Structure of mycobacterial respiratory complex I

Area of Science:

• Biochemistry
• Structural Biology
• Microbiology

Background:

• Oxidative phosphorylation, involving the electron transport chain (ETC) and ATP synthase, is a target for treating mycobacterial infections.
• The mycobacterial ETC is branched, with multiple dehydrogenases and oxidases.
• Proton-pumping NADH dehydrogenase (Complex I) is typically in low abundance and considered dispensable in mycobacteria.

Purpose of the Study:

• To investigate the structure and function of Complex I in mycobacteria.
• To understand the role of Complex I under specific growth conditions.
• To identify novel subunits and interactions within the mycobacterial Complex I assembly.

Main Methods:

• Culturing *Mycobacterium smegmatis* under carbon-limited conditions.
• Isolation of rotenone-sensitive Complex I.
• Cryo-electron microscopy (cryo-EM) for structural determination.
• Biochemical analysis of enzyme subunits and lipid interactions.

Main Results:

• Carbon limitation significantly increased Complex I abundance in *M. smegmatis*.
• Cryo-EM revealed the structure of Complex I, including the "orphan" protein MSMEG_2064 as a subunit.
• MSMEG_2064 occupies a site analogous to the redox-sensing NDUFA9 in eukaryotic Complex I.
• The structure identified a bound purine nucleoside triphosphate and acyl phosphatidylinositol dimannoside.
• Menaquinone was observed in two positions within the quinone channel, suggesting conserved binding mechanisms.

Conclusions:

• Complex I is more abundant and structurally significant in mycobacteria than previously thought, especially under nutrient limitation.
• The identified subunits and lipid interactions provide insights into mycobacterial energy metabolism and potential drug targets.
• The conserved menaquinone binding mechanism highlights potential therapeutic strategies against mycobacterial infections.