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.
© 2021. The Author(s).
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Triphosphatases / chemistry*
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Adenosine Triphosphatases / genetics
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Adenosine Triphosphatases / metabolism
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Amino Acid Sequence
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Binding Sites
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Cell Membrane / chemistry
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Cell Membrane / enzymology
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Cloning, Molecular
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Cryoelectron Microscopy
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Gene Expression
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Genetic Vectors / chemistry
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Genetic Vectors / metabolism
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Humans
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Hydrophobic and Hydrophilic Interactions
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Isoenzymes / chemistry
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Isoenzymes / genetics
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Isoenzymes / metabolism
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Lysophospholipids / chemistry*
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Lysophospholipids / metabolism
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Membrane Transport Proteins / chemistry*
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Membrane Transport Proteins / genetics
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Membrane Transport Proteins / metabolism
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Models, Molecular
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Phosphatidylethanolamines / chemistry*
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Phosphatidylethanolamines / metabolism
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Phosphatidylserines / chemistry*
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Phosphatidylserines / metabolism
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Phospholipid Transfer Proteins / chemistry*
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Phospholipid Transfer Proteins / genetics
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Phospholipid Transfer Proteins / metabolism
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Protein Binding
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Protein Conformation, alpha-Helical
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Protein Conformation, beta-Strand
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Protein Interaction Domains and Motifs
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Protein Multimerization
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Saccharomyces cerevisiae / enzymology*
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae Proteins / chemistry*
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism
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Sequence Alignment
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Sequence Homology, Amino Acid
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Substrate Specificity
Substances
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Isoenzymes
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Lysophospholipids
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Membrane Transport Proteins
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Phosphatidylethanolamines
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Phosphatidylserines
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Phospholipid Transfer Proteins
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Recombinant Proteins
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Saccharomyces cerevisiae Proteins
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lysophosphatidylserine
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phosphatidylethanolamine
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Adenosine Triphosphatases
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ATP9A protein, human
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NEO1 protein, S cerevisiae