Flipping the script on flippases

By Wendy Bindeman

Head and upper torso photograph of Todd Graham. He is wearing a white shirt, blue blazer, and eye-glasses. — Todd Graham

Todd Graham, Stevenson Chair of Biological Sciences and professor of cell and developmental biology, and external collaborators recently published a paper describing the structure of a yeast “flippase” called Neo1. The research was led by three co-first authors: Bhawik Jain, a postdoc from the Graham lab, Lin Bai from Peking University, and Qinglong You from the Van Andel Institute. It was published on October 13, 2021, in Nature Communications.

We sat down with Graham to learn more about this exciting new research.

What problem does your research address?
The cell membrane is made up of a structure called the phospholipid bilayer. It is a complex structure, and its organization is integral to its function. The different types of lipids or fats in the membrane are often asymmetrically distributed between the inner and outer leaflets.

There are three major families of enzymes that help regulate this asymmetry: “scramblases,” “floppases,” and “flippases.” Flippases are responsible for selectively transporting phospholipids from the extracellular side to the cytosolic side of the membrane. Most flippases are type IV P-type ATPase enzymes (P4 ATPases). Our manuscript addressed the question of how this group of transporters evolved the ability to recognize and transport phospholipids by using a yeast flippase called Neo1 as a case study.

What was unique about your approach to the research?
Most P4 ATPases are alpha-beta heterodimers, which means that they have two different subunits. The alpha subunit provides the ATPase activity while the beta subunit provides structural support and participates in regulatory interactions and recognition of substrates. In contrast, P4B ATPases (a subtype of the P4 ATPases) lack a beta subunit, so it was unclear how these proteins transport substrate. In our paper, we provided the first structures of a P4B ATPase, Neo1, in different conformational states. We also tested the functional significance of our observations through mutagenesis and cellular assays.

Bhawik Jain, postdoctoral fellow in the Graham lab and co-first author on the manuscript.

What were your findings?
The primary conclusion was that the fundamental mechanisms for phospholipid transport are conserved in evolutionarily distant P4 ATPases. Most of the amino acid residues important for substrate binding and transport are conserved between the monomeric Neo1 and the dimeric P4A ATPases. The substrate-binding amino acid residue provided by the beta subunit in P4A ATPases is simply replaced by a similarly functioning residue in Neo1. In addition, the P4B ATPases appear to be an evolutionary intermediate between the dimeric P4A ATPases and a third ATPase type called Ca⁺⁺ ATPases. Comparing structures of enzymes from each of these three P-type ATPase subgroups points to key changes that were needed (evolutionarily) to allow the alpha subunit to interact with the beta subunit in order to form the standard alpha-beta heterodimer structure.

Two diagrams/structures of Neo1. The top part of both diagrams sits on a gray banner that says “membrane.” The rest of the proteins sit on the bottom (white) portion labeled “cytosol.” Left: a space-filling rendering of the protein. Right: a ribbon cartoon of Neo1 with the different alpha helices labeled 1-10 (including 1a, 1b, 4a, 4b, and 6a, 6b). A symbol between both structures says 180˚ and indicates that the protein on the right was rotated 180˚ compared to the protein on the left with respect to the y-axis. Both proteins have sections that are color coded and labeled according to the caption. The protein on the right has a cluster of red and blue spheres in the center of the P-domain; one of the spheres is labeled “Asp-503." — Left: a cryo-electron microscopy 3D model of Neo1, the yeast flippase studied in this paper. Right: an atomic model/ribbon cartoon of Neo1, but rotated 180˚ compared to the image on the left. The different domains of Neo1 are indicated by the following colors: green, TMD (transmembrane domain); yellow, A-domain; dark blue, P-domain; teal, N-domain; orange, N-terminus.

What do you hope will be achieved with the research results in the short and long terms?
The short-term goal was to better understand structure/function relationships for the P4 ATPase protein family. In the long term, studies like these may help us understand the mechanism of various diseases. Humans express 14 members of the P4 ATPase family, and defects in these proteins are linked to severe neurological diseases, liver disease, immune deficiency, and metabolic disease; a human P4B ATPase is specifically linked to a new and rare neurodevelopmental disorder. These structure/function studies are helping us understand the human health consequences of variants in these proteins.

Where is this research taking you next?
The yeast P4B ATPase Neo1, which we used as a model in this paper, plays a role in vesicle-mediated protein trafficking in the secretory pathway. Our studies indicate that Neo1 transports two different lipids across the membrane. We now have the ability to “mutationally tune” or alter the substrate specificity of Neo1, which will allow us to determine if transport of a particular lipid substrate by Neo1 is crucial for its role in vesicle-mediated protein transport.

What are the societal/environmental/economic benefits of this research?
These studies could provide novel therapeutic strategies for the treatment of diseases caused by loss-of-function mutations in the P4 ATPases.

Funding
This work was supported by the National Institutes of Health and the Van Andel Institute.

Go Deeper
The article “Structural basis of the P4B ATPase lipid flippase activity” was published in the journal Nature Communications on October 13, 2021.