The Rad50 binding domain (RBD) of Mre11
To map the Mre11 RBD, we generated a series of P. furiosus Mre11 (pfMre11) deletion constructs and tested their ability to co-express and co-purify with coiled-coil truncated pfRad50 constructs (). Mre11 C-terminal truncations, which left the N-terminal core nuclease domain intact, revealed that the 342–379 region contained residues essential for binding histidine-tagged pfRad50 (pfRad50-NC). A C-terminal Mre11 construct (residues 348–426) also bound Rad50 (). To finely map the Mre11 RBD, we expressed and co-purified Mre11–Rad50 complexes of untagged Rad50 with shortened coiled-coils connected by intramolecular "Gly-Gly-Ser-Gly-Gly" sequences (pfRad50-link1) with predicted minimal Mre11 RBD regions containing a histidine-tag. Our shortest Mre11 construct, residues 348–381 (Mre11RBD), bound tightly to and co-purified with pfRad50-link1. Collectively, these data delineate a major physical Mre11–Rad50 interaction for Mre11 residues 348–379 and a corresponding Rad50 binding site within the first coiled-coil ~6 heptad repeats, proximal to the ATPase core.
The four-helix architecture of the Mre11–Rad50 interface
To define the Mre11–Rad50 interface structure, we solved two independent X-ray crystal structures, to 2.1 Å and 3.4 Å resolution, of pfMre11RBD
bound to pfRad50-NC (), which reveal the same interface. Our 2.1 Å structure provides high-resolution details about this interface (). The Mre11 RBD consists of two helices (RBD-αI and RBD-αJ, named sequentially from nuclease core labeling10
) that interact with the Rad50 coiled-coil base through a conserved hydrophobic surface patch. This 4-helix interaction differs from classical 4-helix bundle interfaces, such as in human Mn superoxide dismutase14
and typical coiled-coil packing such as in bacterial pili15,16
, as Mre11 helices pack almost orthogonally to the two Rad50 coiled-coil helices. The Mre11RBD
–Rad50 interface includes 72% of the 32 Mre11RBD
residues and has a ~970 Å2
buried surface area (BSA). Ten Mre11RBD
hydrophobic core residues account for 75% of the total BSA, and this strong interface spans ~20 Å across and ~30 Å up the Rad50 coiled-coils. The conserved interface in human Mre11–Rad50 likely involves Mre11 RBD residues Gln435–Lys475, and Rad50 coiled-coil regions around Arg184–Lys204 and Lys1,098–Asp1,129.
Data collection and refinement statistics
Mre11 RBD is flexibly linked to the Rad50 coiled-coils
Our sequence alignments and Disopred2 (ref.17
) disorder predictions show residues spanning from 333 at the end of previous Mre11 nuclease coordinates9,10
to reside 348 of our interface structure have high sequence divergence, suggesting intrinsic disorder (). In fact, residues 334–342 were present and disordered in our previous structures (PDB codes 3DSC, 3DSD, 1II7)9,10
, but we missed the significance of this observation. Thus, combined results reveal that a flexible tether links the Mre11 nuclease and RBD domains.
Comparison of Mre11RBD–Rad50 structures from two different crystal forms furthermore reveals that the Rad50 coiled-coils can adopt dramatically variable orientations relative to the ATPase domains in the nucleotide free form (). These changes are highlighted by core Mre11RBD–Rad50 superimpositions; this region superimposes well, but the Rad50 ATPase domain can rotate substantially with respect to the coiled-coils. These domain motions uncover intrinsic flexibility in the hinge region at the base of the coiled-coil N-terminal α-helix, which is adjacent to the Mre11RBD–Rad50 interface. In our structures this region has limited contacts with the N-terminal half (N-lobe) of the Rad50 ATPase domain and adopts dramatically different conformations, imparting a 30° twist and 15 Å shift relative to the coiled-coils. Conversely, the base of the C-terminal helix of the coiled-coil is rigid, and makes extensive contacts with the C-terminal half (C-lobe) of the Rad50 ATPase core.
