Construction of Asymmetrical Hexameric Biomimetic Motors with Continuous Single-Directional Motion by Sequential Coordination

Abstract

The significance of bionanomotors in nanotechnology is analogous to mechanical motors in daily life. Here the principle and approach for designing and constructing biomimetic nanomotors with continuous single-directional motion are reported. This bionanomotor is composed of a dodecameric protein channel, a six-pRNA ring, and an ATPase hexamer. Based on recent elucidations of the one-way revolving mechanisms of the phi29 double-stranded DNA (dsDNA) motor, various RNA and protein elements are designed and tested by single-molecule imaging and biochemical assays, with which the motor with active components has been constructed. The motor motion direction is controlled by three operation elements: (1) Asymmetrical ATPase with ATP-interacting domains for alternative DNA binding/pushing regulated by an arginine finger in a sequential action manner. The arginine finger bridges two adjacent ATPase subunits into a non-covalent dimer, resulting in an asymmetrical hexameric complex containing one dimer and four monomers. (2) The dsDNA translocation channel as a one-way valve. (3) The hexameric pRNA ring geared with left-/right-handed loops. Assessments of these constructs reveal that one inactive subunit of pRNA/ATPase is sufficient to completely block motor function (defined as K = 1), implying that these components work sequentially based on the principle of binomial distribution and Yang Hui's triangle.

Keywords: bionanomotors; inter-subunit communication; one-way traffic; real-time recording; single-molecule imaging.

Figure 1

Illustration of the motor components of the phi29 dsDNA packaging motor. (A) Side view and (B) Top view of the phi29 dsDNA packaging motor composed of the three co-axial rings of the dodecameric connector, hexameric pRNA ring, and hexameric ATPase ring.

Figure 2

(A) Sequence of pRNA monomer with CCA bulge (upper panel) or 5’/3’ end (lower panel) highlighted in the box. The illustrations of the corresponding 3D structures are shown. The bars symbolize the end-truncation, and the empty squares symbolize the CCA-deletion. (B) Viral assembly activity of pRNA hexamers with different designs. Each color symbolizes one purified dimer or trimer. The uppercase D and T symbolize the normal dimer and trimer, respectively. The lower case d and t symbolize the mutant dimer and trimer, respectively.

Figure 3

(A) Illustration of the internal loop in the phi29 portal protein (left) and SPP1 portal protein (right), showing their function as a ratchet for the one-way translocation of the DNA during genome packaging. (B) Illustration of procapsids with side view of the mutant connectors based on PDB: 1h5w. (C) Comparison of procapsid activity with equal amount of procapsid protein.

Figure 4

ATP binding and hydrolysis activity assay of gp16 arginine finger mutant and inter-subunit interactions of ATPase. (A) Capillary Electrophoresis assay for the binding affinity test of different gp16 with dsDNA. (B) Interactions between gp16 arginine finger mutants with gp16 wild-type are shown by the band shift of both ATPase and DNA in the gel. (Green: mCherry channel; Blue: eGFP channel; Red: Cy5 channel). (C) Both dimers and monomers exist in gp16 ATPase rings. In 15%–35% glycerol gradient, one peak for eGFP-gp16 R146A (a) and two peaks for eGFP-gp16 wild-type (b) were observed after parallel ultracentrifugation, indicating that dimer formation is interrupted by the mutation of arginine finger. The fractions derived from the gradient have been applied to EMSA (c) and in vitro assembly activity assay, confirming the formation of dimers mediated by the arginine finger. The isolated gp16 dimer fraction (Fr. 18) showed significantly reduced activity compared to the monomers (Fr. 22) (d), supporting the previous finding that the addition of fresh gp16 monomer is required for re-initiating DNA packaging intermediates.^[66]

Figure 5

Illustration of inter-subunit interaction inside of gp16 ATPase. Gp16 ATPase hexameric ring was constructed (left panel), and the interaction between two adjacent subunits (right panel) has been shown with the arginine finger highlighted in the red sphere and Walker domain (represented by E119 residue in Walker B domain) highlighted in the blue sphere. The interaction of arginine finger with the upstream adjacent subunit is supported by the relative location of the related domains.

Figure 6

ATPase coordination with a series of conformational changes during DNA binding and ATP hydrolysis as regulated by the arginine finger, resulting in the asymmetrical configuration of ATPase (A). The asymmetrical structures have also been found in many other biomotors, including V1-ATPase^[74] (B) and MCM2-7 protein (EM accession:EMD-5429)^[80] (C).

Figure 7

Single-molecule detection of the continuous ATPase translocation on dsDNA. (A) The gp16 complex labeled with Cy3^[56] moved along the dsDNA chain tethered between two polylysine coated beads. (B) The motion of the Cy3-gp16 complex was analyzed by Image J and (C) a kymograph was generated to demonstrate the motion of the ATPase.

Figure 8

Single-molecule detection of continuous DNA translocation in the phi29 biomotor. Comparison of the trajectories and travel distance of microspheres attached to the end of DNA in two different packaging intermediates Beads I (A) and Beads II (B). Beads I showed random Brownian motion. Bead II also showed Brownian motion at the beginning, however, such motion ceased after around 150 seconds, indicating the end of the translocation event. The stalling of the dsDNA serves as evidence of uni-directional translocation. (C) Sequential images of a fluorescent microsphere attached to DNA, corresponding to when Beads II tends to stop as indicated in the figure.