hiv-1 infection starts with the binding of the viral surface - protein gp120 to the human t - cell receptor cd4 via a hydrophobic pocket on gp120 , which binds effectively to phe43 in cd4 ( 1 ) . this initial association is followed by membrane fusion and transfer of the viral genetic material into the cell , thus enabling the reproduction of the virus whilst destroying the host . the crucial role of the gp120-cd4 interaction has been demonstrated by an experiment leading to hiv-1 vulnerable rats after insertion of human cd4-cdr2 protein sequences ( 2 ) , while the impact of phe43 was confirmed by significant loss of binding affinity of cd4 for gp120 after mutation around the binding site ( 3 , 4 ) . therefore the amino acid sequence in gp120 is highly conserved and less prone to mutations ( 1 , 2 ) , thus providing a promising target for the development of new drugs ( 5 ) . blocking the cellular entry of the virus is more practical and exhibit less side effects compared to a drug operating at intracellular level , e.g. a protease inhibitor ( 6 ) . bms-378806 inhibits the gp120-cd4 binding , as shown in an enzyme - linked immunosorbent assay ( elisa ) , without inhibitory activity against hiv-1 reverse transcriptase , protease and integrase and it has been proposed as a good candidate against hiv-1 infection ( 7 , 8) . a clinical study of gp120 inhibition has not been conducted so far , however an oligopeptide cd4 mimic has been used to study the inhibition of the hiv-1 entry using a cell - based fusion assay ( 9 ) . several computational studies have been performed on docking and molecular dynamics of small ligands with either gp120 ( 5 , 10 ) or cd4 ( 11 ) to identify suitable inhibitor candidates . another investigation applied molecular dynamics on the gp120-cd4 complex targeting to predict a mimic for the natural phe43 conformation in the complex ( 1 ) . this is the first study that conducts computational protein - protein interactions to propose the design of a therapeutic strategy . pdb files for gp120-cd4 protein complexes of hiv-1 ( 12 ) from up to 2010 were downloaded and checked for the presence of the indicated hydrophobic interaction via phe43 . as none of the structures had additional molecules at the binding site , they were stripped from all antibodies , water and ligands to lower the computational time . in order to avoid crystal packing and antibody induced conformational effects , the protein complexes were minimized in gromacs ( 13 ) using steepest - descent , followed by conjugate gradient approach to an energy convergence of 0.01 kj / mol . out of 22 crystal structures , only 1rzk ( 14 ) and 1g9n ( 15 ) remained intact and were used for the study . the root mean square deviations ( rmsd ) of the structures before and after minimization were computed using the swiss - pdbviewer ( 16 ) . the phe43 cap was mutated in the original protein complex using the swiss - pdbviewer to his43 ( neutral and protonated - generated by adt ( 17 ) ) , trp43 and tyr43 . docking studies applied a keylock protocol with receptor ( gp120 ) and ligand ( cd4 ) in ten replicates on autodock4 ( 18 ) , patchdock & firedock ( 19 , 20 ) , zdock ( 21 ) and cluspro ( 22 ) . protein binding energies were determined within the software packages for autodock and patchdok / firedock , while zdock and cluspro output files were minimized in gromacs and the energy was compared with the accumulated energy of the individual proteins . docking studies were performed with both rigid and flexible receptors and conformational flexible ligand at position 43 . the amino acid selection for flexible domains of the receptor was based on closest proximity , including thr108 , asp190 , glu192 , ile193 , asn239 , met240 , trp241 and gly287 for 1g9n and asp190 , glu 192 , asn239 and trp241 for 1rzk ( figs . stick representation shows the binding sites containing flexible amino acid residues in the hydrophobic pocket of gp120 and the cd4 phe43 cap . for 1g9n eight amino acid residues are in close proximity to the phe43 cap ( a ) , while only four amino acid residues fulfill the criteria in 1rzk ( b ) . final docking output files were analyzed for hydrogen bonds using swiss - pdbviewer with default settings . firedock provides an additional analysis tool to quantify long and short range electrostatic interactions , van der waals bindings and p - p stacking