closed conformation , are located in the two regions involved in the controlling of the rotation of the scissile strand during the relaxation step .
the x - ray diffraction study describes a single salt bridge direct contact between lys369 and glu497 ( 5 ) . in the simulation in the absence of dna , a new interaction is observed from 900 and 2500  ps between the lateral chain of lys369 and the carbonylic main - chain group of glu497 , with a distance of 3   . after this time , the distance between these two groups increases to 5   and the lateral chain of asn366 and the asp500 main chain start to interact , their distance being 3   ( see figure 7 , in supplementary data ) .
it is interesting to note that this new inter - lip interaction is also observed in the last part ( from 1 to 3.5  ns ) of the simulation of the protein in the presence of dna ( 13 ) .
a perturbation of the inter - lip interactions found at the end of simulation a , occurring between charged or polar groups ( glu497coo ; glu497co ; asn366od1asp500nh ) , has been attempted by increasing the ionic strength of the system . in details , 21 na and cl atoms
have been added to the system , which has been simulated for 500 ps ( simulation b ) . however , since the energetic stability of the inter - lip interactions is not sufficiently perturbed by the increase of ionic strength ( see figure 7 in supplementary data ) , a not physical perturbation ( i.e. change of electrostatic charges ) has been introduced in the system , in order to create a destabilization within a simulation time compatible with the currently computational resources .
it is interesting to note that the introduction of a positive charge replacing the negative one at the carboxylic group of glu497 is not enough to push the two lips far away from each other .
in fact , the introduction of the positive charge on the coo group of glu497 leads to an increase of the glu497lys369 distance because of electrostatic repulsion ( black line in figure 7 in supplementary data , simulation c ) . however , lys369 changes its molecular partner and forms a strong interaction with the glu497 main - chain carbonyl group , as evidenced by the decrease of the   relative distance of the glu497 main - chain and lys369 lateral chain , respectively ( red line in figure 7 in supplementary data , simulation c ) .
the same occurs for the interaction between the lateral chain of asn366 and the main - chain of asp500 ( green line in figure 7 in supplementary data ) .
therefore , the destabilization at the level of the salt bridge between glu497 and lys369 lateral chains , due to the introduction of the positive charge on the glu497 lateral chain , is compensated for by the strengthening of other lip1lip2 interactions that allow the protein to maintain a stable closed configuration . only after the introduction of two additional positive charges , one on the carbonyl group of glu497 and the other one on the lateral chain of asn366 ,
the two lips start to separate each other , as can be observed by monitoring the above - mentioned distances in simulation d ( figure 7 in supplementary data ) .
the system has been allowed to evolve with this additional perturbation for 200  ps . after this time
, the positive charges have been eliminated , bringing the modified residues charges back to their initial values .
the minimum distance between the lips , in the first configuration of the following unperturbed simulation ( simulation e ) , is only 6.5   , and the overall rmsd of this structure is 3.5   from the initial x - ray configuration ( deviation of the main - chain atoms fitted on the main - chain atoms themselves ) . the initial configuration and
a dynamic visualization of the md dynamics is available as supplementary data at nar online .
the first important observation is that the protein domains change their relative position , reaching an open conformation able to bind and trap the dna substrate , but the secondary and tertiary structures of the single domains are very well conserved during the entire simulation period .
this is an important observation , since it implies that the different domains act as independent and internal coherent units that maintain their overall structure , although strongly changing their relative orientation .
core subdomains i and ii ( colored yellow and blue , respectively ) show coordinated motion , separated from that of core subdomain iii ( colored red ) .
this last subdomain , on the other hand , remains strongly interacting with the c - terminal domain ( colored cyan ) throughout the simulation .
a quantitative analysis of the relative protein domain motion is represented in figure 3 , where the average per - residue rmsd has been calculated , overlapping the protein on the core subdomains i and ii ( black line ) , the core subdomain iii ( red line ) , the linker ( green line ) , or the c - terminal domain ( blue line ) .
the analysis indicates a very strong and coherent motion of the core subdomains i  ii with respect to the other protein domains , with maximum rmsd ( about 100   ) when the protein is fitted on the linker domain .
the destabilization of the lip interactions permits a rotation of the core subdomains i  ii nose - cone helices , initially orientated parallel to the linker domain ( figure 2a ) , of nearly 90 after 2.5  ns ( figure 2b ) and of 180 after 5  ns ( figure 2c ) .
this motion permits the opening of the two halves that embrace the dna , formed by core subdomains i 
ii on one side and core subdomain iii and c - terminal domain on the other side .
ii continues during the last 5  ns of the simulation time , although with a lower extent .
