Clamp loaders are AAA+ ATPases that load sliding clamps onto DNA to enable high-speed DNA replication. Using deep mutagenesis, we mapped mutational sensitivity of the T4 bacteriophage clamp loader and clamp. Most residues not directly involved in catalysis or binding tolerate mutation, but Gln118 in the AAA+ module is an exception. It forms a hydrogen-bonded junction within a helical segment we term the central coupler, linking ATPase sites to DNA and the clamp. Suppressor mutations and molecular dynamics show this junction maintains rigidity, critical for allosteric signaling. Its conservation across diverse AAA+ ATPases suggests that hydrogen-bonded networks commonly couple ATP hydrolysis to mechanical work.
We also examined catalytic residues. In T4 clamp loaders, ATP hydrolysis depends on an aspartate in the DEAD-box motif and an interfacial arginine. Replace of the aspartate by cysteine reduces activity but can be rescued by distant single-site substitutions. Cryo-EM reveals an inactive state with blocked DNA binding and disassembled catalytic sites. Restorative mutations cluster in regions that undergo conformational change during the on-off transition, suggesting that they shift equilibrium toward activation by increasing DNA affinity.
These results highlight the structural plasticity and evolutionary potential of clamp loaders. Distant mutations fine-tune conformational states and inter-subunit communication, enabling recovery of function through diverse paths. This shows how molecular machines evolve new functions through accessible mutations.