energies . the rmsds of docked conformations with respect to the minimized crystal structure of the corresponding protein complex were calculated using swiss - pdbviewer for the amino acid around the binding site , which were used as criteria for the gap - cap interaction between cd4 and gp120 to complement the qualitative visual analysis of the docking structure . molecular dynamics were applied on both individual proteins as well as on the protein complexes . the initially minimized structures were soaked in spc explicit water solvent ( 23 ) and minimized in gromacs by steepest - descent and conjugate gradient approach to energy convergence of 0.01 kj / mol . md was performed for 5ns under constant pressure of one atmosphere with a pressure coupling constant of 1.0 ps and at 310 k with a temperature coupling constant of 0.1 ps at 1 femtosecond time step . the g_lie module in gromacs was used to calculate the gibbs free energy of binding ( dgbind ) of aqvist ( 24 ) as shown in eq . 1:1dgbind = a(evdw , bound- evdw , free)+b(ecoul , bound - ecoul , free ) , 
  the approach is based on the assumption that a receptor - ligand protein - protein interaction can be estimated by determining the difference in solvent ( water ) interaction energy of the free ligand ( efree ) and the complex ( ebound ) . the remaining variable , i.e. the receptor and its water interaction , can be ignored , as it remains constant in the comparison , thus only affects the total energy , but not the difference between the ligands . the rmsds and potential energies were calculated by gromacs modules g_rms and g_energy , respectively . pdb files for gp120-cd4 protein complexes of hiv-1 ( 12 ) from up to 2010 were downloaded and checked for the presence of the indicated hydrophobic interaction via phe43 . as none of the structures had additional molecules at the binding site , they were stripped from all antibodies , water and ligands to lower the computational time . in order to avoid crystal packing and antibody induced conformational effects , the protein complexes were minimized in gromacs ( 13 ) using steepest - descent , followed by conjugate gradient approach to an energy convergence of 0.01 kj / mol . out of 22 crystal structures , only 1rzk ( 14 ) and 1g9n ( 15 ) remained intact and were used for the study . the root mean square deviations ( rmsd ) of the structures before and after minimization were computed using the swiss - pdbviewer ( 16 ) . the phe43 cap was mutated in the original protein complex using the swiss - pdbviewer to his43 ( neutral and protonated - generated by adt ( 17 ) ) , trp43 and tyr43 . docking studies applied a keylock protocol with receptor ( gp120 ) and ligand ( cd4 ) in ten replicates on autodock4 ( 18 ) , patchdock & firedock ( 19 , 20 ) , zdock ( 21 ) and cluspro ( 22 ) . protein binding energies were determined within the software packages for autodock and patchdok / firedock , while zdock and cluspro output files were minimized in gromacs and the energy was compared with the accumulated energy of the individual proteins . docking studies were performed with both rigid and flexible receptors and conformational flexible ligand at position 43 . the amino acid selection for flexible domains of the receptor was based on closest proximity , including thr108 , asp190 , glu192 , ile193 , asn239 , met240 , trp241 and gly287 for 1g9n and asp190 , glu 192 , asn239 and trp241 for 1rzk ( figs . stick representation shows the binding sites containing flexible amino acid residues in the hydrophobic pocket of gp120 and the cd4 phe43 cap . for 1g9n eight amino acid residues are in close proximity to the phe43 cap ( a ) , while only four amino acid residues fulfill the criteria in 1rzk ( b ) . final docking output files were analyzed for hydrogen bonds using swiss - pdbviewer with default settings . firedock provides an additional analysis tool to quantify long and short range electrostatic interactions , van der waals bindings and p - p stacking energies . the rmsds of docked conformations with respect to the minimized crystal structure of the corresponding protein complex were calculated using swiss - pdbviewer for the amino acid around the binding site , which were used as criteria for the gap - cap interaction between cd4 and gp120 to complement the qualitative visual analysis of the docking structure . molecular dynamics were applied on both individual proteins as well as on the protein complexes . the initially minimized structures were soaked in spc explicit water solvent ( 23 ) and minimized in gromacs by steepest - descent and conjugate gradient approach to energy convergence of 0.01 kj / mol . md was performed for 5ns under constant pressure of one atmosphere with a pressure coupling constant of 1.0 ps and at 310 k with a temperature coupling constant of 0.1 ps at 1 femtosecond time step . the g_lie module in gromacs was used to calculate the gibbs free energy of binding ( dgbind ) of aqvist ( 24 ) as shown in eq . 