the region that permits these relative domain motions is the n - terminal portion of helix 8 ( residues 434444 , figures 2d and 4 ) , and can be identified also by the intersection of the black and red lines in figure 3 .
the c - terminal portion of helix 8 , on the contrary , remains strongly interacting with helix 16 located in the c - terminal domain , during the whole opening dynamics ( figure 4 ) .
these two regions remain in close contact because of the presence of a cluster of hydrophobic residues . on the opposite site of this portion of helix 16
is located arg590 , a residue belonging to the catalytic pentad ( 27 ) that , due to these hydrophobic interactions , is maintained in an orientation similar to the one found in the crystallographic structures .
actually , all five active site residues maintain , for the whole simulation time , an orientation very similar to the one found in the closed configuration detected through x - ray diffraction ( 5 ) .
the similar orientation can be appreciated in figure 5a where the active site region of the last frame of simulation e ( gray ) is overimposed on the x - ray structure ( red ) . as a matter of fact ,
the largest deviation of the active site backbone atoms from the 1a36 crystallographic structure is only 1.7   .
the recent 3d structure of deinococcus radiodurans ( dra ) topoisomerase ib , the only one crystallized in an open conformation in the absence of dna , has indicated that the five active site residues are preassembled , being in a position nearly identical to those found in the dna - bound human htop1 ( 10 ) . in line ,
the orientation of the active site residues of the human enzyme in our simulation is also very similar to that of the bacterial enzyme , as shown in figure 5b , where the last frame of simulation e ( gray ) is overimposed to the structure of dra ( blue ) . 

figure 3.average per - residue rmsd of htop1 for the final unperturbed simulation e represented as a function of the residue number .
the average per - residue rmsd have been calculated overlapping the protein on the initial position of the core subdomains i and ii ( black line ) , the core subdomain iii ( red line ) , the linker ( green line ) or the c - terminal domain ( blue line ) . 

figure 4.snapshot of the final configuration of simulation e , showing only the core subdomain iii , the c - terminal , and the linker domain regions of the protein .
helices 8 , 9 and 16 and the catalytic arg590 are highlighted in the figure to evidentiate the hydrophobic interactions between the c - terminal portion of helix 8 and helix 16 .
figure 5.(a ) superimposition of the active site region of the last frame of simulation e ( gray color ) with the human enzyme x - ray structure ( red color ) .
( b ) superimposition of the active site region of the last frame of simulation e ( gray color ) with the deinococcus radiodurans enzyme x - ray structure ( blue color ) .
average per - residue rmsd of htop1 for the final unperturbed simulation e represented as a function of the residue number .
the average per - residue rmsd have been calculated overlapping the protein on the initial position of the core subdomains i and ii ( black line ) , the core subdomain iii ( red line ) , the linker ( green line ) or the c - terminal domain ( blue line ) .
snapshot of the final configuration of simulation e , showing only the core subdomain iii , the c - terminal , and the linker domain regions of the protein .
helices 8 , 9 and 16 and the catalytic arg590 are highlighted in the figure to evidentiate the hydrophobic interactions between the c - terminal portion of helix 8 and helix 16 .
( a ) superimposition of the active site region of the last frame of simulation e ( gray color ) with the human enzyme x - ray structure ( red color ) .
( b ) superimposition of the active site region of the last frame of simulation e ( gray color ) with the deinococcus radiodurans enzyme x - ray structure ( blue color ) . in our simulation , despite the huge inter - domain protein motion that spans up to 100   , the active site residues fully maintain their relative orientation , indicating that , in the human enzyme also , the active site is preassembled and in the appropriate position to cleave the dna substrate , at variance with what has been proposed for the vaccinia virus enzyme , where only the dna binding would trigger the active site assembly ( 28 ) .
the presence of a preassembled active site suggests that , in the open conformation , the enzyme is ready to interact with dna , and the only conformational change that the protein must undergo is the one needed to embrace the substrate . in line , electrostatic calculation of the open protein conformation shows that the protein displays a vast positive region ( blue in figure 6 ) with the active site resides located nearly at its center , perfectly suited to interact with the negatively charged supercoiled dna substrate . 

