In the biophysical analysis of BRCT domain of 53BP1 and BRCA1, Ekblad and coworkers reported that the BRCT domain of 53BP1 is slightly more stable than that of BRCA1 (Ekblad, et al.,2004
). Molecular dynamics simulations were performed to investigate the stability of these two proteins. The RMSD of the Cα atoms of these two proteins over time with respect to the starting minimized structures are very close to each other, as shown in , with BRCA1 slightly larger than 53BP1, which is consistent with the experimental report. The conserved subunit repeat 1 has the closest stability to 53BP1 and BRCA1 (). The more disordered α2B of BRCA1 contributes to the slightly more flexible repeat 2 vs the corresponding region of 53BP1, as shown in . The inter-repeat linker is not as conserved as other regions. 53BP1 with the β-hairpin-like structure is more stable than BRCA1 with loop LA followed by the α-helix structure at the linker position, as shown in . Note that the linker region is one of the binding regions of 53BP1 with p53. Structural diversity and flexibility at this region may contribute to a weaker binding of BRCA1 to p53 than 53BP1.
Figure 2 Cα RMSDs for the BRCT domain and its sub-domain of 53BP1 (blue) and BRCA1 (red) as well as 53BP1-p53 (blue) and BRCA1-p53 (red) complexes. (a) Cα RMSDs of 53BP1 are slightly lower than of BRCA1 for the whole BRCT domain including repeat (more ...)
53BP1-p53 and BRCA1-p53 complexes
The interaction regions of 53BP1 with p53 include part of the p53 DNA binding region and 53BP1 N-terminal repeat as well as the linker region. Specifically, loops L2, L3 and H1 helix of p53 interact with the linker, α4A and α3A of 53BP1, respectively. One of the DNA binding features, H2, is not involved in the 53bp1-p53 interaction. A model was built based on the 53BP1-p53 crystal structure and BRCA1 NMR structure, as shown in . Molecular dynamics simulations were performed for both 53BP1-p53 and BRCA1-p53 complexes. In spite of the relatively small contact area with p53, the interface of 53BP1-p53 complex is rather stable. The RMSD value of the interaction region vs the initial structure is around 2Å (blue in ). The BRCT structures are similar in BRCA1 and 53BP1, however, the simulations suggest that the interaction region of the BRCA1-p53 complex is much less stable (, in red).
The interface sequence alignment of BRCA1 and 53BP1 is shown in . 53BP1 has ten residues that contact with p53, including three in α3A, four in α4A, and three in the linker region. BRCA1 retains eight of these ten residues to interact with p53, but the two contact positions in the linker region are different because of the distinct linker structures of 53BP1 and BRCA1. Among those eight same-position residues, the least conserved residue is P1849/R1737. 53BP1 Proline 1849 interacts with Arginine 248 of p53 by favorable stacking interaction; however, there is a charge conflict between the corresponding R248-R1737 destabilizing the interaction of BRCA1 and 53BP1. BRCA1 K1724 in α3A also has a charge conflict with p53 R181.
In addition to the sequence distinction at the interface, there are two structural differences that may also affect the interaction of BRCA1 with p53. The structure between β3A and α2A is one of them, with a 6 residue loop in 53BP1 but an 11 residue loop in BRCA1. The longer loop of BRCA1 reaches p53, providing BRCA1 an additional possible contact with p53 as compared to 53BP1, including interactions of Arg248-Glu1694 and Met243-Phe1695.
The linker region structure is also different between 53BP1 and BRCA1. In 53BP1 the linker is composed of two β strands followed by a loop, whereas BRCA1 has a loop followed by α helix. Due to the structural difference, corresponding residues in the BRCA1 linker region cannot interact with p53 as in 53BP1. Instead, in the BRCA1-p53 complex two other residues, R1744 and K1750, contact with R248 and Q167 of p53, respectively. The R1744-R248 charge conflict destabilizes the BRCA1-p53 interface.
In the stable 53BP1-p53 complex most interacting residues retain their interaction over time, as shown in . Three 53BP1 domains bind to p53. These interface residue pair distances suggest that the most stable interaction is α3A and α4A of 53BP1 with the H1 helix and L3 loop of p53, respectively. The only exception is that the distance of residue pair R248-L1847 increases after 9ns. The least stable interaction is at p53 L2 with the 53bp1 linker region. There are two contacts in this region, Gln165-D1861 and Gln167-Q1863. The minimum distance of both contacts fluctuated and the contact is lost after 8 ns simulation.
