Structure and Function Study of Phi29 DNA packaging motor

Linear double-stranded DNA (dsDNA) viruses package their genomes into preformed protein shells via nanomotors using ATP as an energy source. The central hub of the bacteriophage 429 DNA-packaging motor contains a 3.6-nm channel for dsDNA to enter during packaging and to exit during infection. The negatively charged interior channel wall is decorated with a total of 48 positively charged lysine residues displayed as four 12-lysine rings from the 12 gp10 subunits that enclose the channel. The standard notion derived frommany models is that these uniquely arranged, positively charged rings play active roles in DNA translocation through the channel. In this study, we tested this prevailing view by examining the effect of mutating these basic lysines to alanines, and assessing the impact of altering the pH environment. Unexpectedly, mutating these basic lysine residues or changing the pH to 4 or 10, which could alter the charge of lysines, did not measurably impair DNA translocation or affect the one-way traffic property of the channel. The results support our recent findings regarding the dsDNA packaging mechanism known as the ‘‘push through a one-way valve’’. INTRODUCTION A key step in the life cycle of tailed double-stranded DNA (dsDNA) viruses is the packaging of their genomes into preformed protein shells, termed procapsids, via ATPdriven nanomotors (1–6). This has been observed for 429 (7–10), T4 (11–13), l (2,14), T3 (4,15,16), T7 (17), ε15 (18), P22 (19,20), SPP1 (5), P1 (21), PRD1 (22), herpes simplex virus (23,24), human cytomegalovirus (25,26), and adenovirus (27). The 429 nanomotor packages its genomic DNA into a procapsid consisting of four proteins: gp7 (the scaffold protein), gp8 (the capsid protein), gp8.5 (the fiber protein), and gp10 (the connector) (28,29). The nanomotor is composed of three components: the connector at the vertex of procapsid (30), a packaging RNA (pRNA) hexameric ring that binds to the N-terminus of the connector (9,31–35), and a ring of ATPase gp16 (1) linked by the pRNA (36,37). The connector serves as the nucleation site for procapsid assembly and determines the shape and size of the procapsid (38–40). The 429 DNA packaging motor, which was first assembled in vitro 25 years ago (7), is the most extensively studied motor system and one of the most powerful molecular motors constructed to date (41,42). Elucidating the molecular mechanism of this motor may aid in the development of biomimetics and synthetic nanodevices (43–46) to deliver therapeutics to targets such as the heart, eyes, and other organs in the body. As a result, the 429 DNA packaging motor has attracted great attention from scientists in the fields of virology, molecular biology, structural biology, nanotechnology, biophysics, biomaterials, nanomachines, RNA biochemistry, and therapeutics. Although the protein sequences of many viral connectors share little homology, the global structures are morphologically similar (19,30,47,48), suggesting that viral DNA packaging may utilize similar mechanisms. Extensive studies reveal that the structure of the 429 connector is a coneshaped ring surrounded by 12 subunits of gp10 (30,48– 52). The wider end, which is buried inside the procapsid, is 13.6 nm, and the narrower, protruding end is 6.8 nm. The narrowest part of the connector channel is 3.6 nm, which allows dsDNA to enter during DNA packaging and to exit during host infection. Because the highly negative charged channel wall is decorated with four lysine rings (K200, K209, K234, and K235; Fig. 1) scattered inside the channel, these basic lysine residues were proposed to interact with the negative phosphate backbone of dsDNA during DNA packaging (30). An active role for the connector and a potential function of the lysine residues in DNA packaging have also been proposed in many DNA-packaging models (30,51,53). Here, we focused on dissecting the role of these interior lysine residues in 429 DNA packaging. The positively charged lysine residues were substituted with neutral residues and the pH environment was changed to either neutralize or enhance the charges. Contrary to expectations (30,51,53), neither the change in pH nor the exchange of the lysine residues affected the genome packaging by the active ATP-driven motor or the translocation of the dsDNA driven by electric current through membrane-embedded connectors. Submitted September 2, 2011, and accepted for publication November 14, 2011. Huaming Fang and Peng Jing contributed equally to this work. *Correspondence: guop@purdue.edu This is an Open Access article distributed under the terms of the Creative Commons-Attribution Noncommercial License (http://creativecommons. org/licenses/by-nc/2.0/), which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Editor: Robert Nakamoto. 2012 by the Biophysical Society 0006-3495/12/01/0127/9 $2.00 doi: 10.1016/j.bpj. 2011.11. 4013 Biophysical Journal Volume 102 January 2012 127–135 127