figure 6.a snapshot of the open conformation of the final unperturbed simulation e represented with the same domain colors as figure 2 ( left panel ) , together with its electrostatic potential mapped onto its molecular surface ( right panel ) .
red color represents negative and blue positive potential ( 5 to + 5 kbt ) .
electrostatic potentials were calculated using the program grasp ( 29 ) . a snapshot of the open conformation of the final unperturbed simulation e represented with the same domain colors as figure 2 ( left panel ) , together with its electrostatic potential mapped onto its molecular surface ( right panel ) .
red color represents negative and blue positive potential ( 5 to + 5 kbt ) .
in the first 3500  ps of simulation in the absence of dna , defined here as simulation a , the protein is maintaining a conformation very close to the crystallographic state , being the largest rmsd calculated on the main chain atoms of 3.5   .
this value is very close to that found for the protein simulated in complex with the dna substrate for an identical simulation time length ( 13 ) .
this result indicates that the closed configuration of the protein is very stable , independently of the presence of dna , and that the intra - domain and inter - domain protein interactions are sufficient to maintain the closed conformation .
also , the overall flexibility of the protein with or without dna is quite similar , as can be observed in figure 1 , where the per - residue rmsf in the absence of dna , evaluated for the last 3000  ps of simulation a , is compared to the rmsf evaluated on the same time window for the simulation previously carried out on the protein  dna complex ( 13 ) .
the maxima and minima of the rmsf are identical in the two simulations , being very similar to the protein regions characterized by the largest flexibility .
in fact , in the absence of dna , the linker and the loop connecting -strands 12 and 13 centered on gly520 are the regions characterized by the largest fluctuations , as already observed for the enzyme in the closed conformation in the presence of dna ( 12 ) .
differences are observed in the absolute values of the rmsf of definite regions , such as the  nose - cone helices ( helices 5 and 6 in core subdomains ii and i , respectively ) and the small loop at the n - terminal of the linker domain ( residues pro636-met645 ) , which show larger rmsfs in the simulation without dna than those observed in the simulation of topo70 in complex with dna ( figure 1 ) . residues 634640 are known to be highly flexible , and are not detected in several topoisomerase 
dna complex crystals because of their thermal fluctuations , while they are seen in the structure of the topo70dna  topotecan ternary complex ( 26 ) .
this region seems to display a scale of disorder , being more ordered in the ternary topo70dna  topotecan complex , less ordered in the binary topo70dna complex and even more disordered in the absence of dna . on the contrary ,
the maximum fluctuations , located in the loop of the linker domain between helices 18 and 19 , are less ample in the simulation without the dna . 

figure 1.average per - residue rmsf represented as a function of the residue number of htop1 in the absence of dna for the initial unperturbed simulation a ( full line ) in comparison with the ones previously calculated for the protein in complex with dna ( dashed line ) . 

figure 2.four snapshots of the final unperturbed simulation e taken at different times , showing the opening of the protein .
core subdomains i , ii and iii are rendered in yellow , blue and red , respectively . linker and c - terminal domains are rendered in green and cyan , respectively .
the hinge residue glu445 is indicated by an arrow in panel d. average per - residue rmsf represented as a function of the residue number of htop1 in the absence of dna for the initial unperturbed simulation a ( full line ) in comparison with the ones previously calculated for the protein in complex with dna ( dashed line ) .
four snapshots of the final unperturbed simulation e taken at different times , showing the opening of the protein .
core subdomains i , ii and iii are rendered in yellow , blue and red , respectively . linker and c - terminal domains are rendered in green and cyan , respectively .
the hinge residue glu445 is indicated by an arrow in panel d. per - residue - rmsds ( data not shown ) give higher values in the simulation in the absence of dna , for the linker domain and the nose - cone helices , which move away from the space previously occupied by dna .
both these regions have been shown to form several direct and water - mediated contacts with the dna phosphate groups in the strands downstream of the scissile base ( 5,11,13 ) .
it is worth noting , therefore , that the greater changes of dynamics and structure , in the absence of dna but in the closed conformation , are located in the two regions involved in the controlling of the rotation of the scissile strand during the relaxation step .
the x - ray diffraction study describes a single salt bridge direct contact between lys369 and glu497 ( 5 ) . in the simulation in the absence of dna , a new interaction is observed from 900 and 2500  ps between the lateral chain of lys369 and the carbonylic main - chain group of glu497 , with a distance of 3   . after this time , the distance between these two groups increases to 5   and the lateral chain of asn366 and the asp500 main chain start to interact , their distance being 3   ( see figure 7 , in supplementary data ) .
it is interesting to note that this new inter - lip interaction is also observed in the last part ( from 1 to 3.5  ns ) of the simulation of the protein in the presence of dna ( 13 ) .
a perturbation of the inter - lip interactions found at the end of simulation a , occurring between charged or polar groups ( glu497coo ; glu497co ; asn366od1asp500nh ) , has been attempted by increasing the ionic strength of the system . in details , 21 na and cl atoms
have been added to the system , which has been simulated for 500 ps ( simulation b ) . however , since the energetic stability of the inter - lip interactions is not sufficiently perturbed by the increase of ionic strength ( see figure 7 in supplementary data ) , a not physical perturbation ( i.e. change of electrostatic charges ) has been introduced in the system , in order to create a destabilization within a simulation time compatible with the currently computational resources .