Mre11 RBD mutations disrupt Mre11–Rad50 interactions in vivo
Based on sequence alignments of Mre11 orthologs (), mutations were introduced into S. pombe Mre11 (also known as Rad32) to test the functional significance of the Mre11 RBD. Hydrophobic Cys-Leu (CL) residues in RBD-αI and Cys-Val (CV) residues in RBD-αJ were changed to charged Arg-Arg (RR) residues, either separately or in combination. Collectively, these residues at the Mre11 RBD core mediate hydrophobic interactions to both of the Rad50 coiled-coil α-helices and also between RBD-αI and RBD-αJ. Thus, their substitution to charged Arg residues should disrupt the interface. Indeed, two-hybrid analyses showed that Mre11 and Rad50 have a robust interaction, but this was severely diminished by the RR mutation in either RBD-αI or RBD-αJ (). Importantly, our Mre11 RBD mutations did not impair Mre11 homodimeric or Nbs1 interactions, indicating specific disruption of the major Mre11–Rad50 interface.
A conserved interface links eukaryotic Mre11 and Rad50
The Mre11 RBD is critical for DSBR in fission yeast
To test whether the Mre11 RBD mutants show increased DNA damage sensitivity, we examined responses to four genotoxins: 1) ionizing radiation (IR), which directly makes DSBs; 2) UV light, which creates DNA photoproducts that can be processed into DSBs; 3) camptothecin (CPT), a topoisomerase inhibitor that causes replication fork breakage when the replisome encounters a topoisomerase–CPT complex; and 4) hydroxyurea (HU), which stalls replication forks by inhibiting ribonucleotide reductase required for dNTP synthesis. The mre11
alleles replace genomic mre11
) and encode a C-terminal myc-tag. These strains were compared to mre11Δ
and myc-tagged mre11-WT
control strains. This myc-tag does not noticeably impair Mre11 function9
. Immunoblotting showed that the Mre11 RBD mutants were expressed at levels comparable to wild type ().
The Mre11–Rad50 interaction interface coordinates DSBR in S. pombe
In agreement with their poor abilities to interact with Rad50 in two-hybrid assays (), the Mre11 RBD-αI (mre11-CL454RR
) and RBD-αJ (mre11-CV479RR
) mutants resembled mre11Δ
in being very sensitive to IR, UV, CPT and HU (). These mutants also formed smaller colonies than wild type, indicating defects in repair of spontaneous DNA damage. Serial dilution assays performed with UV, HU or CPT shows that the Mre11 RBD mutants are slightly more resistant than mre11Δ
cells. This small difference might be because the Mre11 RBD-αI and RBD-αJ mutants retain residual interactions with Rad50 that were not detected by yeast two-hybrid analysis; thus we did survival assays on an mre11-RRRR
allele that should completely disrupt the interface. This allele appeared identical to the mre11-CL454RR
alleles in serial dilution assays () and in IR survival assays the mre11-RRRR
strain is slightly more resistant than mre11Δ
( and Supplementary Fig. 2
). Collectively, these data show that the Mre11 RBD forms an interface that is critical for DSBR, although weak function is maintained when it is mutated.
ExoI nuclease can compensate for Mre11 RBD mutant phenotypes
To see if Mre11 interface mutants impact DNA end-processing in fission yeast, we tested if ExoI nuclease can compensate for Mre11. In the IR survival assays the mre11-RRRR
strain is slightly more sensitive than the previously characterized mre11-H134S
mutant (). Genetic and biochemical studies indicate that the mre11-H134S
genotoxin sensitivity is caused by a defect in ssDNA endonuclease activity9
that is suppressed by inactivating the Ku70–Ku80 complex, which can bind and block ends. This rescue requires Exo1 exonuclease, indicating that Mre11 endonuclease activity is critical for generating single-strand overhangs that are competent for HR repair18,19
. To assess whether the mre11-RRRR
mutant is defective in DNA end-processing, we created mre11-RRRR
strains lacking Ku80 and/or Exo1. We found that the pku80Δ
mutation suppressed the slow growth phenotype as well as the IR, CPT, UV and HU sensitivities of mre11-RRRR
cells (). This supports a model in which Ku promotes non-homologous end-joining by binding to DSB ends and inhibits Exo1-dependent resection19
. Accordingly, the extreme genotoxin sensitivity of the mre11-RRRR pku80Δ exo1Δ
strain showed that this suppression was dependent on Exo1 activity. Indeed, the exo1Δ
mutation substantially exacerbated the mre11-RRRR
phenotypes. These results indicate that the mre11-RRRR
phenotypes primarily reflect an inability of Mre11–Rad50 to process DNA ends for DSBR by HR.
