Effect of multi-drug resistance ABC transporter activity on their conformation-induced redistribution on membranes
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00:00
Diagram
Transcript: English(auto-generated)
00:15
Thanks for selecting me for a short talk. I'm a post-doc researcher in Institute Curie
00:23
working with Patricia Becerra. I'm working in a collaboration with Daniel Levy and Maxim Daum from Institute Curie. I'm going to introduce you my ongoing work on conformational dynamics related distribution on membrane. It's a crosstalk between conformation dynamic of transmembrane protein and biophysical properties
00:43
of the membrane, such as curvature or tensions. As we all know, the cell membranes are two-dimensional pseudo-seed in which transmembrane proteins are embedded, peripheral membrane proteins.
01:04
Some are transmembrane proteins, such as ABC transporters are among the major class of the transporters involved in transporter activity, lipid flippages activity. Mostly, they are involved in a multi-drug resistance in a cancer cell, especially also they
01:21
are resistant to the bacterial cell. I'm working with the bacterial ABC transporter, which has open conformation. ATP-driven conformational change induces or exports the drug, which has open conformation.
01:41
ATP binds, closes, and then the drug translocated outside the cell. So it has two shapes. One is a conical. One is a cylindrical. What happens when protein goes in the conformational dynamics inside the membrane? It means that the transmembrane part motion
02:03
is conveyed to my bilayer. So bilayer curvature is also changing upon dynamics. So what happens, or what is a crosstalk when the conformation change is happening to the membrane? And what happens when we have a certain membrane biophysical fixed parameter to the conformational dynamics?
02:23
So it's both crosstalks. Recently, Daniel Levy Group has shown that the open conformation, which is a conical, has a spherical arrangement. They form ring-like structure, whereas when
02:40
you inhibit by orthovanadate, the cycle gets arrested, ATP cycle, and you have a cylinder-shaped protein, you usually end up getting a ribbon-like structure. So what happens here, that whenever there is a protein inclusion, which
03:00
has a curvature in a conical shape, you have a membrane strain. Whereas when the protein is having a cylindrical shape, which has no curvature, they have no membrane strain. Usually, so the conformational change from conical, cylindrical, and I'm
03:23
going to talk what is on the effect of the membrane. So a bit about the physics. Membrane is a flat membrane. You can define the bending of the membrane. When you have insertion of the protein, then this spontaneous curvature induced by the protein
03:43
comes into play. And this bending energy, you can reduce as effective a spontaneous curvature of protein derived the recruitment of the protein in the curved surface to minimize the bending energy of the membrane.
04:01
And that's how the curved protein try to enrich into the curved membrane. And that's derived the membrane curvature to protein sortings of the curved molecules, like conical transmembrane proteins or helical insertions. Our lab developed a tool to study
04:21
how we can play around the membrane curvature and protein sorting. We grow giant granular vesicles. This is a minimal in vitro reconstituted systems in purified minimal components, where you have lipid membrane.
04:43
You reconstitute your protein. And you pull the tube, nanotube, with the optical tweezer. And you can play around. You have the almost flat surface. You have a curved surface. You can go from 100 nanometer to 10 nanometer. You can control the membrane tensions.
05:00
You can play around with the force. So this is a very good system. And we can calculate the protein enrichment from flat to the curved surface. Our lab has shown that the KBAP, which is a conical shape, has a preference for the curved surface,
05:21
whereas the aquaporin, which is a cylindrical shape, doesn't have any preference for the flat or curved surface. But what happens is that the protein has the both shape conical. And it's going in a dynamic state. So I'm going to take open conformation,
05:42
close conformation, and in dynamic state. Let's see what happens. But first, I need to reconstitute my protein. We reconstituted open conformation. This is the most challenging task. And then we reconstituted the close conformation.
06:02
I want to remind here that when we do the reconstitutions by electroformation, we usually having both leaflet or symmetric reconstitutions of the protein. In both cases, I'm going to use the symmetric reconstituted system. Let's talk about the close conformation, which we got it
06:22
in presence of orthovanadate. This is the fixed conformation. We reconstituted this protein. We pulled the tube. And we started increasing the tension, you can see from the movie. When we increase the tension, we modulate the radius.
06:42
And as we modulate the radius, you can see here that the protein is sorting. There is no protein. As you're increasing the tension, you're modulating, decreasing the radius. And then the protein sorting is started. So relative enrichment calculated.
07:02
And then the radius we calculated from the fluorescence, we can't calculate directly from the tension. There is a relation from tension to the radius. But we have the proteins, so the correlation doesn't go very well. And we plotted our protein enrichment versus curvature. And it depends on the protein density on the surface first.
07:23
And also, protein enrichment has a curvature preference around 20 nanometer here. So lesser the protein density on the surface, easier to flow through the neck. So we have almost protein is flowing from flat surface
07:47
to the curved surface. Lipid is flowing from outside, so there is a mixing. And this allows the reduction in bending energy due to the spontaneous curvature that follows. And after fitting our curve with the model,
08:05
we deduced the curvature of the protein around inverse of 6 nanometer. Initially, we presume that the protein is cylindrical, but it's not cylindrical. It has a simultaneous curvature.
08:21
And this goes well with the crystalline structure of the other ABC transporter. And so here, what we propose is that the protein which are inside out are sorting out into the tube, which has a preferred curvature from the inside.
