Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase

Milena Timcenko; Thibaud Dieudonné; Cédric Montigny; Thomas Boesen; Joseph A. Lyons; Guillaume Lenoir; Poul Nissen

doi:10.1016/j.jmb.2021.167062

Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase

Milena Timcenko, Thibaud Dieudonné, Cédric Montigny, Thomas Boesen, Joseph A. Lyons, Guillaume Lenoir, Poul Nissen^*

^*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

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Abstract

P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of cell membranes. The enzymatic cycle of P-type ATPases is divided into autophosphorylation and dephosphorylation half-reactions. Unlike most other P-type ATPases, P4-ATPases transport their substrate during dephosphorylation only, i.e. the phosphorylation half-reaction is not associated with transport. To study the structural basis of the distinct mechanisms of P4-ATPases, we have determined cryo-EM structures of Drs2p-Cdc50p from Saccharomyces cerevisiae covering multiple intermediates of the cycle. We identify several structural motifs specific to Drs2p and P4-ATPases in general that decrease movements and flexibility of domains as compared to other P-type ATPases such as Na⁺/K⁺-ATPase or Ca²⁺-ATPase. These motifs include the linkers that connect the transmembrane region to the actuator (A) domain, which is responsible for dephosphorylation. Additionally, mutation of Tyr380, which interacts with conserved Asp340 of the distinct DGET dephosphorylation loop of P4-ATPases, highlights a functional role of these P4-ATPase specific motifs in the A-domain. Finally, the transmembrane (TM) domain, responsible for transport, also undergoes less extensive conformational changes, which is ensured both by a longer segment connecting TM helix 4 with the phosphorylation site, and possible stabilization by the auxiliary subunit Cdc50p. Collectively these adaptions in P4-ATPases are responsible for phosphorylation becoming transport-independent.

Original language	English
Article number	167062
Journal	Journal of Molecular Biology
Volume	433
Issue	16
ISSN	0022-2836
DOIs	https://doi.org/10.1016/j.jmb.2021.167062
Publication status	Published - Aug 2021

Keywords

cryo-EM
Drs2p-Cdc50p
membrane protein
phosphatidylserine

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title = "Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase",

abstract = "P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of cell membranes. The enzymatic cycle of P-type ATPases is divided into autophosphorylation and dephosphorylation half-reactions. Unlike most other P-type ATPases, P4-ATPases transport their substrate during dephosphorylation only, i.e. the phosphorylation half-reaction is not associated with transport. To study the structural basis of the distinct mechanisms of P4-ATPases, we have determined cryo-EM structures of Drs2p-Cdc50p from Saccharomyces cerevisiae covering multiple intermediates of the cycle. We identify several structural motifs specific to Drs2p and P4-ATPases in general that decrease movements and flexibility of domains as compared to other P-type ATPases such as Na+/K+-ATPase or Ca2+-ATPase. These motifs include the linkers that connect the transmembrane region to the actuator (A) domain, which is responsible for dephosphorylation. Additionally, mutation of Tyr380, which interacts with conserved Asp340 of the distinct DGET dephosphorylation loop of P4-ATPases, highlights a functional role of these P4-ATPase specific motifs in the A-domain. Finally, the transmembrane (TM) domain, responsible for transport, also undergoes less extensive conformational changes, which is ensured both by a longer segment connecting TM helix 4 with the phosphorylation site, and possible stabilization by the auxiliary subunit Cdc50p. Collectively these adaptions in P4-ATPases are responsible for phosphorylation becoming transport-independent.",

keywords = "cryo-EM, Drs2p-Cdc50p, membrane protein, phosphatidylserine",

author = "Milena Timcenko and Thibaud Dieudonn{\'e} and C{\'e}dric Montigny and Thomas Boesen and Lyons, {Joseph A.} and Guillaume Lenoir and Poul Nissen",

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doi = "10.1016/j.jmb.2021.167062",

language = "English",

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Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase. / Timcenko, Milena; Dieudonné, Thibaud; Montigny, Cédric et al.
In: Journal of Molecular Biology, Vol. 433, No. 16, 167062, 08.2021.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

TY - JOUR

T1 - Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase

AU - Timcenko, Milena

AU - Dieudonné, Thibaud

AU - Montigny, Cédric

AU - Boesen, Thomas

AU - Lyons, Joseph A.

