Abstract
V1-ATPases are highly conserved ATP-driven rotary molecular motors found in various membrane systems. We recently reported the crystal structures for the Enterococcus hirae A3B3DF (V1) complex, corresponding to the catalytic dwell state waiting for ATP hydrolysis. Here we present the crystal structures for two other dwell states obtained by soaking nucleotide-free V1 crystals in ADP. In the presence of 20 μM ADP, two ADP molecules bind to two of three binding sites and cooperatively induce conformational changes of the third site to an ATP-binding mode, corresponding to the ATP-binding dwell. In the presence of 2 mM ADP, all nucleotide-binding sites are occupied by ADP to induce conformational changes corresponding to the ADP-release dwell. Based on these and previous findings, we propose a V1-ATPase rotational mechanism model.
Highlights
V1-ATPases are highly conserved ATP-driven rotary molecular motors found in various membrane systems
The central axis rotates at 120° per ATP molecule with three dwell states: waiting for ATP binding (ATP-binding dwell) at 0°, waiting for Pi release (Pi-release dwell) at 65°, and waiting for ATP hydrolysis at 90°
The crystal structures of the nucleotide-free and nucleotide-bound A3B3 and V1 complexes revealed conformational changes of the A3B3 complex induced by the binding of nucleotides and the DF axis (Supplementary Fig. 1), suggesting that the E. hirae V1-ATPase (EhV1) structure corresponds to the catalytic dwell waiting for ATP hydrolysis in the rotary cycle[31]
Summary
The titration experiment of ADP into 2AMP-PNP-bound EhV1 with 21 mM AMP-PNP (saturated concentration) showed that the exothermicity was remarkably lower than that into nucleotide-free EhV1 (Fig. 8b,d), and no noticeable exothermic signals were observed for titrations up to an ADP/EhV1 molar ratio of 600 (2.4 mM ADP) (Supplementary Fig. 10); It is predicted the exothermic signal should be very small because the apparent Kd values for ADP to 2AMP-PNP-bound EhV1 were estimated very high[42,43] This finding suggests that ADP is not able to bind to the ‘empty’ form of 2ATPV1 owing to low affinity, as in the case of AMP-PNP. These findings suggest that EhV1 is able to bind AMP-PNP and ADP at two or three binding sites competitively and to reversibly change the conformations
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