Conceived and designed the experiments: AA VI. Performed the experiments: AA. Analyzed the data: AA PS VI. Contributed reagents/materials/analysis tools: AA AU. Wrote the paper: AA PS VI.
The authors have declared that no competing interests exist.
Abasic (AP) sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1) cleaves the phosphodiester backbone 5′ to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-β). While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-β, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-β based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-β located downstream of APEX1 (3′ to the damaged site) and three with pol-β located upstream of APEX1 (5′ to the damaged site). Molecular dynamics (MD) simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (∼−10.0 kcal/mol) to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-β at the 3′-side of APEX1 are those most likely to occur
Oxidative damage to DNA happens in every cell as a consequence of the life process. Such damage can inhibit DNA replication and RNA transcription; if not repaired, it can lead to cancer. Consequently, all cells contain an important mechanism for identification and repair of oxidative lesions. Two proteins figure prominently: AP endonuclease 1, which cleaves the damaged site, and DNA polymerase beta, which inserts a new nucleotide to replace the damaged one. While several biochemical studies indicate interaction between the two proteins, the details of the interaction remain unknown. Here, we develop and apply a new methodology to predict the most likely protein-protein interface between the two proteins. The methodology relies on the assumption, which is validated by experimental evidence, that both proteins must bind to DNA in order to interact. Analysis of the simulated protein behavior in water allowed us to suggest how protein interaction might be coupled to conformational changes in DNA polymerase beta. Further comparative analysis identified coordinated mutations of specific residues at the predicted interfaces. This method can be applied to predict interaction details for any protein pair as long as the proteins in the pair are associated with DNA during the interaction.
Loss of a nucleobase without cleavage of the DNA backbone results in formation of an abasic (AP) site. AP sites arise frequently in normal DNA from a variety of causes: spontaneous hydrolysis of nucleobases, DNA damaging agents or DNA glycosylases that remove specific abnormal bases, such as uracil,
Biological experiments to examine whether APEX1 and pol-β interact have been carried out using several different methodologies
We present detailed theoretical analysis of possible complexes between APEX1 and pol-β, at which EMSA analysis or the yeast two-hybrid system can only hint. The analysis predicts a communicative interaction between the two proteins under the assumption that the initial interaction occurs when both proteins are seated on the DNA helix. Crystal structures of the individual proteins bound to DNA are known
A model of the initial complex was built by aligning the DNAs that were co-crystallized in complexes with APEX1 or with pol-β. The DNA strand in the co-crystal with APEX1 has a 30 degree bend at the AP site; therefore, in the initial complex we aligned the DNA in the pol-β co-crystal with 5′- or 3′-sides of the damaged DNA in the APEX1 co-crystal. Several potential interacting complexes from either side were considered (see schematic diagram in
Comments underneath each picture reflect the conclusion made about each complex after analysis.
We presumed that protein-protein interaction occurs when both proteins are associated with DNA, but that only one protein at a time performs its function at the damaged site. Since pol-β follows APEX1 in the BER pathway, it is likely to sense the cleaved DNA site through APEX1 bound to DNA. Therefore, the first priority was to correctly represent the interaction of APEX1 with DNA in the complex rather than that of pol-β. Consequently, we kept only DNA from the APEX1 co-crystal for subsequent molecular dynamics. The 90 degree bend found in the pol-β co-crystal will not be present in the initial complex.
Crystal structures for pol-β are available in three different conformations: one that allows for the incoming nucleotide to correctly hydrogen bond in preparation for insertion
For this orientation (see schematic diagram in
(A) Alignment of DNAs co-crystallized with pol-β and APEX1. X stands for the abasic site and x stands for lesion. Notations c1, c2, and c3 mark the alignments used to produce three initial complexes. (B) View of the 3′ complex structure. APEX1 is on the right and pol-β is on the left. The area of protein-protein interaction is circled.
