Key hinge residues control tuberculosis topoisomerase I motion

Tuberculosis is caused by the bacterium Mycobacterium tuberculosis, and the enzyme topoisomerase I (MtbTOP1) is essential for that bacterium’s survival. Despite its importance, scientists still do not fully understand how MtbTOP1 changes shape as it carries out its job of relaxing negatively supercoiled DNA. These shape changes require coordinated movement between different parts of the protein, including opening and closing a gate that lets DNA pass through a toroidal cavity inside the enzyme. To probe this mystery, researchers led by Yuk-Ching Tse-Dinh set out to identify flexible hinge regions that might control those inter-domain rearrangements. They started from the available MtbTOP1 crystal structure and used the online server PACKMAN to search for likely hinge segments. PACKMAN predicted a candidate region spanning PRO506 to LEU526 at the border between domains D2 and D4, with a reported p-value <0.05. That prediction focused attention on a short stretch of the protein where movement could enable the opening and closing required during catalysis, setting the stage for experimental testing of specific residues in that segment.

Among residues in the predicted PRO506 to LEU526 region, the team noted a highly conserved ARG516 that makes contact with DNA inside the protein’s toroidal cavity. Structural analysis suggested ARG516 helps hold domains together by interacting with GLU207 in D4 and ASP691 in D5, linking three parts of the enzyme during conformational changes. To test the functional role of these contacts, the researchers introduced alanine substitutions at specific positions to produce mutant topoisomerases and then examined their biochemical behavior. The biochemical experiments showed that mutating GLU207 and ARG516 led to a significant loss of DNA relaxation activity, indicating the enzyme could no longer carry out its catalytic function effectively. Importantly, these mutations did not affect the enzyme’s ability to bind DNA or to cleave it, pointing to a specific defect in the domain rearrangements needed for relaxation rather than a general loss of DNA interaction or catalytic chemistry. These results are consistent with ARG516 and GLU207 acting as hinge residues that enable the necessary inter-domain movements.

The findings clarify one piece of how MtbTOP1 works: a short hinge region around PRO506 to LEU526, and particularly interactions involving ARG516 and GLU207, appears to be important for the conformational gymnastics that let the enzyme relax supercoiled DNA. By showing that specific substitutions disrupt relaxation without blocking DNA binding or cleavage, the study supports a model in which precise inter-domain rearrangements—not just the active site chemistry—are essential for catalysis. This deeper mechanistic insight helps disentangle the sequence of events inside MtbTOP1 as it opens and closes its gate around DNA. Understanding these dynamics at the residue level gives researchers a clearer map of which parts of the protein control motion versus chemistry, and it provides targeted hypotheses for future structural, biochemical, and possibly therapeutic research aimed at interfering with the enzyme’s function in Mycobacterium tuberculosis. The work highlights the value of combining computational hinge prediction with focused mutagenesis and biochemical testing to reveal functional roles for specific residues.