The type IV pilus assembly motor PilB is a robust hexameric ATPase with complex kinetics

Area of Science:

• Biochemistry
• Molecular Biology
• Microbiology

Background:

• Bacterial type IV pilus (T4P) are essential nanomachines involved in various cellular processes.
• T4P assembly is driven by the PilB ATPase, with a proposed symmetrical rotary mechanism.

Purpose of the Study:

• To perform the first kinetic studies on the PilB ATPase to test the proposed rotary mechanism.
• To investigate the kinetic properties of PilB from *Chloracidobacterium thermophilum*.

Main Methods:

• Genome sequence analysis of *C. thermophilum* to identify *pilB* gene.
• Heterologous expression and purification of PilB protein.
• Steady-state kinetic analysis of wild-type and mutant PilB ATPase activity across a range of ATP concentrations.

Main Results:

• *C. thermophilum* PilB forms a hexamer and exhibits robust ATPase activity with complex kinetics.
• Observed substrate inhibition at high ATP concentrations suggests regulation of T4P assembly.
• A less active mutant PilB showed similar ATP inhibition, supporting protomer co-ordination.

Conclusions:

• Kinetic data support a co-ordinated, symmetrical rotary mechanism for PilB ATPase activity.
• Bacterial intracellular ATP levels may intricately regulate T4P assembly and function.
• This study provides the first biochemical evidence for the proposed PilB catalytic mechanism.