Time (ns) | Interface Area (Å2) | Interface Area (% of Surface area) | Planarity RMSD (Å) | Length & Breadth (Å) | Polar Atoms (% of interface) | Non-polar Atoms (% of Interface) | Gap Volume (Å3) | Gap Volume/Interface Area | Hydrogen Bonds at Interface | Salt Bridges in Interface | Binding Energy (kcal/mol) | Area of segment #1 (Å2) | Area of segment #2 (Å2) | Area of segment #3 (Å2) |
0.1 | 565 | 4.2 | 2.7 | 37 & 24 | 47 | 53 | 8655 | 7.5 | 6 | 0 | −6.6 | 142 | 238 | 185 |
0.2 | 644 | 4.7 | 3.7 | 40 & 25 | 50 | 50 | 8731 | 6.7 | 3 | 0 | −6.6 | 159 | 281 | 204 |
0.3 | 530 | 3.8 | 3.1 | 37 & 24 | 49 | 51 | 8480 | 7.9 | 2 | 0 | −2.8 | 140 | 227 | 163 |
0.4 | 521 | 4.0 | 2.9 | 27 & 24 | 43 | 57 | 8894 | 8.3 | 5 | 0 | −8.3 | 166 | 191 | 164 |
0.5 | 544 | 4.1 | 3.0 | 35 & 21 | 43 | 57 | 9681 | 8.5 | 6 | 0 | −5.0 | 132 | 226 | 186 |
0.6 | 585 | 4.4 | 2.8 | 36 & 25 | 40 | 60 | 10060 | 8.4 | 5 | 0 | −6.1 | 148 | 230 | 207 |
0.7 | 540 | 4.1 | 3.0 | 35 & 26 | 49 | 51 | 9465 | 8.6 | 5 | 0 | −10.0 | 89 | 239 | 212 |
0.8 | 450 | 3.4 | 2.6 | 34 & 22 | 54 | 46 | 11862 | 12.8 | 3 | 0 | −5.8 | 108 | 215 | 127 |
0.9 | 478 | 3.6 | 2.7 | 40 & 24 | 51 | 49 | 10089 | 10.3 | 4 | 0 | −9.5 | 137 | 194 | 147 |
1.0 | 632 | 4.8 | 3.4 | 39 & 24 | 49 | 51 | 9879 | 7.7 | 5 | 0 | −7.3 | 247 | 200 | 185 |
Values are calculated for interface in APEX1 after 0.1 ns intervals of MD simulation. To calculate the ratio of “gap volume/interface area,” the sum of interface areas in APEX1 and pol-β is used in denominator. The free energy of binding was calculated with FOLD-X server.
In order to resolve steric overlaps and ensure optimal atom packing at the protein-protein interface, an MD simulation was applied to the complex. The MD simulation continued for 1 ns (see
During the entire simulation a stable complex was observed with interface area reaching 644 Å2 and binding energy as low as −10 kcal/mol. The interface area fluctuated by ∼30% while the shape of interface stayed essentially the same (see column ‘Length&Breadth’). Similarly, the interface atomic content did not change and consisted of slightly less than 50% of polar and slightly more than 50% non-polar atoms. On average the interface had 4 short-lived hydrogen bonds. Out of 21 different hydrogen bonds observed during the simulation at most 6 could be observed at any time. The most stable hydrogen bonds observed throughout the simulation contained the sidechain atoms of Asn222 and mainchain oxygen of Gln31 and mainchain nitrogen of His34. Estimation of binding energies with FOLD-X server revealed that the major contribution to the free energy of binding comes from hydrophobic desolvation and Van der Waals interaction with 1/3 contribution from hydrogen bonding. Thus, we concluded that the major interactions stabilizing the interface were hydrophobic.
