Rotation of artificial rotor axles in rotary molecular motors

Abstract

F₁- and V₁-ATPase are rotary molecular motors that convert chemical energy released upon ATP hydrolysis into torque to rotate a central rotor axle against the surrounding catalytic stator cylinder with high efficiency. How conformational change occurring in the stator is coupled to the rotary motion of the axle is the key unknown in the mechanism of rotary motors. Here, we generated chimeric motor proteins by inserting an exogenous rod protein, FliJ, into the stator ring of F₁ or of V₁ and tested the rotation properties of these chimeric motors. Both motors showed unidirectional and continuous rotation, despite no obvious homology in amino acid sequence between FliJ and the intrinsic rotor subunit of F₁ or V₁ These results showed that any residue-specific interactions between the stator and rotor are not a prerequisite for unidirectional rotation of both F₁ and V₁ The torque of chimeric motors estimated from viscous friction of the rotation probe against medium revealed that whereas the F₁-FliJ chimera generates only 10% of WT F₁, the V₁-FliJ chimera generates torque comparable to that of V₁ with the native axle protein that is structurally more similar to FliJ than the native rotor of F₁ This suggests that the gross structural mismatch hinders smooth rotation of FliJ accompanied with the stator ring of F₁.

Keywords: ATPase; F1; V-ATPase; protein design; rotary molecular motor.

Fig. 1.

Construct of xenogeneic subunits in A₃B₃. (A) Structure models of V₁ and the fusion rotor proteins from subunit D and FliJ. V₁^DΔ58–113 represents V₁ with subunit D, of which the loop domain (58–113) was truncated to leave only the coiled-coil structure. The truncated D and the A and the B subunits are represented in yellow, red, and green, respectively. Cyan in the fusion rotors represents the parts of FliJ. The helical portions derived from V₁-D are represented in yellow, and those from FliJ are represented in blue. Dashed lines of the fusion rotors in ChV₁^D30/J86/D68 and ChV₁^J147/DC21 represent disordered C-terminal 12-aa residues (13). (B) SDS/PAGE analysis of ChV₁. Proteins are visualized by Coomassie blue staining (Upper) or by immunostaining using anti-FliJ to detect the xenogeneic rotors (Lower). The specificity of anti-FliJ was described in ref. .

Fig. S1.

Sequence and structural alignment of V₁-D and FliJ. The amino acid sequences of V₁-D from T.th (Tth_D), Enterococcus hirae (Ehr_D) and Saccharomyces cerevisiae (Sce_D) were aligned with Clustal-W. The FliJ sequences from S. enterica (Sen_J) and Ehr_D were structurally aligned with MATRAS using PDB structures (PDB ID codes 3AJW and 3AON) due to the low sequence similarity between FliJ and V₁-D. The conserved residues are highlighted in red (identical) or pink (strong similarity). The C- and N-terminal α-helices are represented as blue cylinders and β-strands are represented as thick yellow arrows. The deleted region in D^Δ58–113 is enclosed by a thin line. The DC21 is highlighted as outline characters on a black background. The residue numbers of Tth_D are indicated by black arrows.

Fig. S2.

Expression constructs of chimeric V₁. The expression plasmids for ChV₁ were constructed based on the A₃B₃D expression plasmid. The amino acid regions from FliJ are highlighted in blue.

Fig. 2.

Rotation and torque estimation of the chimeric or truncated V₁. (A) Time courses of 290-nm duplex rotation recorded at 250 or 1,000 frames per s. Overall time courses of WT V₁ (black), truncated V₁^DΔ58–113 (red), ChV₁^D30/J86/D68 (green), ChV₁^D30/J86/D29 (purple), and ChV₁^J147/DC21 (sky blue). (B) Thin colored curves show 30 consecutive 120° steps overlaid on top of each other, with the thick blue lines representing the average. The straight red lines indicate linear fit to the cyan curve between 0° to 120°. The slope of the red line, the angular velocity in radians⁻¹, gives the torque N. (C) Torque values estimated from the instantaneous rotary speed in consecutive 120 steps.

Fig. S3.

Rotation time courses of chimeric V₁s and WT V₁ (black line) at 4 mM ATP, respectively.

Fig. 3.

Construct of xenogeneic subunits in α₃β₃. (A) Construct of F₁-FliJ chimeras. Side views showing the central FliJ [blue; Protein Data Bank (PDB) ID code 3AJW] and pair of α (green) and β subunits (red) of thermophilic Bacillus PS3 F₁-ATPase (PDB ID code 4XD7). The helical portions of F₁-γ and those of FliJ are colored in orange and blue, respectively. The C-terminal unstructured portions of FliJ are represented by dashed lines. The linker portion containing the thrombin recognition site is represented by the purple line. (B) SDS/PAGE analysis of ChF₁. Proteins are visualized by Coomassie blue staining.

Fig. S4.

Sequence alignment of F₁-γ and FliJ. The amino acid sequences of F₁-γ from thermophilc Bacillus PS3 (PS3_γ), E. coli (Eco_γ), and bovine mitochondria (Bov_γ) were aligned with Clustal-W. The FliJ sequence (Sen_J) and Bov_γ were structurally aligned with MATRAS using PDB structures (PDB ID codes 3AJW and 1E79) due to the low sequence similarity between FliJ and F₁-γ. The conserved residues are highlighted in red (identical) or pink (strong similarity). The C- and N-terminal α-helices are represented as blue cylinders and β-strands are represented as thick yellow arrows.

Fig. S5.

Expression construct of F₁-FliJ chimeras. The expression plasmids for the series of ChF₁s were constructed based on the α₃β₃γ expression plasmid by fully or partially substituting γ subunit with FliJ sequence. The amino acid regions from FliJ and F₁-γ subunit are highlighted in blue and orange, respectively.

Fig. 4.

Rotation of F₁-FliJ chimeras. (A) Time courses of rotation of WT F₁ (black), ChF₁(21γ) (red), ChF₁(17γ) (green), ChF₁(9γ) (purple), and ChF₁(0γ) (sky blue). (B) Rotation rates of WT F₁ (n = 8), ChF₁(21γ) (n = 13), ChF₁(17γ) (n = 12), ChF₁(9γ) (n = 8), and ChF₁(0γ) (n = 10) estimated from the fastest portions of consecutive revolutions over 10 s. Filled circles indicate rotation rates for individual molecules. Columns show the means. The error bars indicate SDs. (C) Torque values estimated from the averaged rotation rate of WT F₁ and the series of ChF₁ shown in B considering the torque of WT F₁ is 40 pN⋅nm. Columns show the means. The error bars indicate SDs.

Fig. S6.

Rotation time courses of WT F₁ and F₁-FliJ chimeras. The rotations of the series of ChF₁s were more noisy than those of WT F₁.

Fig. S7.

Structural alignment of FliJ and V₁-D or F₁-γ. FliJ (blue; PDB ID code 3AJW) was superimposed on the D subunit of V₁ (yellow; PDB ID code 3W3A) or γ subunit of F₁ (orange; PDB ID code 4XD7). Structural superpositions were calculated using MATRAS server. Rmsds between FliJ and the D subunit of V₁ or γ subunit of F₁ are 3.2 Å or 4.4 Å, respectively.