Structural basis of the P4B ATPase lipid flippase activity.

Abstract

P4 ATPases are lipid flippases that are phylogenetically grouped into P4A, P4B and P4C clades. The P4A ATPases are heterodimers composed of a catalytic α-subunit and accessory β-subunit, and the structures of several heterodimeric flippases have been reported. The S. cerevisiae Neo1 and its orthologs represent the P4B ATPases, which function as monomeric flippases without a β-subunit. It has been unclear whether monomeric flippases retain the architecture and transport mechanism of the dimeric flippases. Here we report the structure of a P4B ATPase, Neo1, in its E1-ATP, E2P-transition, and E2P states. The structure reveals a conserved architecture as well as highly similar functional intermediate states relative to dimeric flippases. Consistently, structure-guided mutagenesis of residues in the proposed substrate translocation path disrupted Neo1's ability to establish membrane asymmetry. These observations indicate that evolutionarily distant P4 ATPases use a structurally conserved mechanism for substrate transport.