Analysis of the complex allowed identification of potential interacting residues of APEX1 and pol-β (see
The initial complex is shown in
Seg | Residues in APEX1 | Interface Area (percentage) | Residues in pol-β | Interface Area (percentage) |
1 | Leu179 | 0–10 | Arg299 | 0–5 |
Leu182 | 4–7 | Leu301 | 0–6 | |
Glu183 | 0–5 | Val303 | 0–14 | |
Gln186 | 0–11 | Thr304 | 0–11 | |
Pro234 | 0–7 | 2–10 | ||
6–10 | 5–15 | |||
Gln238 | 0–6 | Ala307 | 0–5 | |
2 | 2–12 | 7–14 | ||
9–16 | 4–10 | |||
Pro223 | 2–5 | 16–26 | ||
5–17 | ||||
Gly225 | 2–7 | |||
3 | Asn272 | 0–5 | 6–11 | |
6–12 | Arg112 | 0–8 | ||
13–17 | 10–13 | |||
Asn | 1–7 |
The residues assembling the majority of the interface are highlighted in bold. The columns “Seg” refers to the discrete interface segments where the listed residues are located (see
The 3′-complex with pol-β in open conformation (PDB-file 9ici) was constructed in the same fashion as the 3′-complex with pol-β in the closed conformation (see schematic diagram on
Time (ns) | Interface Area (Å2) | Interface Area (% of Surface area) | Planarity RMSD (Å) | Length & Breadth (Å) | Polar Atoms (% of interface) | Non-polar Atoms (% of Interface) | Gap Volume (Å3) | Gap Volume/Interface Area | Hydrogen Bonds at Interface | Salt Bridges in Interface | Binding Energy (kcal/mol) |
0.1 | 453 | 3.4 | 2.2 | 25 & 23 | 48 | 52 | 9600 | 10.3 | 2 | 0 | −5.4 |
0.2 | 530 | 3.8 | 2.1 | 26 & 18 | 41 | 59 | 11612 | 10.7 | 1 | 0 | −5.2 |
0.3 | 569 | 4.1 | 2.1 | 26 & 19 | 47 | 53 | 10579 | 9.2 | 2 | 0 | −3.4 |
0.4 | 603 | 4.3 | 2.2 | 29 & 21 | 52 | 48 | 8305 | 6.7 | 3 | 0 | −10.0 |
0.5 | 573 | 4.1 | 2.3 | 29 & 17 | 51 | 49 | 12766 | 10.7 | 6 | 0 | −7.1 |
0.6 | 644 | 4.5 | 2.4 | 31 & 23 | 48 | 52 | 11227 | 8.4 | 5 | 0 | −5.2 |
0.7 | 639 | 4.3 | 2.8 | 29 & 19 | 44 | 56 | 13134 | 10.0 | 5 | 0 | −9.6 |
0.8 | 645 | 4.3 | 2.5 | 34 & 19 | 49 | 51 | 11145 | 8.5 | 3 | 0 | −6.5 |
0.9 | 687 | 4.5 | 3.4 | 31 & 34 | 46 | 54 | 13799 | 10.0 | 4 | 0 | −7.7 |
1.0 | 732 | 4.8 | 3.6 | 42 & 30 | 49 | 51 | 13752 | 9.4 | 3 | 0 | −5.2 |
Values are calculated for interface in APEX1 after 0.1 ns intervals of MD simulation. To calculate the ratio of “gap volume/interface area” the sum of interface areas in APEX1 and pol-β is used in denominator. The free energy of binding was calculated with FOLD-X server.
For most of the simulation time the interface consisted of a single surface patch formed from segments #2 and #3 of the complex with closed conformation of pol-β plus several peripheral residues: Leu44, Glu217, Ile218, Asn259, Pro261, Tyr262, Tyr264 in APEX1 and Ala32, Arg40, Thr93, Val115, Glu117 in pol-β. In the last 0.1 ns of simulation another patch appeared between residues 177–183 in APEX1 and 231–233 in pol-β. Since its area was less than 50 Å2, we neglected it in subsequent analysis. The interface atomic content was similar to the one observed for complex with pol-β in closed conformation, i.e, it consisted of slightly less than 50% polar and slightly more than 50% non-polar atoms. At the same time there were fewer hydrogen bonds at the interface suggesting that hydrophobic interaction was even more important for interface stabilization than in the complex with pol-β in closed conformation.
For this orientation (see schematic diagram on
(A) Alignment of DNAs co-crystallized with pol-β and APEX1. X stands for the abasic site and x stands for lesion. Notations c4, c5, and c6 mark the alignments used to produce three initial complexes. (B) View of the 5′ complex structure. APEX1 is on the left and pol-β is on the right. The area of protein-protein interaction is circled.
