Structural Basis for the Mechano-Chemical Coupling and Inter-Subunit Coordination of Ring ATPase

Abstract

Members of the Additional Strand Conserved Glutamate (ASCE) superfamily perform a great variety of biological tasks. The gene product 16 (gp16) ring ATPase, one member of the ASCE superfamily, is the active component of the bacteriophage Phi29 packaging motor. Three decades of extensive studies in this system via biochemical and single molecule methods have achieved one of the most comprehensive mechanochemical characterizations of an ASCE ring ATPase to date. The current kinetic understanding of the gp16 ring ATPase provides a solid foundation to build a parallel structural interpretation of its DNA translocation mechanism. It has been shown that the motor translocates DNA using a burst-dwell mechanism and exhibits multiple levels of coordination among the catalytic cycles of individual subunits. Underlying mechanisms, such as inter-subunit communication and proper timing of the cycle by one of the five subunits have been proposed to explain such mechanism. Highly conserved residues such as the arginine finger and the gamma-phosphate sensor as well as important motor-DNA interactions are thought to be responsible for these features; however, the structural mechanism used by this motor is yet to be determined. In this work, we investigate the role of these structural elements in the dynamics of the gp16 ring ATPase by observing DNA translocation in real time using high-resolution optical tweezers and targeted mutagenesis. Our study provides important information regarding the structural design of the gp16 ring ATPase that drives inter-subunit coordination and its coupling to perform DNA translocation. Our results are relevant for other ring NTPases in the ASCE superfamily that share similar structural elements.