F ₁ F _o -ATPase, a rotary ATPase, is widespread in the membranes of mitochondria, chloroplasts, and bacteria and converts ATP energy with a proton motive force across the membrane by its physical rotation. Homologous protein complexes play roles in ion and protein transport. Mycoplasma mobile , a pathogenic bacterium, was recently suggested to have a special motility system evolutionarily derived from F ₁ -ATPase.

Rotary ATPases, including F ₁ F _O -, V ₁ V _O -, and A ₁ A _O -ATPases, are molecular motors that exhibit rotational movements for energy conversion. In the gliding bacterium, Mycoplasma mobile , a dimeric F ₁ -like ATPase forms a chain structure within the cell, which is proposed to drive the gliding motility. However, the mechanisms of force generation and transmission remain unclear. We determined the electron cryomicroscopy (cryo-EM) structure of the dimeric F ₁ -like ATPase complex. The structure revealed an assembly distinct from those of dimeric F ₁ F _O -ATPases. The F ₁ -like ATPase unit associated by two subunits GliD and GliE was named G ₁ -ATPase as an R ₁ domain of rotary ATPases. G ₁ -β subunit, a homolog of the F ₁ -ATPase catalytic subunit, exhibited a specific N-terminal region that incorporates the glycolytic enzyme, phosphoglycerate kinase into the complex. Structural features of the ATPase displayed strong similarities to F ₁ -ATPase, suggesting a rotation based on the rotary catalytic mechanism. Overall, the cryo-EM structure provides insights into the mechanism through which G ₁ -ATPase drives the Mycoplasma gliding motility.

Mycoplasma pneumoniae is a human pathogen that glides on host cell surfaces by a repeated catch and release mechanism using sialylated oligosaccharides. At a pole, this organism forms a protrusion called an attachment organelle composed of surface structures, including an adhesin complex and an internal core structure. To clarify the structure and function of the attachment organelle, we focused on a core component, P65, which is essential for stabilization of the adjacent surface and core proteins P30 and HMW2, respectively. Analysis of its amino acid sequence (405 residues) suggested that P65 contains an intrinsically disordered region (residues 1–217) and coiled-coil regions (residues 226–247, 255–283, and 286–320). Four protein fragments and the full-length P65 were analyzed by size exclusion chromatography, analytical centrifugation, circular dichroism spectroscopy, small-angle X-ray scattering, limited proteolysis, and negative staining electron microscopy. The results showed that P65 formed a multimer composed of a central globule with 30 and 23 nm axes and four to six projections 14 nm in length. Our data suggest that the C-terminal region of P65 is responsible for multimerization, while the intrinsically disordered N-terminal region forms a filament. These assignments and roles of P65 in the attachment organelle are discussed.

Rotary ATPases, including F ₁ F _o - and V/A-ATPases, are molecular motors that exhibit rotational movements for energy conversion ¹ . In the gliding bacterium, Mycoplasma mobile , a dimeric F ₁ -like ATPase forms a chain structure with the glycolytic enzyme, phosphoglycerate kinase (PGK), within the cell ² , which is proposed to drive the bacterial gliding motility ^2–4 . However, the mechanisms of force generation and transmission remain unclear. Here, we present a 3.2 Å resolution structure of the dimeric ATPase complex obtained using electron cryomicroscopy (cryo-EM). Notably, the structure revealed an assembly distinct from that of known dimeric forms of F ₁ F _o -ATPase ⁵ , despite containing conserved F ₁ -ATPase structures. The two ATPase units were interconnected by GliD dimers, previously identified as MMOB1620 ^2,6 . Gliβ, a homologue of the F ₁ -ATPase catalytic subunit ⁶ , exhibited a specific N-terminal region that incorporates PGK into the complex. Structural conformations of the catalytic subunits, catalytically important residues, and nucleotide-binding pattern of the catalytic sites of the ATPase displayed strong similarities to F ₁ -ATPase, suggesting a rotation based on the rotary catalytic mechanism conserved in rotary ATPases ^1,7–10 . Overall, the cryo-EM structure underscores an evolutionary connection in the rotary ATPases and provides insights into the mechanism through which F ₁ -like ATPase drives bacterial gliding motility.

Mycoplasma mobile is a parasitic bacterium that forms gliding machinery on the cell pole and glides on a solid surface in the direction of the cell pole. The gliding machinery consists of both internal and surface structures. The internal structure is divided into a bell at the front and chain structure extending from the bell. In this study, the internal structures prepared under several conditions were analyzed using negative-staining electron microscopy and electron tomography. The chains were constructed by linked motors containing two complexes similar to ATP synthase. A cylindrical spacer with a maximum diameter of 6 nm and a height of 13 nm, and anonymous linkers with a diameter of 0.9–8.3 nm and length of 5–30 nm were found between motors. The bell is bowl-shaped and features a honeycomb surface with a periodicity of 8.4 nm. The chains of the motor are connected to the rim of the bell through a wedge-shaped structure. These structures may play roles in the assembly and cooperation of gliding machinery units. Significance: Mycoplasma mobile , a parasitic bacterium, glides on solid surfaces at speeds of up to 4.0 μm per second through a specialized mechanism. The gliding machinery, located at one pole of the cell, is composed of surface legs and internal motor structures. The force generation unit within the internal structure evolved from ATP synthase. This study aimed to clarify the entire architecture of the gliding machinery using electron tomography.

Mycoplasma pneumoniae is a human pathogen that glides on host cell surfaces by a repeated catch and release mechanism using sialylated oligosaccharides. At a pole, this organism forms a protrusion called an attachment organelle composed of surface structures, including an adhesin complex and an internal core structure. To clarify the structure and function of the attachment organelle, we focused on a core component, P65, which is essential for stabilization of the adjacent surface and core proteins P30 and HMW2, respectively. Analysis of its amino acid sequence (405 residues) suggested that P65 contains an intrinsically disordered region (residues 1-217) and coiled-coil regions (residues 226-247, 255-283, and 286-320). Four protein fragments and the full-length P65 were analyzed by size exclusion chromatography, analytical centrifugation, circular dichroism spectroscopy, small-angle X-ray scattering, limited proteolysis, and negative staining electron microscopy. The results showed that P65 formed a multimer composed of a central globule with 30 and 23 nm axes and 4 to 6 projections 14 nm in length. Our data suggest that the C-terminal region of P65 is responsible for multimerization, while the intrinsically disordered N-terminal region forms a filament. These assignments and roles of P65 in the attachment organelle are discussed.