Mechanical inhibition of isolated Vo from V/A-ATPase for proton conductance

Abstract

<jats:p>V-ATPase is an energy converting enzyme, coupling ATP hydrolysis/synthesis in the hydrophilic V1 domain, with proton flow through the Vo membrane domain, via rotation of the central rotor complex relative to the surrounding stator apparatus. Upon dissociation from the V1 domain, the Vo domain of the eukaryotic V-ATPase can adopt a physiologically relevant auto-inhibited form in which proton conductance through the Vo domain is prevented, however the molecular mechanism of this inhibition is not fully understood. Using cryo-electron microscopy, we determined the structure of both the holo V/A-ATPase and isolated Vo at near-atomic resolution, respectively. These structures clarify how the isolated Vo domain adopts the auto-inhibited form and how the holo complex prevents formation of the inhibited Vo form.</jats:p>

Cross-linking mass spectrometry uncovers protein interactions and functional assemblies in synaptic vesicle membranes

“…50 out of 74) satisfies a cross-linking distance of ≤30 Ȧ (Supplementary Data 4) as expected for the BS3 crosslinker 46 . Importantly, long-distance cross-links (>30 Ȧ) are located in flexible subunits such as the peripheral stalks or subunit a1 (Supplementary Data 4) 47,48 . Assuming random crosslinking of identified cross-linked lysine residues shows a broader distribution with longer cross-linking distances ( Supplementary Fig.…”

Structural snapshots of V/A-ATPase reveal a new paradigm for rotary catalysis

“…We previously determined the Cryo-EM structures of the wild-type V/A-ATPase containing an ADP in the catalytic site of AB closed 16, 17 . The V/A-ATPase bound to the inhibitory ADP exhibits no ATPase activity until the ADP is removed 13, 16, 24 .…”

“…We previously determined the Cryo-EM structures of the wild-type V/A-ATPase containing an ADP in the catalytic site of AB closed 16, 17 . The V/A-ATPase bound to the inhibitory ADP exhibits no ATPase activity until the ADP is removed 13, 16, 24 . Partial ADP removal from AB closed is possible by dialysis against an EDTA-phosphate buffer, but it is difficult to obtain a homogenous nucleotide-free V/A-ATPase after such a treatment, due to the high binding affinity of the ADP to AB closed (Supplementary Table 1).…”

“…The V 1 moiety of Tth V/A-ATPase (A 3 B 3 D 1 F 1 ) is an ATP driven rotary motor where the central DF shaft rotates inside the hexameric A 3 B 3 containing three catalytic sites, each composed of an AB dimer. The V o domain (E 2 G 2 d 1 a 1 c 12 ) is composed of stator parts including the a subunit and two EG peripheral stalks and the d 1 c 12 rotor complex, which consists of a central rotor complex with the DF subunits of V 1 14–16 . When ATP hydrolysis by A 3 B 3 powers the DF shaft, the reverse rotation of the central rotor complex drives proton translocation in the membrane-embedded V o domain (Fig.…”

“…According to the binding change mechanism of ATP synthesis 6 , the three catalytic sites in ATP synthases are in different conformations but interconvert sequentially between three different conformations as catalysis proceeds. Indeed, our previous structure demonstrated that the A 3 B 3 hexamer in the V/A-ATPase adopts an asymmetrical structure composed of three different AB dimers, termed open (AB open ), semi-closed (AB semi ), and closed (AB closed ) 16, 17 . The asymmetrical structure of the V 1 implies that sequential interconversion between these three different AB dimers is synchronized with rotation of the central DF shaft.…”