Simultaneous ligand binding to intact and partially formed ATP-binding sites in the hexameric termination factor Rho

Thermodynamic coupling between ligand-binding sites affords macromolecular machines a means to coordinate processive function. Because these machines may be compositionally complex, quantifying and interpreting ligand-binding events can be experimentally difficult. Biophysical methods that convolve binding events into a one-dimensional metric, which suffice for monomeric macromolecules that bind to a single ligand, are insufficient to adequately describe the complexity of binding to oligomeric systems. Confounding factors include structural heterogeneity that may invalidate basic assumptions used to interpret the measurements. In this communication, we use native mass spectrometry to measure ATP binding to a hexameric helicase, the Escherichia coli termination factor Rho. Providing new insights into classical and more recent biochemical experiments, we observe and quantify ATP binding to hexameric and low-order complexes. Moreover, we observe superstoichiometric binding consistent with ATP binding to partially formed binding sites at the edges of the open washer structure. Such detailed insights are likely critical to understanding the mechanisms by which a broad range of macromolecular machines harness the free energy from ligand binding, hydrolysis, and exchange to coordinate their ligand-dependent functions.