High Degree of Coordination and Division of Labor among Subunits in a Homomeric Ring ATPase

Ring NTPases of the ASCE superfamily perform a variety of cellular functions. An important question about the operation of these molecular machines is how the ring subunits coordinate their chemical and mechanical transitions. Here, we present a comprehensive mechanochemical characterization of a homomeric ring ATPase—the bacteriophage φ29 packaging motor—a homopentamer that translocates double-stranded DNA in cycles composed of alternating dwells and bursts. We use high-resolution optical tweezers to determine the effect of nucleotide analogs on the cycle. We find that ATP hydrolysis occurs sequentially during the burst and that ADP release is interlaced with ATP binding during the dwell, revealing a high degree of coordination among ring subunits. Moreover, we show that the motor displays an unexpected division of labor: although all subunits of the homopentamer bind and hydrolyze ATP during each cycle, only four participate in translocation, whereas the remaining subunit plays an ATP-dependent regulatory role. [Display omitted] ► A complete mechanochemical model for a homomeric ring NTPase is presented ► Nucleotide analogs are used to locate chemical transitions in the mechanical cycle ► Nucleotide binding, hydrolysis, and product release are all highly coordinated ► All five subunits hydrolyze ATP in each cycle, but only four translocate DNA A complete model for the mechanochemical cycle of the bacteriophage φ29 DNA translocase is presented, which includes coordinated ADP release and ATP binding events during the dwell phase, followed by sequential ATP hydrolysis during the burst. Four of the five subunits drive translocation, whereas the remaining subunit plays an ATP-dependent regulatory role, indicating that homomeric ring ATPases can display functional asymmetry.