Figure 3 Variations of distances between interface residue pairs from (a) 53BP1-p53 and (b) BRCA1-p53 complexes. The top plots are residue pairs between p53 and 53BP1/BRCA1 α4 region residues. The bottom plots are residues pairs between p53 and 53BP1/BRCA1 (more ...)
The interacting residues distances of BRCA1-p53 complex along time are shown in . All residue pairs in α3A fall apart from p53 after 8ns, if not earlier, as shown at the top. The interaction of BRCA1 α3A with p53 H1 helix is lost after 2ns, due to the K1724-R181 charge repulsion. The BRCA1 linker-p53 L2 loop contact region fluctuated during the 10 ns due to the flexibility of the linker. The most stable residue pair, M243-F1695 in p53 L3 loop and BRCA1 LA loop, does not exist in 53BP1. In short, all three contact regions between 53BP1 and p53 lose contact with p53 in BRCA1 after 10 ns simulation, and the only region that retains contact is the LA loop region. By comparing snapshots at 0 ns and 10 ns (, top), it appears that the linker region has dramatically moved away from p53 L3 loop toward p53 H2 helix. There are two possible reasons: the charge repulsion between BRCA1 Arg1744 in the linker region with p53 Arg248 in the L3 loop and the charge attraction between BRCA1 Arg1744 with p53 Glu285. The distance between these two residue pairs is shown in . The Arg248-Arg1744 distance increased sharply after 5 ns, while the Glu285-Arg1744 distance started to decrease after 5 ns. It is conceivable therefore that the charge repulsion of p53 L3 loop drives the flexible linker loop toward the charge attraction of the H2 helix.
Figure 4 Snapshots of the BRCA1-p53 interface from the wild type, F1695L and D1733G at 0 ns and 10 ns. The top panel is the wild type BRCA1-p53 interface, where the R1744 at the linker region drifts away from crowded LA loop region from 0 ns to 10 ns. The middle (more ...)
The other possible reason for the loss of the contact of BRCA1 linker with the p53 L2 loop can be attributed to the bulky LA loop. The longer LA loop, including a phenylalanine residue, of BRCA1 makes the contact region more crowded blocking the rotation of the charged residue in the p53 L3 loop to avoid the positive charge rich region of the BRCA1 linker, driving away the flexible linker region.
As discussed in the previous section, we attribute the weaker interaction of BRCA1 with p53 to the electrostatic repulsion between α3A, α4A as well as the linker region of BRCA1 and p53, the steric hindrance of BRCA1 LA loop, and the flexible loop of the linker of tandem BRCT. To investigate whether these electrostatic and steric factors can be modulated allowing the formation of a stable complex, thirteen simulations of mutants in α3A, α4A, LA and in the linker region have been performed. Among these thirteen mutants, mutations of 1724, 1733, 1737 and 1744 were designed to investigate the effect of electrostatic repulsion. Mutations of 1694 and 1695 are on the LA loop, which provides a unique contact region of BRCA1 with p53 comparing to 53BP1, are designed for probing the steric hindrance of the BRCA1 LA loop. A mutant with two mutations at 1737 and at 1744 was also designed to compare with the single point mutations at each of these sites. Four of these thirteen mutants, F1695L, N1730S, D1733G, and F1734S are carcinogenic; these are the only known carcinogenic mutations of BRCA1 which are in contact with residues with p53 in our modeled complex. Interestingly, F1695 and D1733 are not conserved between BRCA1 and 53BP1, whilst N1730 and F1734 are conserved. The results of simulations are shown in and .
Cα RMSDs of interface for BRCA1 mutants interacting with p53. The top plots are mutants of the LA loop (left) and α3 region (right). The middle plots are mutants of α4 region. The bottom plots are mutants of linker region.
Figure 6 Cα RMSFs relative to the average structures of the last 5 ns of the trajectories for the BRCA1 mutants. The top plots are mutants of LA loop (left) and α3 region (right). The middle plots are mutants of α4 region. The bottom plots (more ...)
In α3A, there is an electrostatic repulsion between positively charged residues K1724 and R181 of p53. Two mutants, K1724E and K1724H, were simulated. K1724E is expected to stabilize the interface. Since the corresponding residue in 53BP1 is histidine, K1724H mutant was also simulated. (top, right) shows that K1724E stabilizes the interface, whilst K1724H has a slightly smaller RMSD value along time than the wild type BRCA1 in the first 5 ns, but after 5 ns the RMSD of K1724 increases and has no significant difference from that of wild type BRCA1. The RMSF plot () shows that the residue fluctuation at the interface of K1724E significantly decreases, especially in the linker region. This again is not the case for K1724H. This result is consistent with our expectation that eliminating positive charge repulsion in α3A region can stabilize the BRCA1-p53 interface.