1:1dgbind = a(evdw , bound- evdw , free)+b(ecoul , bound - ecoul , free ) , 
  the approach is based on the assumption that a receptor - ligand protein - protein interaction can be estimated by determining the difference in solvent ( water ) interaction energy of the free ligand ( efree ) and the complex ( ebound ) . the remaining variable , i.e. the receptor and its water interaction , can be ignored , as it remains constant in the comparison , thus only affects the total energy , but not the difference between the ligands . the rmsds and potential energies were calculated by gromacs modules g_rms and g_energy , respectively . pdb files for gp120-cd4 protein complexes of hiv-1 ( 12 ) from up to 2010 were downloaded and checked for the presence of the indicated hydrophobic interaction via phe43 . as none of the structures had additional molecules at the binding site , they were stripped from all antibodies , water and ligands to lower the computational time . in order to avoid crystal packing and antibody induced conformational effects , the protein complexes were minimized in gromacs ( 13 ) using steepest - descent , followed by conjugate gradient approach to an energy convergence of 0.01 kj / mol . out of 22 crystal structures , only 1rzk ( 14 ) and 1g9n ( 15 ) remained intact and were used for the study . the root mean square deviations ( rmsd ) of the structures before and after minimization were computed using the swiss - pdbviewer ( 16 ) . the phe43 cap was mutated in the original protein complex using the swiss - pdbviewer to his43 ( neutral and protonated - generated by adt ( 17 ) ) , trp43 and tyr43 . docking studies applied a keylock protocol with receptor ( gp120 ) and ligand ( cd4 ) in ten replicates on autodock4 ( 18 ) , patchdock & firedock ( 19 , 20 ) , zdock ( 21 ) and cluspro ( 22 ) . protein binding energies were determined within the software packages for autodock and patchdok / firedock , while zdock and cluspro output files were minimized in gromacs and the energy was compared with the accumulated energy of the individual proteins . docking studies were performed with both rigid and flexible receptors and conformational flexible ligand at position 43 . the amino acid selection for flexible domains of the receptor was based on closest proximity , including thr108 , asp190 , glu192 , ile193 , asn239 , met240 , trp241 and gly287 for 1g9n and asp190 , glu 192 , asn239 and trp241 for 1rzk ( figs . stick representation shows the binding sites containing flexible amino acid residues in the hydrophobic pocket of gp120 and the cd4 phe43 cap . for 1g9n eight amino acid residues are in close proximity to the phe43 cap ( a ) , while only four amino acid residues fulfill the criteria in 1rzk ( b ) . final docking output files were analyzed for hydrogen bonds using swiss - pdbviewer with default settings . firedock provides an additional analysis tool to quantify long and short range electrostatic interactions , van der waals bindings and p - p stacking energies . the rmsds of docked conformations with respect to the minimized crystal structure of the corresponding protein complex were calculated using swiss - pdbviewer for the amino acid around the binding site , which were used as criteria for the gap - cap interaction between cd4 and gp120 to complement the qualitative visual analysis of the docking structure . molecular dynamics were applied on both individual proteins as well as on the protein complexes . the initially minimized structures were soaked in spc explicit water solvent ( 23 ) and minimized in gromacs by steepest - descent and conjugate gradient approach to energy convergence of 0.01 kj / mol . md was performed for 5ns under constant pressure of one atmosphere with a pressure coupling constant of 1.0 ps and at 310 k with a temperature coupling constant of 0.1 ps at 1 femtosecond time step . the g_lie module in gromacs was used to calculate the gibbs free energy of binding ( dgbind ) of aqvist ( 24 ) as shown in eq . 1:1dgbind = a(evdw , bound- evdw , free)+b(ecoul , bound - ecoul , free ) , 
