Structural Biology of Rad50 ATPase

Abstract

To clarify the key role of Rad50 in DNA double-strand break repair (DSBR), we biochemically and structurally characterized ATP-bound and ATP-free Rad50 catalytic domain (Rad50cd) from Pyrococcus furiosus. Rad50cd displays ATPase activity plus ATP-controlled dimerization and DNA binding activities. Rad50cd crystal structures identify probable protein and DNA interfaces and reveal an ABC-ATPase fold, linking Rad50 molecular mechanisms to ABC transporters, including P glycoprotein and cystic fibrosis transmembrane conductance regulator. Binding of ATP gamma-phosphates to conserved signature motifs in two opposing Rad50cd molecules promotes dimerization that likely couples ATP hydrolysis to dimer dissociation and DNA release. These results, validated by mutations, suggest unified molecular mechanisms for ATP-driven cooperativity and allosteric control of ABC-ATPases in DSBR, membrane transport, and chromosome condensation by SMC proteins.

Dynamics and Structural Changes Induced by ATP Binding in SAV1866, a Bacterial ABC Exporter

“…This construction was preferred to that of a docking of an ATP molecule in the binding pocket as carried out in a MD simulation study of BtuCD. 44 According to Dawson and Locher 17 the binding mode of AMP-PNP in SAV1866 is consistent with that of ATP in the NBD dimer of Rad50 45 and the electronic density corresponding to sodium ion is close to where magnesium and sodium ions were identified in isolated NBDs. 45,46 Second, the ATP structure built from AMP-PNP in our system is bound in a similar fashion as in two other crystal structures, that of Hlyb (PDB code 1XEF) and of TAP1 (PDB code 2IXF).…”

Structure and mechanism of ATP-dependent phospholipid transporters

“…The Walker A motif establishes extensive interactions with the phosphate group of an ATP molecule [15,16]. The Walker B motif contains an aspartate involved in the coordination of water and Mg 2+ at the catalytic site and a catalytic glutamate required for ATP hydrolysis [16][17][18][19]. A similar role in coordination has a conserved glutamine at the Q-loop located between the Walker sequences [16,[19][20][21].…”

“…A conserved aromatic amino acid residue in the A-loop upstream of the Walker A motif is involved in interacting with the π-stack of the adenosine ring of ATP [22][23][24]. Downstream of Walker B are the D-loop and the H (or switch)-loop that contain a conserved aspartate and a conserved histidine, respectively, involved in the coordination of the γ-phosphate either through interaction with a water molecule (D-loop) [17,19,20,25,26] or through direct hydrogen bonding (H-loop) [19,27]. Finally, the ABC signature is a helical subdomain located in between the Walker sequences of each NBD containing the amino acid sequence LSGGQ, also involved in ATP binding [28][29][30][31][32][33].…”

“…Finally, the ABC signature is a helical subdomain located in between the Walker sequences of each NBD containing the amino acid sequence LSGGQ, also involved in ATP binding [28][29][30][31][32][33]. When the transporter binds ATP, the two NBDs arrange in a head-totail manner, generating the so-called "closed-dimer" conformation, in which two nucleotide molecules are sandwiched in the interface between the two NBDs and interact with residues from the Walker A, Walker B, H-and Q-loops of one NBD and the D-loop and ABC signature of the other [15,17,34]. Due to this arrangement some ABC transporters show an allosteric behavior for hydrolysis of the two ATP molecules, presumably controlled by the D-loop [26,33,35].…”

Exploiting Reaction Intermediates of the ATPase Reaction to Elucidate the Mechanism of Transport by P-glycoprotein (ABCB1)

“…The structures of several ABC domains show the existence of a "nucleotide sandwich dimer," where two ATP molecules bring the two NBDs together (55)(56)(57). A mutation in the conserved Walker B carboxylate (E171Q) of the ABC domain of MJ0796 was used to obtain a "sandwich dimer" in which two ATP molecules were occluded, one at each ATP site (56).…”