MD was applied to the 5′-complex in order to resolve steric overlaps and ensure optimal atom packing at the protein-protein interface. The MD simulation continued for 1 ns (see
The 5′-complex with pol-β in open conformation was constructed by replacing the structure of pol-β in the c4 complex (see
Time (ns) | Interface Area (Å2) | Interface Area (% of Surface area) | Planarity RMSD (Å) | Length & Breadth (Å) | Polar Atoms (% of interface) | Non-polar Atoms (% of Interface) | Gap Volume (Å3) | Gap Volume/Interface Area | Hydrogen Bonds at Interface | Salt Bridges in Interface | Binding Energy (kcal/mol) |
0.1 | 818 | 5.9 | 3.1 | 30 & 23 | 51 | 49 | 8963 | 5.4 | 5 | 0 | −10.0 |
0.2 | 801 | 5.7 | 3.1 | 27 & 24 | 51 | 49 | 9757 | 5.9 | 6 | 1 | −13.5 |
0.3 | 789 | 5.6 | 3.9 | 32 & 25 | 57 | 43 | 7156 | 4.4 | 8 | 1 | −6.7 |
0.4 | 775 | 5.4 | 3.0 | 29 & 22 | 49 | 51 | 8847 | 5.5 | 6 | 1 | −6.6 |
0.5 | 757 | 5.2 | 3.1 | 29 & 24 | 47 | 53 | 7978 | 5.2 | 8 | 0 | −10.9 |
0.6 | 693 | 4.8 | 3.0 | 29 & 23 | 46 | 52 | 7762 | 5.4 | 8 | 0 | −6.1 |
0.7 | 783 | 5.4 | 3.8 | 33 & 24 | 54 | 46 | 7337 | 4.5 | 8 | 0 | −7.4 |
0.8 | 667 | 4.5 | 3.9 | 33 & 23 | 45 | 55 | 7622 | 5.4 | 5 | 0 | −5.8 |
0.9 | 729 | 4.8 | 2.9 | 27 & 23 | 52 | 48 | 8707 | 5.9 | 6 | 0 | −6.1 |
1.0 | 654 | 4.4 | 2.7 | 26 & 22 | 51 | 49 | 8235 | 6.1 | 6 | 0 | −5.2 |
Values are calculated for interface in APEX1 after 0.1 ns intervals of MD simulation. To calculate the ratio of “gap volume/interface area” the sum of interface areas in APEX1 and pol-β is used in denominator. The free energy of binding was calculated with FOLD-X server.
Residues in APEX1 | Interface Area (percentage) | Residues in pol-β | Interface Area (percentage) |
Glu101 | 0–6 | 6–10 | |
Asn102 | 0–5 | Leu287 | 5–8 |
Ser120 | 0–5 | Glu288 | 0–8 |
Ala121 | 0–6 | Lys289 | 1–7 |
5–11 | Phe291 | 0–5 | |
12–14 | Thr292 | 3–6 | |
6–10 | Asn294 | 2–5 | |
Glu126 | 2–6 | Arg299 | 0–7 |
Tyr144 | 0–8 | 5–12 | |
Glu149 | 0–6 | Gly302 | 3–5 |
Glu150 | 0–7 | 13–18 | |
Asp152 | 3–6 | 5–14 | |
12–17 | Gly305 | 3–8 | |
Glu154 | 0–5 | 13–18 | |
Gly155 | 5–8 | ||
Val157 | 4–7 | ||
Arg181 | 0–5 |
The residues assembling the majority of the interface are highlighted in bold. The column “Interface Area” displays range of interface area represented by a residue during MD simulation (see
The 90 degree bend of DNA found in the co-crystals of pol-β reflects the state when pol-β is seated on the cleaved AP-site. Since details of its binding to DNA are unknown we considered the possibility that pol-β can bind to a straight DNA, displace APEX1 and occupy the site of the lesion. In order to explore this possibility we, extended the DNA in the pol-β co-crystal with a straight 12-mer template DNA taken from the PDB. We then constructed complexes in the same way as we did for the complexes described above, i.e., by aligning DNAs from pol-β and APEX1 co-crystals.