AU - Lenoir, Guillaume

AU - Nissen, Poul

PY - 2021/8

Y1 - 2021/8

N2 - P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of cell membranes. The enzymatic cycle of P-type ATPases is divided into autophosphorylation and dephosphorylation half-reactions. Unlike most other P-type ATPases, P4-ATPases transport their substrate during dephosphorylation only, i.e. the phosphorylation half-reaction is not associated with transport. To study the structural basis of the distinct mechanisms of P4-ATPases, we have determined cryo-EM structures of Drs2p-Cdc50p from Saccharomyces cerevisiae covering multiple intermediates of the cycle. We identify several structural motifs specific to Drs2p and P4-ATPases in general that decrease movements and flexibility of domains as compared to other P-type ATPases such as Na+/K+-ATPase or Ca2+-ATPase. These motifs include the linkers that connect the transmembrane region to the actuator (A) domain, which is responsible for dephosphorylation. Additionally, mutation of Tyr380, which interacts with conserved Asp340 of the distinct DGET dephosphorylation loop of P4-ATPases, highlights a functional role of these P4-ATPase specific motifs in the A-domain. Finally, the transmembrane (TM) domain, responsible for transport, also undergoes less extensive conformational changes, which is ensured both by a longer segment connecting TM helix 4 with the phosphorylation site, and possible stabilization by the auxiliary subunit Cdc50p. Collectively these adaptions in P4-ATPases are responsible for phosphorylation becoming transport-independent.

AB - P4-ATPases define a eukaryotic subfamily of the P-type ATPases, and are responsible for the transverse flip of specific lipids from the extracellular or luminal leaflet to the cytosolic leaflet of cell membranes. The enzymatic cycle of P-type ATPases is divided into autophosphorylation and dephosphorylation half-reactions. Unlike most other P-type ATPases, P4-ATPases transport their substrate during dephosphorylation only, i.e. the phosphorylation half-reaction is not associated with transport. To study the structural basis of the distinct mechanisms of P4-ATPases, we have determined cryo-EM structures of Drs2p-Cdc50p from Saccharomyces cerevisiae covering multiple intermediates of the cycle. We identify several structural motifs specific to Drs2p and P4-ATPases in general that decrease movements and flexibility of domains as compared to other P-type ATPases such as Na+/K+-ATPase or Ca2+-ATPase. These motifs include the linkers that connect the transmembrane region to the actuator (A) domain, which is responsible for dephosphorylation. Additionally, mutation of Tyr380, which interacts with conserved Asp340 of the distinct DGET dephosphorylation loop of P4-ATPases, highlights a functional role of these P4-ATPase specific motifs in the A-domain. Finally, the transmembrane (TM) domain, responsible for transport, also undergoes less extensive conformational changes, which is ensured both by a longer segment connecting TM helix 4 with the phosphorylation site, and possible stabilization by the auxiliary subunit Cdc50p. Collectively these adaptions in P4-ATPases are responsible for phosphorylation becoming transport-independent.

KW - cryo-EM

KW - Drs2p-Cdc50p

KW - membrane protein

KW - phosphatidylserine

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U2 - 10.1016/j.jmb.2021.167062

DO - 10.1016/j.jmb.2021.167062

M3 - Journal article

C2 - 34023399

AN - SCOPUS:85107557010

SN - 0022-2836

VL - 433

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

IS - 16

M1 - 167062

ER -

Structural Basis of Substrate-Independent Phosphorylation in a P4-ATPase Lipid Flippase

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