Our results provide a structural framework for eukaryotic Mre11 and its complex with Nbs1, revealing the molecular basis of a core part of the MRN complex (). Nbs1 wraps as an extended chain around the Mre11 phosphodiesterase domain with 2:2 (M:N) stoichiometry, but only one of the two Nbs1 completely binds to Mre11 via the NFKxFxK motif as the observed asymmetric binding mode sterically excludes binding of the second Nbs1 NFKxFxK motif to the latching loops. Although we have crystallized only a small portion of Nbs1, the observed complex likely harbors most if not all of the interaction sites between Nbs1 and Mre1150
. However, it is thought that there exists, at least for the human MRN complex, an additional interface between Nbs1 and Rad50, which might map to the region N–terminal to the co–crystallized fragment52
Models for the principle architecture of eukaryotic MRN and MRN dependent DNA double–strand break signaling
Recently, a crystal structure of human Mre11 was published that differs significantly from the dimeric conformation of the S. pombe
Mre11 structure presented here53
, despite an otherwise identical fold (Supplementary Fig. 6a
). Whereas in S. pombe
the canonical four helix bundle forms the dimer interface19,54
, the human Mre11 dimer is connected by a disulfide bond between helix aC from each monomer (Supplementary Fig. 6b,c
). The responsible cysteines are not conserved between H. sapiens
and S. pombe,
or even all vertebrates (e.g. Xenopus laevis
). Thus, at this time, it is unclear what functional state the conformation of the human Mre11 structure in the absence of Nbs1 displays.
The extended interface of Nbs1 along the phosphodiesterase domains of Mre11 gives insights into the molecular pathology of A–TLD. A–TLD associated point mutations along these interfaces validate the observed interactions and show that binding between Mre11 and Nbs1 is mediated by several distributed, independent interaction sites. (). Thus, single point mutations are unlikely to completely disrupt the complex, explaining the hypomorphic phenotype of different A–TLD variants. Furthermore, the mitotic repair and recombination functions of MR are largely unaltered by the ScMre11 N113S mutation, if nuclear levels of MR are recovered by an NLS on Mre1141
(). This argues for proficient Mre11 nuclease and Rad50 ATP binding capabilities in of MRN in ATLD3/4. However, the NLS scarcely rescued telomere shortening in the mre11–N113S
, indicating that a stable interaction between the NFKxFxK motif of Xrs2/Nbs1 and the latching loops of Mre11 may be crucial for MRX–mediated telomere maintenance. This is in agreement with a former study in which an acidic point mutation in the Xrs2 NFKxFxK motif led to shortened telomere length50
. One major role of the MRX complex in telomere maintenance is to recruit the checkpoint kinase Tel1 to short telomeres55
, so the phenotypes observed may represent defects in Tel1 recruitment or activation.
In contrast, the arginine finger mutation (Arg76 in S. cerevisiae
and Arg85 in S. pombe
) cannot be rescued by NLS-tagging, suggesting that this mutation induces a fundamental defect in Mre11. As SpMre11cd
R85A still formed dimers and interacted with Nbs1mir
, and prokaryotic Mre11 lack this motif altogether, it is surprising that the arginine finger mutation has such a dramatic phenotype19,54
. Thus, we propose a role of this arginine in orienting the Mre11 dimer in a particular conformation that is important for MRN function. In addition, this arginine finger coordinates a conserved aspartate in the latching loop (Asp109 in S. cerevisiae
and Asp118 in S. pombe
), mutated in NBSLD, and the analogous mutation in S. cerevisiae
resulted in significantly shortened telomeres, We assume that this stems from a partly destabilized Mre11 latching loop coordination and Xrs2 interaction in mre11–D109G
, which impairs the Xrs2 dependent telomere maintenance functions of the complex.
The asymmetric bridging of Mre11 dimers by a single Nbs1 subunit, paired with the uncovered intrinsic flexibility of the eukaryotic Mre11 dimer, is perhaps the most significant and unexpected finding of our structural and functional analyses. Nbs1 side chains in direct contact with Mre11 at the latching loops are more or less invariant across species41,56
, so this asymmetric bridging appears to be a conserved feature of the Nbs1–Mre11 interaction. We do not know what the other “free” NFKxFxK motif does in the complex. One possibility is that one Nbs1 controls the functional architecture of MR while the other may interact with other repair proteins or DNA. This could provide an asymmetry that may reflect the necessarily asymmetric protein interactions at a DNA end.
Because the Mre11 dimer structure is bridged by the pseudo–symmetric NFKxFxK motif, it is tempting to speculate that conformational changes in the Mre11 dimer and Mre11–Nbs1 interaction are important for MRN function. In support of this, the dimer interface residues of bacterial and archaeal Mre11 undergo conformational changes upon Rad50 dependent ATP binding 19,20,57,58
. Additionally, DNA bound archaeal Mre1159
and S. pombe
Mre11 structures have different angles in the Mre11 dimer, which might represent different functional states of the protein (Supplementary Fig. 6d,e
). An Mre11 dimer angle rotation, controlled by Nbs1 on one side and by DNA and/or Rad50 plus ATP on the opposing side of Mre11, might be sensed by ATM via the C–terminal tails of Nbs1, adjacent to the NFKxFxK motif (). This region of Nbs1 contains an ATM interaction motif30
which directly stimulates ATM in Xenopus
While the mechanistic link between Rad50 and DNA binding to Mre11 requires further studies, our data suggest that Mre11 dimer flexibility and its control by Nbs1 could be an important part of MRN function.