  the approach is based on the assumption that a receptor - ligand protein - protein interaction can be estimated by determining the difference in solvent ( water ) interaction energy of the free ligand ( efree ) and the complex ( ebound ) . the remaining variable , i.e. the receptor and its water interaction , can be ignored , as it remains constant in the comparison , thus only affects the total energy , but not the difference between the ligands . the rmsds and potential energies were calculated by gromacs modules g_rms and g_energy , respectively . the selection of pdb crystal structures was based on a continuous peptide structure after minimization . 1rzk and 1g9n are the only structures that do not expose unnatural binding distances due to missing amino acids . minimization of the protein complexes only led to minor changes in rmsds , i.e. below 2  , as shown in table 1 . the results for the docking binding energy and compliance with the gap - cap interaction are summarized in table 2 . the binding energy corresponds to best out of ten runs that complies with the gap - cap interaction . only if such structure was not found , the global energy minimum was selected . docking conformation ( a ) reflects the proper hydrophobic gap - cap interaction , while ( b ) refers to a non - gap - cap binding , as listed in table 2 . root mean square deviations ( rmsds ) of the minimized structures with respect to the initial crystal structure for 1rzk and 1g9n protein complexes with their respective amino acid mutants as calculated by swiss - pdbviewer . docking energies [ kj / mol ] and gap - cap conformation fractions for 1rzk and 1g9n using autodock 4 , cluspro , zdock and firedock . values in parenthesis indicate the rate of proper pocket gap - cap conformations over a total of ten dockings . best docking conformation refers to lowest energy with highest gap - cap conformation fraction , when possible ; lowest docking energies and highest gap - cap conformation fractions are highlighted ( in asterisks ) . firedock and cluspro provided both , proper conformations and energies for the dockings , while zdock appeared to be disfavored by endothermic energies the rmsd of the docked conformation with respect to the minimized crystal structure was used to supplement the visual conformation inspection . table 3 lists the rmsd results for all atoms at the binding site . for qualified gap - cap conformations firedock results indicate easiest bindings for small aromatic amino acids ( phenylalanine and histidine ) but stronger binding for the larger aromatic tryptophan . root mean square deviations ( rmsds ) of the docked conformations ( 1 - 10 for firedock ) for 1rzk and 1g9n protein complexes ( directed and blind approaches ) with reference to the minimized crystal structures as calculated by swiss - pdbviewer . x : not analyzed due to non - gap - cap conformation when inspected by eyes ; italic numbers refer to rmsds for structures that are likely to fail the visual inspection . bold numbers indicate proper gap - cap interaction ; criteria : rmsd  1.0  
 table 4 shows a breakdown of the total binding energy for firedock . the sums of individual energies exceed the total binding energy in table 2 indicating protein folding . the strong binding of gp120 and trp43 mutated cd4 corresponds with large van der waals interaction and p - p - stacking . this finding matches previous reports on interactions of phe-43 with its neighboring trp and tyr ( 25 , 10 ) . no intermolecular hydrogen bond in the gap - cap region could be found , except a hydrogen bond between the phenolic hydroxyl group of cd4 tyr43 and the asncarbonyl in gp120 . contribution of different interaction types ( electrostatic , hydrogen bonding , vdw and - stacking ) [ kj / mol ] to the total binding energy for protein - protein complexes of gp120 and ( mutated ) cd4 in firedock . the data for the directed binding of the tyr43 mutated cd4 ( boxed ) were not considered due to the non - gap - cap based protein - protein binding . molecular dynamics investigations of the minimized