Tercer y cuarto escenario de práctica: Elementos para entender y construir la caja

Even though the archaeal bipartite Rad50 ATP binding cassette had been crystallized in its ATP-bound state previously (Hopfner et al., 2000b) and the sequence of this domain is about 25% identical and about 45-50% similar to the bacterial T. maritima, the structure lacked the important coiled-coil domain and could not give insights into how

5. Discussion 89 ATP might impact on the orientation of the coiled-coils as well as the interaction with Mre11. Therefore Rad50´s nucleotide binding domain (NBD) together with approx. 50 amino acids of the Mre11 binding coiled-coil region and Mre11´s helix-loop-helix (HLH) domain was crystallized to gain insights into the structural framework.

The high resolution structure of TmMre11HLH:Rad50NBD bound to the non- hydrolyzable ATP analog AMPPNP offers a detailed view of nucleotide coordination and hydrolysis. The resulting electron density was of high quality and nicely visualized the protein-protein interaction between Rad50´s coiled-coil and Mre11´s C-terminal HLH domain (designated as interface 1) (Figure 32). Interface 1 is stabilized mainly by hydrophobic patches which are highly conserved from bacteria to higher eukaryotes. Moreover mutations in interface 1 resulted in DNA damage sensitivity in S. cerevisiae

yeast strains examined in vivo by the response to genotoxins (Lammens et al., 2011). The large interaction area of 1334 Å2_{, the genetic studies and the high similarity of this}

interaction region to the nucleotide free, open state of MR (PDB entry: 3QG5) (Bemeleit, 2007; Lammens et al., 2011), emphasizes the biological relevance of this anchor point between Mre11 and Rad50 in DSB repair.

Figure 32: Schematic representation of (A) the T. maritima Mre11:Rad50NBD _{complex in its nucleotide}

free state (PDB entry: 3QG5) and (B) T. maritima Mre11HLH_:Rad50NBD _{bound to AMPPNP. Mre11 is}

colored in blue, Rad50 is colored in orange. The macromolecular interfaces 1 and 2 are highlighted by black boxes. Important domains are annotated.

The flexible linker that connects Mre11´s nuclease module with the HLH domain in the nucleotide free MR complex potentially facilitates large conformational changes based on ATP and/or DNA binding (Figure 32). Besides interface 1, tight Rad50 NBD engagement upon ATP binding results in an approx. 50° rotation of the signature motif helix in respect to the Walker motifs, consequently resulting in a 50° rotation of the coiled-coil-HLH-

5. Discussion 90 interacting region with respect to the N-terminal ABC-ATPase domain (lobe I) (Figure 10). As a consequence ATP-binding and therefore the formation of the engaged NBDs strongly affects the angle between the two coiled-coils protruding from the DNA binding catalytic head, consistent with scanning force microscopy of human MRN (Moreno- Herrero et al., 2005), where DNA binding was shown to alter the angle between two coiled-coils by about 60°. Comparing the angle between the coiled-coils in the nucleotide free, open conformation (~120°) and the ATP-bound, closed state (~60°), there is also a difference of about 60°. This suggests that the movement in the catalytic head from the nucleotide free MR complex to the ATP-bound conformation with engaged NBDs is the molecular basis for the observed mesoscale movements of the MRN coiled-coils upon DNA binding. The ATP-driven domain rotation in P. furiosus Mre11HLH(RBD):Rad50NBD was suggested to be transduced to a ~30 Å linear pull on the linker region connecting the HLH domain with the nuclease module of Mre11 (Williams et al., 2011). Therefore, the repositioning of the coiled-coils with respect to the ATPase core could presumably transmit conformational changes to substrate-specific domains of Mre11 therefore stimulating DNA binding and processing activity (Hopfner and Tainer, 2003). Furthermore, the high resolution structure of the TmMre11HLH:Rad50NBD:AMPPNP complex enabled the alignment of the tertiary ATPase domain structures between P. furiosus (PDB entry: 1F2U) and T. maritima. Both structures are quite similar, with overall rms deviations of 2.05 Å, highlighting the similarity of morphological features in bacteria and archaea.

So far, two different states of Rad50´s nucleotide binding domains are known, the nucleotide unbound NBDs which resembles also the conformation in its ADP-bound state (Hopfner and Tainer, 2003) and the ATP-bound state, which results in reorientation of the signature motif helix and therefore mesoscale movements of the coiled-coil domain. To analyze if Rad50´s ATPase mechanism is affected in a third state, for instance the transition state like in P-Type ATPases where phosphorylation of a specific amino acid leads to a conformational change and therefore to the pumping power of the ATPase (Kuhlbrandt, 2004), Mre11HLH:Rad50NBD of T. maritima was crystallized bound to ADP orthovanadate. Since the orthovanadate ion has similar size and charge to inorganic phosphate, it can adopt its trigonal bipyramidal coordination, therefore mimicking the phosphate ion transition state expected during phosphoryl transfer. Based on these features, vanadate is a valuable tool for studying enzyme mechanism (Smith and Rayment, 1996).

5. Discussion 91 Structural comparison of the Rad50 ATP binding region in AMPPNP-bound and ADP[VO4]3--state revealed no large conformational changes. These results suggest that

ATP hydrolysis is not synchronized with a force-generating step leading to a stimulation of enzymatic action. This is in opposition to the proposed model where Rad50 might enter an adenylate kinase cycle as a result of occurring conformational changes upon ATP hydrolysis, which would necessarily reduce the distance between the two ATP binding sites from approx. 35Å to approx. 16Å (Bhaskara et al., 2007). However, Rad50 could stimulate the nuclease activity of Mre11 either by ATP-binding and/or ATP-release.

In summary, the Mre11:Rad50 complex has to undergo large open-to-closed conformational changes upon ATP binding. Linkage between the Rad50 coiled-coils and Mre11´s HLH domain in interface 1 seems to be essential as an anchor point. In addition, the conformational changes driven by nucleotide binding lead to an alteration of the coiled- coil domains with respect to the ATPase core which is suitable to mediate communication within and between MR(N) complexes. In contrast, phosphoryl transfer does not seem to play a role in energy transfer followed by conformational alterations or stimulation of the adjacent substrate domain.