08:44
And let's take an example of the open conformation. Here, I pull the tube, and I waited for 15 minutes. I'm not changing any tension. I'm not modulating my radius. Protein itself is modulating.
09:01
It enriches to the curved surface once you provide. And it remodulates the radius, and it reaches up to 30 nanometer. So this almost enrichment to the tube is almost 30 times. And it automatically modulates from 100 nanometer
09:24
to 30 nanometer. Always, it reaches 30 nanometer. You can see here, sometime you have huge clusters and phase segregation. There might be. So here, the protein is sorting which are outside to the leaf light.
09:44
Here, the protein is not sorting from inside. And here, our curvature for this one is 30 nanometer from the previous cryo EM image. They came up with a radius of 50 nanometer. So you have to keep in mind that this radius which
10:04
we are calculating, which is this transmembrane domain and interplay of the lipid bilayer. This is coming from the extracellular domain also. So we have to keep in mind.
10:21
There might be protein-protein interaction. We are not ruling out because we have a huge. We don't know is it a cluster or not. And there is a crowding effect because we have almost 50 times enrichment in the tube. Now, let's take an example of ABC transporter dynamics
10:42
where in presence of ATP, you can see when it's open, there is one curvature. When it's closed, then you have another curvature. So the sign of the curvature, membrane curvature, is changing upon the ATP cycling. So in this experiment, what I did
11:01
is I pulled the tube in open conformation. There is a protein enrichment. And then I added the ATP on the tube from here. And then what we observed that the protein which was enriched in the tube has no, they just went back to the flat surface.
11:22
And this is, you can see, and this decreases with the time. And then it goes to a steady state. So in dynamic state, our protein, you have to remember that we have a symmetric reconstitution. So when I add the ATP, only outside molecules
11:42
are in a dynamic state. Inside molecules are open always. And when I add, usually when they are in the tube, outside protein is in wrong side.
12:01
It doesn't have a preferred curvature. So they move to the side. And the good part is it's impossible to check in this giant enamel vesicles the activity of the protein. So here we've shown that the protein is active first.
12:21
In conclusion, I want to say that the ABC transporter has a dynamic, which is the close conformation has membrane curvature around inverse of 90 nanometer. It has a conical shape. It's not a cylindrical shape.
12:43
Oppo form modulates the membrane curvature by itself. And it reaches almost to 30 nanometer. My protein is active. And it's in dynamic state because of the flexibility and negative curvature preference, they move out to the surface.
13:08
And this is, so BMRA, in my experiment, I proposed that the BMRA stay longer in a post-hydrotic conformation during the cyclic state. And our observation is orthogonal to the rest
13:22
of the experiments. They propose is that the ABC transporter usually stays in open conformation a longer time. Thanks to Patricia Vassaro, to our groups,
13:42
and the funding agencies. And also, I want to mention the Daniel, who, Suzanne, who is preparing the proteoliposome, and also the other collaborator, Maxim Dam. Thank you very much.
14:02
So what would happen if you drive the ATP hydrolysis inside of the GOV by, let's say, optical methods, right? And catch ATP. So now you're adding the ATP from inside rather than from outside.
14:20
So one is a technical part. When we, OK, when I'm going to add the ATP inside, then the protein which are going to cycle
14:40
into the inner leaflet of the bilayer. In that case, they have the wrong preference of the curvature. So usually, they will just, so in this case,
15:06
if they will start pumping out, so there is a physics also, there is a, they will cluster first.
15:22
That's for sure, because there are two, not all the protein are going to be synchronized in one conformation. So probably one conformation will derive, like population of the conformation will drive to have a cluster. So we will have two sort of clusters.
15:40
But again, it's in a dynamic state, so it's very hard to say what's going to happen, frankly. We can have two quick questions. All right, Alekse. Is there any evidence that in the cell you have some sorting based on the conformational state or the state of the protein?
16:07
OK, in vivo observations. There are some of the observations where you have the curved membranes, like you have some of the transporters there. And they are the curved membranes, so they usually,
16:24
but as such, there is no direct evidence that the transporters are clustering. It's just a theoretical model proposed in 1986 by, and that says that if the protein are curved, they will cluster. So that's a logical conclusion in physics
16:41
that if they are curved, they will cluster. So, Mila, you had a question? Oh, yeah. So around the same line, what is the implication for in vivo? Because what's the density of this protein in normal cells and there will be other normal cells,
17:00
so what can you think about what's the physiological importance of this if there is immediate? So when the cell is in dynamic state, you have a lot of curved surfaces, like whenever the cell is dividing or you have the producer of the cells.
17:22
So usually, I'm not correlating my concentration with the in vivo, but the sorting at the curved surface is in sense that it need a transport because these ABC transporters are the lipid flippages also.
17:40
So they usually would cluster at the neck or the curved membrane, and depending on the scenario, they want to have export of the molecules or metabolites or anything, they will do that functions or even in the curved membrane,
18:01
people proposes that the lipid need to be flipped, they do these functions properly. But I'm not correlating the concentration in vitro with the in vivo. No, but it's hard because in the cell, there will be also not just one protein in the membrane, it will be grounded with other proteins, but each one will want to have its own curvature.
18:25
Okay, thank you very much.