Architecture of the M2R2-head
To test the Mre11RBD
–Rad50 complex flexibility implied from crystal structures, we examined M2
-head solution conformations with small angle X-ray scattering (SAXS). SAXS combined with crystal structure restraints can accurately define flexible conformations and ensembles in solution, and can also identify existing structures that most closely match the measured experimental scattering20
. Experimental SAXS curves of M2
-head preparations () show dramatic scattering curve changes supporting a flexible to more ordered transition: featureless without ATP (−ATP) to defined peaks and troughs with ATP (+ATP). Further, the radius of gyration decreases from 46.5 to 41.0 Å upon ATP binding, dimensions resembling M2
-head regions within intact pfMre11–Rad50 EM images12
. These results, along with a compaction observed in the pair distribution (p(r)) plot (Supplementary Fig. 3
), show that the M2
-head transitions from a more open to compacted states upon ATP binding. This supports our hypothesis that ATP binding in the M2
-head leads to Rad50 dimerization, which would close the M2
-head to form a globular, toroidal structure.
To model the conformational flexibility implied for the −ATP data by the featureless curve and overall architecture of the M2
-head in the absence and presence of ATP, we used molecular dynamics (MD) and minimal ensemble searches (MES)21
to find M2
-head structural models that best fit the data. We find the predominant M2
-head architecture without ATP is a partially open state; yet, improved fit to the data by a mixture of open, partially open and closed conformations shows the inherent flexibility of the complex without nucleotide (). With ATP we expected to see mixed ATP-bound and free states in solution. So to accurately model M2
-head complex with ATP, we used MES with closed ATP-bound M2
-head models, based on our crystal structures described below, combined with models identified for the −ATP data (). The results suggest that 89% of the M2
-head is in a closed state, with the Mre11 dimer and ATP-induced Rad50 forming a toroidal, globular structure.
-head toroidal structure from our SAXS analyses is independently supported through direct comparisons of M2
-head scattering curves with SAXS curves calculated from the Protein Data Bank, using the database for rapid search of structural neighbors (DARA)22
. DARA analysis shows that the M2
-head quaternary assembly is most structurally conserved with Topoisomerase-II and the DNA mismatch repair protein MutS, another ABC-ATPase superfamily member (Supplementary Fig. 3
). Both of these proteins form toroidal-like structures, supporting the accuracy of M2
-head architecture observed from our MD and MES modeling. Notably, the architectural similarity of M2
-head with MutS reveals a striking convergent evolution of quaternary assembly modes for divergent members of the ABC-ATPase superfamily.
ABC signature helices couple nucleotide state to domain movements
To test if this Mre11RBD
–Rad50 interface is impacted upon nucleotide binding in the Rad50 ABC-ATPase core, we used our Rad50 constructs that intramolecularly link the ABC-ATPase N- and C-lobes with adjacent coiled-coil regions (): these constructs facilitated ternary structure solution of Mre11RBD
to 3.3 Å () and Rad50-link2–AMP:PNP–Mg2+
to 1.9 Å resolution (). Superimposition of our nucleotide-free and -bound structures of Rad50 containing coiled-coil regions and morphing between crystallographically-defined states reveals both global conformational changes (Supplementary Movies 1
) and their underlying basis (). As Rad50 AMP:PNP structures with and without bound Mre11RBD
superimpose well, we used the higher resolution structure for most analyses.
