Directed evolution of a sphingomyelin flippase reveals mechanism of substrate backbone discrimination by a P4-ATPase

Abstract

Phospholipid flippases in the type IV P-type ATPase (P4-ATPases) family establish membrane asymmetry and play critical roles in vesicular transport, cell polarity, signal transduction, and neurologic development. All characterized P4-ATPases flip glycerophospholipids across the bilayer to the cytosolic leaflet of the membrane, but how these enzymes distinguish glycerophospholipids from sphingolipids is not known. We used a directed evolution approach to examine the molecular mechanisms through which P4-ATPases discriminate substrate backbone. A mutagenesis screen in the yeast Saccharomyces cerevisiae has identified several gain-offunction mutations in the P4-ATPase Dnf1 that facilitate the transport of a novel lipid substrate, sphingomyelin. We found that a highly conserved asparagine (N220) in the first transmembrane segment is a key enforcer of glycerophospholipid selection, and specific substitutions at this site allow transport of sphingomyelin.sphingomyelin | P4-ATPase | membrane asymmetry | directed evolution T he asymmetry of the membrane bilayer is a fundamental feature of the eukaryotic plasma membrane (1). Phospholipid (PL) species such as phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidylethanolamine (PE) dominate the cytofacial leaflet, whereas phosphatidylcholine (PC) and various sphingolipids (SLs) populate the exofacial leaflet (2, 3). Organelle membrane asymmetry has proven more difficult to study, and the precise distribution of PLs is still unclear; however, evidence suggests that the organelles of the secretory and endocytic pathways also exhibit asymmetric membranes. For example, PS is initially enriched in the luminal leaflet of the endoplasmic reticulum (ER) but flips to the cytofacial leaflet at the trans-Golgi network (4). This asymmetric membrane architecture has been demonstrated to affect membrane curvature (5-8), secretory function (6, 9-16), membrane polarization (17), and intra-and intercellular signaling (17)(18)(19)(20).PL flippases in the type IV P-type ATPase family (P4-ATPases) help establish membrane asymmetry by using ATP to transport specific glycerophospholipids (GPLs) from the luminal/exofacial to the cytofacial leaflet of the membrane (21, 22). P4-ATPases have been implicated in a host of diverse diseases such as hepatic cholestasis (23-25), aberrant lymphocyte development (26, 27), anemia (27, 28), hearing loss (29), neurologic disease (30-32), and diabetes (33). Eukaryotic organisms express several P4-ATPases that have different subcellular localizations, tissue specificities, and substrate preferences (34). Understanding the substrate preferences of these enzymes and the physical means of substrate discrimination is important for understanding their role in membrane biology, development, and disease.The P-type ATPase family shares a conserved enzyme architecture that can be separated into four primary domains: the actuator (A), the nucleotide-binding (N), the phosphorylation (P), and the transmembrane (TM) domains (35). P4-ATPase substrate selection a...

Decoding P4-ATPase substrate interactions

“…To date, only GPLs have been identified as P4-ATPase substrates, with the headgroup in the sn3 position and the acyl chains in the sn1 and sn2 positions [Figure 3] (Smriti et al , 2007). In their work, Smriti and colleagues propose that the discrimination of PL backbone chirality indicates that the backbone and headgroup may form two contacts for paired selection, a hypothesis that recently received additional support (Roland and Graham, 2016). The precise discrimination of substrate backbone is also clearly illustrated when comparing PC and SM selection.…”

“…It is likely that P4-ATPases were first generated through a gene duplication event with a more ancient ion transporting P-type ATPase family member, yet it is unclear how the P4-ATPases developed the physical capacity to translocate such a large, amphipathic substrate (giant substrate problem). Without a crystal structure, research groups have produced and tested several P4-ATPase homology models using previously crystallized P-type ATPases as templates (Stone et al , 2012, Vestergaard et al , 2014, Roland and Graham, 2016). These studies combined computational modeling with site-directed mutagenesis or forward genetic approaches to each describe important molecular determinants of P4-ATPase substrate transport.…”

“…This study identified an Asn in TM1 that is a major “gatekeeper” of GPL specificity. Specific mutations at this position (Dnf1 – N220S or N220T, but not N220C) significantly enhanced recognition of SM [Figure 4], and combining SM-permissive mutations (Dnf1 –N220S, L1202P) yielded a flippase that preferred SM over PC (Roland and Graham, 2016). N220 is in the same cytofacial cluster of residues (exit gate) now shown to discriminate all three aspects of the PL: headgroup, backbone, and acyl chain occupancy [Figure 3].…”

“…Of particular relevance are a pair of Asn residues in the Dnf1 “exit gate,” N220 of TM1 and N550 of TM3, which have a substantial influence on distinguishing GPL from SL and lyso-PL from diacyl-PL substrates, respectively [Figure 5A, inset]. We speculate that these Asn residues may form an Asn-clamp to select the substrate backbone and ester linkages, while the TM2 and TM4 “exit gate” residues coordinate the headgroup, thereby orienting the acyl chains toward the TM1,3,4 cleft [Figure 5A, inset] (Roland and Graham, 2016). The opposite orientation of the PL, protruding through TM2,4,6, would move this substrate backbone far from this Asn-clamp [Figure 5B, inset] This leads us to favor a model where the hydrophilic “entry” and “exit” gates guide the selection and transport of the PL substrate along the TM1,3,4 groove, while the movement of the “hydrophobic gate” confers directional transport.…”

“…As will be detailed later, this essential enzyme family translocates specific substrates to remodel the membrane bilayer. In addition, P4-ATPases can be mutationally tuned to alter their PL substrate specificity (Baldridge and Graham, 2012, Baldridge and Graham, 2013, Baldridge et al , 2013, Roland and Graham, 2016). These designer P4-ATPases have already proven useful for determining the influence of flipping specific substrates on vesicle trafficking and lateral membrane organization (Xu et al , 2013, Hankins et al , 2015b).…”