At the α4A region, the complexes of D1733R and D1733G mutants with wild type p53 were simulated. Unsurprisingly, both mutants stabilize the interface, as shown in (middle, left). The negatively charged Asp1733 not only attracts p53 R249, it also attracts BRCA1 R1737 from the linker region. Thus, the charge repulsion of R249–R1737 is one of the causes that induce the fluctuation at the linker region. Replacing the negative aspartic acid by neutral glycine or positively charged arginine reduces the fluctuation of the linker region, as shown in .
Simulations of mutants N1730S and F1734S were also performed in the α4A region. Both N1730S and F1734S are carcinogenic mutations; however, N1730 and F1734 are conserved with 53BP1. F1734 interacts with p53 M243; thus, unsurprisingly F1734S does not stabilize the interface. N1730S has slightly smaller RMSD value along time than the wild type BRCA1.
Mutants R1737P and R1737E replace arginine by neutral or negatively charged residues to avoid charge conflict with p53 Arg248. R1744A and R1744E also mutate arginine on the linker region to avoid interaction with E1694 on the LA loop. Interestingly, (bottom, right) shows that R1737P and R1737E have smaller RMSD values than the wild type BRCA1 but the RMSD value of R1744A is close to the wild type. In addition, the RMSF plot suggested that both R1737E and R1737P decrease the fluctuation at the linker region but R1744A increases it, and that R1744E decreases the fluctuation at the linker region but increases the fluctuation in the LA loop. This is not surprising and could be explained by the elimination of charge repulsion of R1737 of BRCA1 with p53 R248, thus stabilizing the linker region, however, losing the electrostatic attraction of R1744 and E1694 destabilizes the system even more. The electrostatic repulsion destabilizes the LA loop. In order to further probe the mutational effects, we performed simulations for the double point mutant R1737E_R1744E and compared the results with the simulations of the corresponding single point mutations. Both RMSD and RMSF results suggested that the difference between single point mutations and double point mutations is not significant.
The contact of the LA loop with p53 is unique to BRCA1 whereas the corresponding loop region of 53BP1 is not long enough for interaction. The BRCA1 LA loop forms the Arg248-Glu1694 and Met243-Phe1695 interactions with the L3 loop of p53. However, the existence of these new contacts destabilizes the contact region of BRCA1 with p53 because Glu1694 on the LA loop not only forms a contact with Arg248 on the p53 L3 loop; it also attracts Arg1744 at the beginning of the BRCT linker. The repulsion between the two arginine residues causes a large conformational change at the interface, including twisting the LA loop and pushing the linker loop toward the direction of p53 H2 helix. The direct consequence of the conformational change is the loss of the contact between the BRCA1 linker with the p53 L2 loop, thus the interaction of p53 and BRCA1 BRCT is weakened.
To test whether the LA loop is responsible for the flexibility of BRCA1-p53 interface, two mutations E1694K and F1695L, have been performed in the LA loop. As shown in , the E1694K avoided the two arginine residue situation, p53 R248 and BRCA1 R1744, stabilizing the linker loop during the first 6 ns, however, the repulsion of the lysine and linker region arginine R1744 finally causes structural fluctuation of the linker region, as shown in the RMSF plot (), increasing the RMSD of E1694K significantly after 6 ns. Although F1695L is not directly connected to the interaction at the linker region, leucine is less likely to cause steric hindrance than the bulky phenylalanine. Arg248 can rotate to form Arg-Asn hydrogen bond instead of interacting with Glu1694. Therefore, repulsion of Arg248-Arg1744 is again avoided and the linker is stabilized.
The binding energy differences, and average RMSD of the complex interface were calculated for each mutant, and are listed in . The more negative the ΔΔG, the stronger the BRCA1-p53 binding it implies; and, smaller values of the RMSD of the complex interface suggest more stable interface. Comparing to wild type BRCA1, mutants F1695L and D1733G have the most negative ΔΔG, suggesting stronger binding with p53 than the wild type BRCA1 in this orientation. In addition, these two mutants have the smallest average RMSD values. Interestingly, these two mutants are the only cancer-related mutants among all the mutants simulated according to the National Human Genome Research Institute (NHGRI) breast cancer mutation database. Considering that the binding energy of F1695L mutants with p53 is more negative and the average RMSD value is smaller than D1733G, we predict that among these thirteen mutants, F1695L could be the mutant candidate to most stabilize the interface of BRCA1-p53 complex in the conformation similar to the 53BP1-p53 complex.
Comparison of the binding energy and interface average Cα RMSD for BRCA1 mutants bound to p53. The mutants with the most negative binding energy are bolded.