Rad50 nucleotide-binding induced conformational changes
structure defines the nucleotide bound state of the unknown half of the M2
-head, with molecular dimensions of 120 × 74 × 62 Å. Globally, the Rad50 ABC-ATPase core dimerizes with AMP:PNP–Mg2+
sandwiched at the crystallographic two-fold interface (), inducing a ~35° rotation of the C-lobe relative to the N-lobe, supporting and extending changes proposed from core structures lacking all coiled-coil regions11
. However, our new nucleotide-bound structures reveal novel positions of two helices, which we term the signature coupling helices, immediately C-terminal to the Rad50 Q-loop. These helices, which are absent from the original nucleotide-bound Rad50 structure, connect the Q-loop to the base of the N-terminal helix of the coiled-coil. Upon nucleotide binding, Rad50 N-lobe rotation drives a π-helix element (π-helix wedge) between the signature coupling helices to splay them apart ( and Supplementary Fig. 4
). The signature coupling helices movement, resembling the opening of an arm at the elbow, acts as a lever exerting force on the base of the N-terminal coiled-coil helix, which our nucleotide-free structures show is a point of flexion. This force repositions the coiled-coils with respect to the ATPase core, impacting the Mre11 RBD position. As shown by structure-based animation (Supplementary Movie 3
), the ATP driven domain rotation is transduced to a ~30 Å linear pull on the Mre11 linker by the Rad50 coiled-coil movement at the Mre11 interface ().
Basic-switches and alternating salt bridges control ATPase rotations
Underlying the global conformational changes described above is a striking, extensive network of >20 charge pairs that switch upon nucleotide binding (). These changes provide a mechanism to physically couple ATPase conformational rotation to coiled-coil and attached Mre11 RBD repositioning. Intriguingly, basic-switch residues (Arg797 and Arg805 in pfRad50) immediately adjacent to the conserved signature motif, which defines the ABC-ATPases superfamily, occupy a conserved helix. We term this the signature helix as it encodes a molecular conformational switch that links signature motif nucleotide recognition to subdomain rotation.
Arg797 hydrogen bonds to main chain signature motif atoms in the nucleotide-free state. Upon nucleotide binding, the signature motif moves to contact the nucleotide. As a consequence, Arg797 detaches from the signature motif and moves to form interaction networks with Glu148 and Asp144 on signature coupling helix-α1 ( and Supplementary Fig. 4
). These new Arg797 interactions can only form after opening and translation of the signature coupling helices following N-lobe rotation and limit further rotation of the first helix. Arg797 movements thus directly link nucleotide recognition by the signature motif to movements of the signature coupling helices that control coiled-coil positioning.
Signature helix Arg805 integrates the signature motif, Q-loop, and domain rotations. Nucleotide binding breaks Arg805 hydrogen bonds to the Asn134 main chain. Arg805 then moves towards the protein surface with concomitant rearrangements of the Q-loop. Arg805 rotates into the signature coupling helices and guides the π–helix to wedge open signature coupling helix-α1 and form new hydrogen bonds to Gln142 and Ile143 main chain carboxyl atoms (). This switch at the junction between the N-lobe, C-lobe, and coiled-coil base, implicates Arg805 as a key coordinator of Rad50 ATPase lobe rotation and its coupling to coiled-coil rearrangements.
Rad50 basic-switches are critical for DSBR in fission yeast
Rad50 ortholog sequence alignments reveal the conservation of basic residues corresponding to pfRad50 Arg797 and Arg805 (). To test the functional significance of these basic-switch residues for DSBR in vivo, we made K1187A, K1187E (pfRad50 Arg797 equivalent), R1195A and R1195E (pfRad50 Arg805 equivalent) mutations in S. pombe Rad50. The rad50 alleles replace genomic rad50 (rad50-WT) and encode a TAP-tag. These strains were compared to rad50Δ and TAP-tagged-rad50-WT control strains. This TAP-tag does not noticeably impair Rad50 function and immunoblotting showed that the Rad50 basic-switch mutants were expressed at wild type levels, with the exception of R1195A that has reduced expression ().
ABC-ATPase superfamily conserved basic-switch residues in Rad50 coordinate DSBR in S. pombe
To test whether the Rad50 basic-switch mutants show increased DNA damage sensitivity, we examined their responses to genotoxins. Serial dilution assays show that Rad50 basic-switch mutants are more sensitive to the clastogen agents than the rad50-WT control strain (). The K1187A variant phenotype is seen mainly with higher doses of clastogens. In contrast, the K1187E and both R1195A and R1195E variants are strikingly sensitive to clastogen agents and are as deleterious as rad50Δ. Thus, these assays reveal the importance of Rad50 signature helix basic-switch residues for DSBR in vivo.