complexes converged energetically but the rmsd indicates partial equilibrium . the gibbs free binding energies , dgbind , for 1rzk indicate slightly higher binding for tryptophan and tyrosine ( table 5 ) , while no significant differences are found for 1g9n . this may be considered as a confirmation of firedock results ( table 2 ) and matches with the significant decrease of affinity for gp120 by tyr43(f43y ) mutated cd4 compared to the trp43(f43w ) analog ( 26 ) . no intermolecular hydrogen bonding between ligand and receptor was found around the binding site . energy profile ( a ) and root mean square deviations ( rmsds of protein backbone ) ( b ) for 1rzk - trp . gibbs free binding energy in solvent water of gp120-cd4 complex and cd4 after 5ns of molecular dynamics simulation . gbind was obtained from the g_lie module applying factors a = 0.181 and = 0.5 . all energies are averaged , lowest binding energies are highlighted ( in asterisks ) . in order to evaluate the prediction of a flexible aromatic inhibitor , an additional docking of gp120 by biphenylalanine ( biphe)-mutated the biphenylalanine was modeled with chem3d and the aromatic moiety was attached to the backbone of the corresponding cd4-phe43 . firedock produced only 1/10 gap - cap docked conformations for 1rzk and 1g9n with docking energies of -103.9 kj mol and -92.3 kj mol , respectively . this refers to best binding for 1rzk , and second best for 1g9n after trp . these results agree well with the experimental result of enhanced binding affinity of gp120 for biphenylalanine bip43-mutated cd4 ( 9 ) . for a drug design , the recommendation is a larger aromatic system with conformational flexibility , to avoid inefficient binding kinetics . hydrophobic interactions and p - stacking are the dominating features in the binding of gp120 and cd4 . while extended aromatic systems on the mutated cd4 can enhance the binding strength , the docking kinetics was reduced , leading to less efficient binding . additional hydrogen bonding based interactions involving a hydrogen donor on the cd4 may further improve the efficiency of inhibition , provided that the hydrogen donor does not affect the hydrophobic interaction . out of the investigated four docking tools , only the network firedock and zdock applications provided good results with respect to the docking conformation , while autodock failed to provide any suitable structure . the missing feature of an integrated energy evaluation in zdock makes firedock the software of choice for protein - protein docking studies . molecular dynamics can only support docking studies qualitatively , as the results depend on the input structure .
background and the purpose of the studythe interactions between hiv-1 gp120 and mutated cd4 proteins were investigated in order to identify a lead structure for therapy based on competitive blocking of the hiv binding receptor for human t - cells . 
 crystal structures of hiv gp120-cd4 complexes reveal a close interaction of the virus receptor with cd4 phe43 , which is embedded in a pocket of the virus protein.methodsthis study applies computer simulations to determine the best binding of amino acid 43 cd4 mutants to hiv gp120 . besides natural cd4 
 , three mutants carrying alternate aromatic residues his , trp and tyr at position 43 were investigated . 
 several docking programs were applied on isolated proteins based on selected crystal structures of gp120-cd4 complexes , as well as a 5 ns molecular dynamics study on the protein complexes . 
 the initial structures were minimized in gromacs to avoid crystal packing effects , and then subjected to docking experiments using autodock4 , firedock , cluspro and zdock . in molecular dynamics , the gibbs free binding energy was calculated for the gp120-cd4 complexes . 
 the docking outputs were analyzed on energy within the respective docking software.results and conclusionvisualization and hydrogen bonding analysis were performed using the swiss - pdbviewer . 
 strong binding to hiv gp120 can be achieved with an extended aromatic group ( trp ) . 
 however , the sterical demand of the interaction affects the binding kinetics . in conclusion , 
 a ligand for an efficient blocking of hiv gp120 should involve an extended but conformational flexible aromatic group , i.e. a biphenyl . 
 a docking study on biphenylalanine-43 confirms this expectation .