No new complexes at the 3′-side of APEX1 could be constructed, since the extended DNA intruded into pol-β. For the same reason no complex at the 5′-side of APEX1 with pol-β in closed conformation could be constructed (see
(A) Alignment of 12-mer template DNA and DNAs co-crystallized with pol-β and APEX1. X stands for the abasic site and x stands for lesion. (B) View of the structure of the complex. APEX1 is on the right and pol-β is on the left. The area of protein-protein interaction is circled.
MD of the complex revealed weak interaction of pol-β and APEX1 (see
Time (ns) | Interface Area (Å2) | Interface Area (% of Surface area) | Planarity RMSD (Å) | Length & Breadth (Å) | Polar Atoms (% of interface) | Non-polar Atoms (% of Interface) | Gap Volume (Å3) | Gap Volume/Interface Area | Hydrogen Bonds at Interface | Salt Bridges in Interface | Binding Energy (kcal/mol) |
0.1 | 592 | 4.3 | 3.3 | 40 & 22 | 48 | 52 | 11652 | 10.0 | 5 | 0 | −0.9 |
0.2 | 520 | 3.8 | 3.4 | 42 & 22 | 46 | 54 | 9425 | 8.9 | 7 | 0 | −5.6 |
0.3 | 504 | 3.5 | 3.4 | 43 & 23 | 42 | 58 | 9418 | 9.2 | 3 | 1 | −3.2 |
0.4 | 620 | 4.3 | 3.4 | 42 & 24 | 47 | 53 | 8806 | 7.0 | 5 | 1 | −4.4 |
0.5 | 671 | 3.9 | 3.8 | 41 & 59 | 41 | 59 | 8276 | 7.0 | 6 | 1 | −2.8 |
0.6 | 591 | 4.0 | 3.2 | 43 & 24 | 41 | 59 | 9664 | 8.1 | 4 | 1 | −3.8 |
0.7 | 582 | 4.0 | 3.1 | 42 & 23 | 46 | 54 | 9092 | 7.6 | 5 | 1 | −2.1 |
0.8 | 639 | 4.3 | 3.5 | 46 & 23 | 49 | 51 | 7459 | 5.7 | 5 | 1 | −8.4 |
0.9 | 701 | 4.7 | 3.6 | 39 & 25 | 47 | 53 | 6829 | 4.8 | 6 | 1 | −6.4 |
1.0 | 651 | 4.4 | 3.5 | 41 & 23 | 52 | 48 | 7150 | 5.5 | 5 | 1 | −5.1 |
Values are calculated for interface in APEX1 after 0.1 ns intervals of MD simulation. To calculate the ratio of “gap volume/interface area” the sum of interface areas in APEX1 and pol-β is used in denominator. The free energy of binding was calculated with FOLD-X server.
Overall the interface consisted of one large and one small segment (
Seg | Residues in APEX1 | Interface Area (percentage) | Residues in pol-β | Interface Area (percentage) |
Small | Lys331 | 0–5 | ||
Asp332 | 5–8 | |||
Ser334 | 0–6 | |||
Large | Glu9 | 6–9 | ||
Glu126 | 5–9 | |||
Gly127 | 1–5 | |||
Gly14 | 0–5 | |||
Asp17 | 4–6 | |||
Arg181 | 4–9 | |||
Glu183 | 0–7 | |||
The residues assembling the majority of the interface are highlighted in bold. The columns “Seg” refers to the discrete interface segments where the listed residues are located. The column “Interface Area” displays range of interface area represented by a residue during MD simulation (see
If APEX1 and pol-β evolved to form a molecular complex so that the specificity of their interaction optimized the function of the BER pathway, then one would expect that the network of inter-residue contacts constrains the protein sequence. In other words, the changes accumulated in the evolution of one of the interacting proteins would be compensated by changes in the other one
In fact, multiple sequence analyses of APEX1 and pol-β revealed correlated mutation at the interface of the two proteins in the 3′-complex (
Only alignment for fragments of interacting regions in the 3′ complexes (with open and closed conformation of pol-β) is shown. Residues at the interfaces are in bold; neighboring residues are in normal font. Interacting residues include residues from segments #1, #2, and #3 and adjacent residues (see text), found at interface only in the complex with open conformation of pol-β. Adjacent residues are termed (where possible) AR. Correlated mutations of interacting residues are highlighted in cyan and orange. Other variations in interacting residues are highlighted in red.
