Abstract
The fungal plasma membrane H+-ATPase Pma1 is a vital enzyme, generating a proton-motive force that drives the import of essential nutrients. Autoinhibited Pma1 hexamers in the plasma membrane of starving fungi are activated by glucose signaling and subsequent phosphorylation of the autoinhibitory domain. As related P-type adenosine triphosphatases (ATPases) are not known to oligomerize, the physiological relevance of Pma1 hexamers remained unknown. We have determined the structure of hexameric Pma1 from Neurospora crassa by electron cryo-microscopy at 3.3-Å resolution, elucidating the molecular basis for hexamer formation and autoinhibition and providing a basis for structure-based drug development. Coarse-grained molecular dynamics simulations in a lipid bilayer suggest lipid-mediated contacts between monomers and a substantial protein-induced membrane deformation that could act as a proton-attracting funnel.
Highlights
The plasma membrane of fungal and plant cells contains a proton-pumping P-type adenosine triphosphatase (ATPase) that maintains the intracellular pH and generates a proton-motive force, driving the import of nutrients by secondary transporters [1]
Focusing on the transmembrane and extracellular regions, which would be least conserved relative to human P-type ATPases and most accessible by drugs, we identified a putative drug-binding pocket in a deep groove between M1, M3, and M4, along a ridge of highly conserved residues, and extending to the extracellular surface (Fig. 6B)
The Pma1 structure reveals that core elements of all known proton- pumping proteins are conserved within the Pma1 monomer: (i) a central proton acceptor/donor (Asp730), (ii) a positively charged residue to control pKa changes of the proton acceptor/donor (Arg695), and (iii) bound water molecules to facilitate rapid proton transport by Grotthus shuttling [44]
Summary
The plasma membrane of fungal and plant cells contains a proton-pumping P-type adenosine triphosphatase (ATPase) that maintains the intracellular pH and generates a proton-motive force, driving the import of nutrients by secondary transporters [1]. The membrane potential generated by the fungal proton pump Pma can reach several hundreds of millivolts [2], which requires tight coupling of ATP hydrolysis to strictly unidirectional proton transport. Unlike any other known P-type ATPase, Pma is a hexamer that localizes to specific ordered microdomains in the fungal plasma membrane [6]. It is an abundant membrane protein, and the hexamers can form tightly packed, paracrystalline arrays in starving or stressed cells, as revealed by early electron microscopy (EM) studies [7, 8]. Pma is rapidly activated by multiple phosphorylation events in the R domain
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