it is interesting to note that the introduction of a positive charge replacing the negative one at the carboxylic group of glu497 is not enough to push the two lips far away from each other .
in fact , the introduction of the positive charge on the coo group of glu497 leads to an increase of the glu497lys369 distance because of electrostatic repulsion ( black line in figure 7 in supplementary data , simulation c ) . however , lys369 changes its molecular partner and forms a strong interaction with the glu497 main - chain carbonyl group , as evidenced by the decrease of the   relative distance of the glu497 main - chain and lys369 lateral chain , respectively ( red line in figure 7 in supplementary data , simulation c ) .
the same occurs for the interaction between the lateral chain of asn366 and the main - chain of asp500 ( green line in figure 7 in supplementary data ) .
therefore , the destabilization at the level of the salt bridge between glu497 and lys369 lateral chains , due to the introduction of the positive charge on the glu497 lateral chain , is compensated for by the strengthening of other lip1lip2 interactions that allow the protein to maintain a stable closed configuration . only after the introduction of two additional positive charges , one on the carbonyl group of glu497 and the other one on the lateral chain of asn366 ,
the two lips start to separate each other , as can be observed by monitoring the above - mentioned distances in simulation d ( figure 7 in supplementary data ) .
the system has been allowed to evolve with this additional perturbation for 200  ps . after this time
, the positive charges have been eliminated , bringing the modified residues charges back to their initial values .
the minimum distance between the lips , in the first configuration of the following unperturbed simulation ( simulation e ) , is only 6.5   , and the overall rmsd of this structure is 3.5   from the initial x - ray configuration ( deviation of the main - chain atoms fitted on the main - chain atoms themselves ) .
the initial configuration and three significant snapshots taken from simulation e are shown in figure 2 .
a dynamic visualization of the md dynamics is available as supplementary data at nar online .
the first important observation is that the protein domains change their relative position , reaching an open conformation able to bind and trap the dna substrate , but the secondary and tertiary structures of the single domains are very well conserved during the entire simulation period .
this is an important observation , since it implies that the different domains act as independent and internal coherent units that maintain their overall structure , although strongly changing their relative orientation .
core subdomains i and ii ( colored yellow and blue , respectively ) show coordinated motion , separated from that of core subdomain iii ( colored red ) .
this last subdomain , on the other hand , remains strongly interacting with the c - terminal domain ( colored cyan ) throughout the simulation .
a quantitative analysis of the relative protein domain motion is represented in figure 3 , where the average per - residue rmsd has been calculated , overlapping the protein on the core subdomains i and ii ( black line ) , the core subdomain iii ( red line ) , the linker ( green line ) , or the c - terminal domain ( blue line ) .
the analysis indicates a very strong and coherent motion of the core subdomains i  ii with respect to the other protein domains , with maximum rmsd ( about 100   ) when the protein is fitted on the linker domain .
the destabilization of the lip interactions permits a rotation of the core subdomains i  ii nose - cone helices , initially orientated parallel to the linker domain ( figure 2a ) , of nearly 90 after 2.5  ns ( figure 2b ) and of 180 after 5  ns ( figure 2c ) .
this motion permits the opening of the two halves that embrace the dna , formed by core subdomains i 
ii on one side and core subdomain iii and c - terminal domain on the other side .
ii continues during the last 5  ns of the simulation time , although with a lower extent .
the region that permits these relative domain motions is the n - terminal portion of helix 8 ( residues 434444 , figures 2d and 4 ) , and can be identified also by the intersection of the black and red lines in figure 3 .
the c - terminal portion of helix 8 , on the contrary , remains strongly interacting with helix 16 located in the c - terminal domain , during the whole opening dynamics ( figure 4 ) .
these two regions remain in close contact because of the presence of a cluster of hydrophobic residues . on the opposite site of this portion of helix 16
is located arg590 , a residue belonging to the catalytic pentad ( 27 ) that , due to these hydrophobic interactions , is maintained in an orientation similar to the one found in the crystallographic structures .
actually , all five active site residues maintain , for the whole simulation time , an orientation very similar to the one found in the closed configuration detected through x - ray diffraction ( 5 ) .
the similar orientation can be appreciated in figure 5a where the active site region of the last frame of simulation e ( gray ) is overimposed on the x - ray structure ( red ) . as a matter of fact ,
the largest deviation of the active site backbone atoms from the 1a36 crystallographic structure is only 1.7   .
the recent 3d structure of deinococcus radiodurans ( dra ) topoisomerase ib , the only one crystallized in an open conformation in the absence of dna , has indicated that the five active site residues are preassembled , being in a position nearly identical to those found in the dna - bound human htop1 ( 10 ) . in line , the orientation of the active site residues of the human enzyme in our simulation is also very similar to that of the bacterial enzyme , as shown in figure 5b , where the last frame of simulation e ( gray ) is overimposed to the structure of dra ( blue ) . 