In the present work we have made detailed predictions about possible interacting complexes of apurinic/apyrimidinic endonuclease (APEX1) and DNA polymerase beta (pol-β). Although it is possible that the two proteins function entirely independently of each other, our predictions were based on the assumption that at concentrations found in the nucleus the proteins interact with each other when handing off the product of APEX1 to pol-β. Experimental data indicate that for interaction to occur the two proteins have to be associated with DNA. Aligning the DNAs in the co-crystallized complexes of APEX1 and pol-β effectively positioned proteins on a DNA. Similarly, shifting the co-crystallized DNAs in either direction enabled us to orient pol-β downstream or upstream of an abasic (AP) site. Five optimal complexes were identified: two with pol-β located downstream of APEX1 (3′ to the lesion) and three with pol-β located upstream of the APEX1 (5′ to the lesion). The complexes are schematically displayed on
Both 3′-complexes were energetically favorable while only one 5′-complex was stable. In particular, interacting surfaces of both proteins in the 3′-complexes open or closed conformation of pol-β repacked during MD simulation analysis to permit sufficient binding to account for complex formation. During the 1 ns of the MD simulation each complex was stable with relatively constant quantitative values of the interfaces and favorable corresponding estimated binding energies (−10 kcal/mol in each complex). On the contrary, MD for the 5′-complex with pol-β in closed conformation revealed an unstable complex that dissociated completely after 0.4 ns. Although, similar MD simulation for the 5′-complex with pol-β in open conformation revealed interactions, interface dynamics and visual inspection led us to conclude that the complex was not realistic and physical measurements were misleading. For this complex a steric trap formed in APEX and entangled a loop of pol-β (residues 299–308).
Comparison of the 3′-complexes and the weak 5′-complex with straight DNA revealed several important differences. The 3′-complexes had on average large interface areas and significantly stronger binding energies than the 5′-complex. Also the interfaces in the 3′-complexes required almost no repacking since the binding energies were low already at the beginning of the MD simulations (see
Both APEX1 and pol-β are truncated at the N-terminus by 42 and 9 residues respectively in their crystal structures. The truncated residues would be unlikely to interfere with the predicted interacting protein surfaces in the 3′-complex. The 42 N-terminal residues of APEX1, if present, would be located at the side of the predicted interface where there is enough space to accommodate them (see
Pol-β binds a cleaved AP site in open conformation
Movement of thumb and 8-kDa subdomains of pol-β changes interface with APEX1 and may trigger complex dissociation. (A) shows 3′ complex APEX1 and pol-β complex with pol-β in open conformation. (B) shows the complex with pol-β in closed conformation. Area of protein-protein interface is circled. Arrows show the direction of thumb and 8-kDa subdomain movement.
Although we do not see complex dissociation in our simulations, we propose two possible scenarios. In the first scenario segment #1 serves as a springboard to displace the APEX1, which eventually leads to dissociation of the complex. Since APEX1 is processive
Of course, APEX1 and pol-β could bind and dissociate from the DNA independently from one another. However Sokhansanj et al.
The structure of APEX1 was taken from PDB-file 1de8
Chain U of 1de8 representing the AP-site containing DNA was used to position the structure of APEX1. Chains C and D of 2fmq representing DNA with lesion were used to position the structure of pol-β. The DNA strands from crystal structures of APEX1 and pol-β identified above were aligned in order to construct possible interacting complexes. Superposition of the proteins was calculated from the alignment by minimizing RMSD between mainchain atoms of aligned nucleotides. The Kabsch algorithm
The length of DNA in co-crystal of pol-β in open conformation was not enough to align to the DNA in APEX1 co-crystal to construct 5′-complex. That is why the 5′-complex was constructed by replacing the structure of pol-β in the 5′-complex with pol-β in the closed conformation. Optimal position of pol-β in open conformation was calculated by aligning its structure to the structure of pol-β in the complex by using protein structure alignment method TOPOFIT
Molecular dynamics simulation was performed with the aid of the Gromacs software package
All parameters of protein-protein interfaces except that for free energy of binding were calculated using Protein-Protein interaction server
BLAST