Substrate Transport and Specificity in a Phospholipid Flippase

Abstract

Type-IV P-type ATPases are lipid flippases which help maintain asymmetric 14 phospholipid distribution in eukaryotic membranes by using ATP hydrolysis to drive unidirectional 15 translocation of phospholipid substrates. Recent Cryo-EM and crystal structures have provided a 16 detailed view of flippases, and we here use molecular dynamics simulations of the yeast flippase 17Drs2p:Cdc50p in an outward open conformation to study the first steps of phospholipid transport. 18 Our simulations show phospholipid binding to a groove and subsequent movement towards the 19 centre of the membrane, and reveal a preference for phosphatidylserine lipids. We find that the 20 lipid head group stays solvated in the groove while the lipid tails stay in the membrane during the 21 (half) transport event. The flippase also induces deformation and thinning of the outer leaflet. 22 Together, our simulations provide insight into substrate binding to lipid flippases and suggest that 23 multiple sites and steps in the functional cycle contribute to substrate selectivity. 24 25 30 et al., 2020), so that phosphatidylserine (PS) and phosphatidylethanolamine (PE) are enriched in 31 the inner (cytosolic) leaflet, while phosphatidylcholine (PC) and sphingomyelin are found more 32 often in the outer (exoplasmic) leaflet. The maintenance or modulation of the asymmetric PL 33 distribution can play a central role in the proper functioning of the cell; for example, an increase in 34 the population of PS in the outer leaflet of the plasma membrane serves as an 'eat-me' signal to 35 induce phagocytosis (Fadok et al., 1992), whereas a decrease of the population of the negatively 36 charged PS on the inner leaflet may result in a release of proteins with positively charged residues 37 from the membrane to the cytosol by an 'electrostatic switch' mechanism (Lipp et al., 2019). 38 It is kinetically unfavourable for PLs to 'flip' (move from the outside to inside) and 'flop' (inside to 39 outside), and it requires energy to maintain asymmetric lipid distributions. Therefore, cells contain 40 1 of 16 Manuscript to be submitted a number of different PL transporters that fall into three broad categories: scramblases, floppases, 41 and flippases (Montigny et al., 2016). Scramblases (such as TMEM16 (Falzone et al., 2018) and a 42 number of G-protein coupled receptors (Menon et al., 2011)) facilitate the movement of PLs in both 43 directions without energy consumption. In contrast, flippases and floppases are unidirectional and 44 ATP-dependent. They use the energy from ATP hydrolysis to flip or flop specific PLs against their 45 concentration gradient. Indeed, PL flip/flop in the presence of ATP-dependent flippases/floppases 46 typically occurs at the time scale of ∼10-100 ms, much faster than that of the spontaneous process 47 which is typically several hours or longer (Kobayashi and Menon, 2018). 48 The majority of lipid flippases constitute the type IV (P4) subgroup of the P-type ATPase super-49 family that includes the well-characterized + / + -ATPas...

Conformational cycle of human polyamine transporter ATP13A2

“…1b and Supplementary Fig. 9), which is essential for substrate transportation by P5Btype ATPases 29 . A unique NTD adjacent to the A domain contains three short hydrophobic helices (denoted H1, H2, and H3) in a triangular shape embedded in the cytosolic leaflet of the lipid bilayer (Fig.…”

“…12a). The solvent exposure of Site 1 is essential for the recruitment of polyamines from the luminal side and facilitates the hydrophilic substrate binding pathway, reminiscent of the E2P state of the P4-type flippase in which the entry site was also found to be fully hydrated to recruit the hydrophilic head group of its phospholipid substrates 29 .…”