CryoEM of endogenous mammalian V-ATPase interacting with the TLDc protein mEAK-7

Area of Science:

• Biochemistry
• Structural Biology
• Cell Biology

Background:

• Vacuolar-type proton pumps (V-ATPases) are crucial rotary proton pumps involved in cellular signaling.
• Understanding V-ATPase interactions with regulatory proteins is key to deciphering their diverse functions.
• mEAK-7 is a protein implicated in nematode dauer formation and mammalian mTOR signaling pathways.

Purpose of the Study:

• To investigate the interaction between porcine kidney V-ATPase and the protein mEAK-7 using cryo-electron microscopy (cryoEM).
• To determine the structural basis for mEAK-7 binding to V-ATPase and its functional consequences.
• To compare the binding of mEAK-7 with other TLDc domain proteins, such as Oxr1p, to V-ATPase.

Main Methods:

• Cryo-electron microscopy (cryoEM) with focused classification was employed to identify endogenous proteins bound to V-ATPase.
• High-resolution cryoEM was used to visualize the structure of porcine kidney V-ATPase in complex with recombinant mEAK-7.
• Comparative analysis of mEAK-7 and Oxr1p binding modes to V-ATPase.

Main Results:

• An additional C subunit and the protein mEAK-7 were identified as V-ATPase binding partners.
• mEAK-7's TLDc domain binds to the V-ATPase stator, while its C-terminal helix interacts with the rotor.
• Unlike Oxr1p, mEAK-7 binding does not inhibit V-ATPase activity; the interaction is labile and sensitive to ATP-induced rotation.

Conclusions:

• V-ATPase binding by TLDc domain proteins can result in varied functional outcomes, from strong inhibition to labile interactions.
• The mEAK-7:V-ATPase interaction is regulated by the enzyme's rotary activity, suggesting a mechanism for dynamic control.
• Structural insights into mEAK-7 binding provide a basis for understanding its role in cellular signaling and V-ATPase regulation.