figure 3.average per - residue rmsd of htop1 for the final unperturbed simulation e represented as a function of the residue number .
the average per - residue rmsd have been calculated overlapping the protein on the initial position of the core subdomains i and ii ( black line ) , the core subdomain iii ( red line ) , the linker ( green line ) or the c - terminal domain ( blue line ) . 

figure 4.snapshot of the final configuration of simulation e , showing only the core subdomain iii , the c - terminal , and the linker domain regions of the protein .
helices 8 , 9 and 16 and the catalytic arg590 are highlighted in the figure to evidentiate the hydrophobic interactions between the c - terminal portion of helix 8 and helix 16 .
figure 5.(a ) superimposition of the active site region of the last frame of simulation e ( gray color ) with the human enzyme x - ray structure ( red color ) .
( b ) superimposition of the active site region of the last frame of simulation e ( gray color ) with the deinococcus radiodurans enzyme x - ray structure ( blue color ) .
average per - residue rmsd of htop1 for the final unperturbed simulation e represented as a function of the residue number .
the average per - residue rmsd have been calculated overlapping the protein on the initial position of the core subdomains i and ii ( black line ) , the core subdomain iii ( red line ) , the linker ( green line ) or the c - terminal domain ( blue line ) .
snapshot of the final configuration of simulation e , showing only the core subdomain iii , the c - terminal , and the linker domain regions of the protein .
helices 8 , 9 and 16 and the catalytic arg590 are highlighted in the figure to evidentiate the hydrophobic interactions between the c - terminal portion of helix 8 and helix 16 .
( a ) superimposition of the active site region of the last frame of simulation e ( gray color ) with the human enzyme x - ray structure ( red color ) .
( b ) superimposition of the active site region of the last frame of simulation e ( gray color ) with the deinococcus radiodurans enzyme x - ray structure ( blue color ) . in our simulation , despite the huge inter - domain protein motion that spans up to 100   , the active site residues fully maintain their relative orientation , indicating that , in the human enzyme also , the active site is preassembled and in the appropriate position to cleave the dna substrate , at variance with what has been proposed for the vaccinia virus enzyme , where only the dna binding would trigger the active site assembly ( 28 ) .
the presence of a preassembled active site suggests that , in the open conformation , the enzyme is ready to interact with dna , and the only conformational change that the protein must undergo is the one needed to embrace the substrate . in line , electrostatic calculation of the open protein conformation shows that the protein displays a vast positive region ( blue in figure 6 ) with the active site resides located nearly at its center , perfectly suited to interact with the negatively charged supercoiled dna substrate . 

figure 6.a snapshot of the open conformation of the final unperturbed simulation e represented with the same domain colors as figure 2 ( left panel ) , together with its electrostatic potential mapped onto its molecular surface ( right panel ) .
red color represents negative and blue positive potential ( 5 to + 5 kbt ) .
a snapshot of the open conformation of the final unperturbed simulation e represented with the same domain colors as figure 2 ( left panel ) , together with its electrostatic potential mapped onto its molecular surface ( right panel ) .
red color represents negative and blue positive potential ( 5 to + 5 kbt ) .
 the open state of human topoisomerase i has been probed by molecular dynamics simulation , starting from the coordinates of the closed structure of the protein complexed with dna , after elimination of the 22-bp dna duplex oligonucleotide . 
 a repulsion force between the two lips of the protein has been introduced for a short time to induce destabilization of the local minimum , after which an unperturbed simulation has been carried out for 10  ns . 
 the simulation shows that the protein undergoes a large conformational change due to rearrangements in the orientation of the protein domains , which however move as a coherent unit , fully maintaining their secondary and tertiary structures . despite movements between the domains as large as 8090   , 
 the catalytic pentad remains preassembled , the largest deviation of the active site backbone atoms from the starting crystallographic structure being only 1.7   . 
 electrostatic calculation of the open protein structure shows that the protein displays a vast positive region with the active site residues located nearly at its center , in a conformation perfectly suited to interact with the negatively charged supercoiled dna substrate . 
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
Initial unperturbed simulation
Charge perturbation
Opening dynamics
SUPPLEMENTARY DATA