POLÍTICAS AGRARIAS ECUATORIANAS DURANTE EL PERÍODO

Due to trapping of the ATP bound engaged Rad50 NBDs by disulfide bridging, it was possible to solve the crystal structure of the T. maritima Mre11:Rad50NBD (S-S) (TmMR

NBD (S-S)_{) complex, trapped in its ATP/ADP bound state.Although ATP was hydrolyzed to}

ADP in the course of disulfide bridging or crystallization, the NBD dimer is structurally similar to the AMPPNP bound form of the Rad50NBD _{dimer. This similarity not only}

emphasizes the relevance of the presented ATP dependent conformational changes of MR, it also suggests that disulfide bond stabilization could be a general approach to trap ABC enzymes in the ATP bound state for structural studies.

Together with the structure of T. maritima Mre11:Rad50NBD in the open nucleotide free state, the illustrated structure in its ATP/ADP bound state allows the study of the ATP dependent conformational cycle of the MR complex (Figure 33). ATP induces a remarkably large transition which leads to an axial rotation of the Rad50 coiled-coils with respect to each other and an inverted orientation of them with respect to Mre11. This conformational switch is consistent with the previously proposed “clamp” model of MR (Williams et al., 2011). Furthermore, SAXS experiments of the bacterial Mre11:Rad50NBD catalytic head domain resulted in a substantial decrease of the radius of gyration (Rg) from

230 Å to 193 Å as well as a more articulated peak at shorter vectors and a significant decrease of the long vectors in the pair distribution function P(r) when ATPS was added to the solution (Lammens et al., 2011). This resembles the SAXS data of the

TmMre11:Rad50NBD complex with BMOE crosslinked and disulfide bonded NBDs, supporting the conclusion that the MR crystal structure in its ATP/ADP bound state

5. Discussion 93 matches the conformation of the complex in solution. In fact, stabilization of MR in open and closed conformations by either interface 2 or ATP suggests an at least two state switch for the bacterial complex, although additional conformations in the presence of DNA cannot be ruled out which will be discussed later on.

Figure 33: Schematic representation of the overall domain movement within the T. maritima

Mre11:Rad50NBD _{complex upon nucleotide binding. Mre11 is colored in blue, Rad50 is colored in orange.}

The interfaces 1 and 2 are highlighted by black boxes. Important domains and motifs are annotated.

Besides the obvious nucleotide-driven conformational change within the whole complex several minor alterations occurred, likely to be important for MR´s function. Helix αGturn is flipped around 90° from the open to the closed state, therefore enabling with its flexibility the large conformational relocation of the Rad50 NBDs. Moreover, the flexible linker connecting Mre11´s capping domain with the HLH motif in the open conformation undergoes a disorder-to-order transition by forming a well-ordered α-helix (αG´) (Figure 33). Still, the most notable result of the closed Mre11:Rad50NBD (S-S) complex is the blocking of Mre11´s dsDNA binding/active site groove by the Rad50 NBD dimer. In fact it is possible that ATP binding to Rad50 regulates MR by sterically controlling access to Mre11s nuclease and DNA binding sites and that the new formed αG´ acts as a kind of flexible spring at the lateral entry side allowing conformational changes to enable access to the active site and/or functions in DNA binding. In addition, Mre11 interacts with Rad50 in

5. Discussion 94 the newly formed interface 2closed in a rather small and polar buried surface area, making conformational changes between the flexible modules not inconceivable (see section 5.6).

At the same time as the structure of TmMre11:Rad50NBD (S-S) in its ATP/ADP bound state was solved, Yunje Cho and coworkers reported a related structure of archaeal Mre11:Rad50NBD bound to the non-hydrolyzable ATP analog ATPS (Lim et al., 2011). The structure of archaeal MR in the absence of ATP is not known at present. However, SAXS analysis based on the archaeal Pyrococcus furiosus (Pf)MRNBD complex indicate as well a two state mechanism with an elongated conformation of MR in its ATP-free, and a closed conformation with engaged NBDs in its ATP bound state (Figure 34). Together with the high degree of similarity between the structures of the ATP/ADP state of bacterial MR and ATPS bound state of archaeal MR it can be suggested that the ATP induced conformational cycle is an evolutionarily conserved feature of the complex. Moreover, the high similarity of the structures rules out crystallization artifacts as a result of site specific mutation and disulfide bridging.

Figure 34: ATP induced conformational cycle of archaeal P. furiosus. (A) Superposition of experimental SAXS curves of P. furiosus MRNBD _{with and without ATPγS illustrate a to T. maritima likewise} conformational change upon ATP binding. This suggests, that also archaeal MR exists in solution as open nucleotide unbound and closed ATP bound conformation. (B) The related electron pair distance distribution function P(r) shows an increase of short distances and a decrease in long distances upon ATPγS binding. Residual long distances match the prediction of a heterogeneous mixture between the open and closed ATP bound complex.

5. Discussion 95