Disease mutations reveal residues critical to the interaction of P4-ATPases with lipid substrates

Rasmus H. Gantzel,Robert S. Molday,Bente Vilsen,Louise S. Mogensen,Jens P. Andersen,Stine A. Mikkelsen,Anna L. Vestergaard

doi:10.1038/s41598-017-10741-z

Rasmus H. Gantzel, Robert S. Molday + Show 5 more

Open Access

https://doi.org/10.1038/s41598-017-10741-z

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Abstract

Phospholipid flippases (P4-ATPases) translocate specific phospholipids from the exoplasmic to the cytoplasmic leaflet of membranes. While there is good evidence that the overall molecular structure of flippases is similar to that of P-type ATPase ion-pumps, the transport pathway for the “giant” lipid substrate has not been determined. ATP8A2 is a flippase with selectivity toward phosphatidylserine (PS), possessing a net negatively charged head group, whereas ATP8B1 exhibits selectivity toward the electrically neutral phosphatidylcholine (PC). Setting out to elucidate the functional consequences of flippase disease mutations, we have identified residues of ATP8A2 that are critical to the interaction with the lipid substrate during the translocation process. Among the residues pinpointed are I91 and L308, which are positioned near proposed translocation routes through the protein. In addition we pinpoint two juxtaposed oppositely charged residues, E897 and R898, in the exoplasmic loop between transmembrane helices 5 and 6. The glutamate is conserved between PS and PC flippases, whereas the arginine is replaced by a negatively charged aspartate in ATP8B1. Our mutational analysis suggests that the glutamate repels the PS head group, whereas the arginine minimizes this repulsion in ATP8A2, thereby contributing to control the entry of the phospholipid substrate into the translocation pathway.

Highlights

P4-ATPases, known as flippases, are lipid pumps that contribute to the phospholipid asymmetry of cellular membranes, thereby being crucial to a number of physiologically important functions
The locations in the modelled bATP8A2 E2P structure of the most important of the residues studied here are indicated in Fig. 2, which indicates the position of I364, previously shown to be a critical element in the transport mechanism of human as well as bovine ATP8A2 (“hydrophobic gate residue”)[14]
A markedly reduced expression level relative to the wild type was seen for the mutants I91A, E897K, and E897R, as well (~30%, Table 1), whereas expression levels were higher for the other mutants including L308F, which showed wild type-like expression

Summary

Introduction

P4-ATPases, known as flippases, are lipid pumps that contribute to the phospholipid asymmetry of cellular membranes, thereby being crucial to a number of physiologically important functions. The largest group of patients with flippase defects suffer from the intrahepatic cholestasis diseases PFIC1 (progressive familial intrahepatic cholestasis type 1) and BRIC1 (benign recurrent intrahepatic cholestasis type 1) caused by mutation of the liver-expressed ATP8B11, 12, 13. It was previously shown that bATP8A2 can be expressed in HEK293T cells and purified by immunoaffinity chromatography in sufficient amounts for detailed studies of its reaction cycle, which is activated by PS and PE, but not by PC6, 15. With this methodology, we set out to study the functional consequences of ATP8B1 liver disease mutations by introducing them in bATP8A2. The two juxtaposed residues E897 and R898 may be part of an exoplasmic facing “entry gate” of the translocation pathway

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