Effect of multi-drug resistance ABC transporter activity on their conformation-induced redistribution on membranes
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Mahalka, Ajay K.
Integral trans-membrane proteins are involved in various cellular functions and their dysfunction is associated with human pathologies. The lipid-protein interactions have been studied to address structure-function relationship of transmembrane proteins at molecular level. However, the effects related to membranes physical properties on trans-membrane proteins have not been well-studied, and not at all when their conformations change. Recent experimental evidence indicates the intrinsic interplay between protein shape and the properties of its membrane environment. It is expected that non-cylindrical proteins tend to cluster and be enriched in curved membranes. Thus, we studied BmrA a bacterial ATP binding cassette (ABC) transporter from B. subtilisinvolved in export of a large diversity of substrates in an ATP dependent manner, fairly homologous to human P-glycoprotein ,. The conformational change in nucleotide-binding domains (NBDs) of BmrA between apo and the post-hydrolytic state (tweezers-like motion) is 5 nm and that is the largest tweezers motion reported till date in the case of trans-membrane proteins. Here we addressed how the conformational dynamics of BmrA influence its membrane properties, in particular its spatial distribution on flat or curved membranes. To decipher the effect of the conformational dynamics of BmrA on its spatial distribution in membranes, depending on membrane curvature, we used cell-sized giant unilamellar vesicles (GUVs) containing either the apo-or closed-conformation BmrA to form membrane nanotubes with controlled radii. We found that, at low protein density, apo-BmrA is highly enriched (50 times) in nanotubes as compared to flat membrane and simultaneously modulates tube radius from 100 nm to 30 nm, due to its high intrinsic curvature. Surprisingly, although the post-hydrolytic closed-conformation BmrA is expected to be cylindrical, we measured an enrichment of this conformation in nanotubes, but about 3 time less pronounced then for apo-BmrA. Eventually, in the presence of ATP, BmrAhas reduced curvature selectivity as compared to the apo form, in agreement with a cycling change of conformation between the apo and the closed forms. This study on reconstituted transmembrane proteins demonstrates that protein distribution on membranes is influenced by the interplayof membrane curvature, effective shape and flexibility of membrane proteins.
 Thanks for selecting me for a short talk. I'm a post-doc researcher in Institute Curie working with Patricia Becero. I'm working in a collaboration with Daniel Levy and Maxim Dom from Institute Curie. I'm going to introduce you my ongoing work on conformational dynamics related distribution on membrane. It's a cross talk between conformational dynamic of transmembrane protein and biophysical properties of the membrane such as curvature or tensors. As we all know that cell membranes are two-dimensional pseudo-seat in which transmembrane proteins are embedded or peripheral membrane proteins. Some are transmembrane proteins such as ABC transporters are the measure class of among the measure class of the transporters involved in transporter activity, lipid flip-ins activity. Especially they are involved in a multi-drug resistance in a cancer cell, especially also they 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. ATP binds, closes and then the drug translocated outside the cell. So it has two shape, 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 is conveyed to my bilayer. So bilayer curvature is also changing upon dynamics. So what happens or what is a cross talk when the conformation changes happening to the group membrane and what happens when we have a certain membrane biophysical fixed parameter to the conformational dynamics. So it's a both cross talks. Recently in Daniel Levy group has shown that the open conformation which is a conical has a spherical arrangement. They form ring like structure whereas when you inhibit by ortho vanadate cycle get arrested ATP cycle and you have a cylinder shape protein you usually end up getting a ribbon like structure. So what happens here that whenever there is a protein inclusion which 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 curvature membrane strain. Usually so the conformational change from conical cylindrical and I'm going to talk what is on the effect of the membrane. To study so bit about the physics membrane is a flat membrane you can have a bending you can define the bending of the membrane. When you have insertion of the protein then this spontaneous curvature induced by the protein comes into the play and this bending energy you can reduce as effective spontaneous curvature of protein is derived the recruitment of the protein in the curved surface to minimize the bending energy of the membrane and that's dependent on that's how the curved protein try to enrich into the curved membrane and that's derived the membrane curvature the protein shortings of the curved molecules like conical transmembrane proteins or helical insertions. Our lab developed a tool to study how we can play around the membrane curvature and protein sorting. We grow a giant enololour vesicles this is a minimal in vitro reconstituted systems in purified minimal components where you have lipid membrane 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 tensons 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 whereas the equiporin which is a cylindrical in shape doesn't have any preference for the flat or curved surface. But what happens that the protein has the both shape conical and it's going in a dynamic state so I'm going to take open conformation, closed 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 closed conformation. I want to remind here that when we do the reconstitutions by electro formation we usually having a both leaflet or symmetric reconstitutions of the protein in both case so I'm going to use this system symmetric reconstituted system. Let's talk about the closed conformation which we got it in presence of ortho vanadette this is the fixed conformation we reconstituted this protein. We pull the tube and we started increasing the tension you can see from the movie. When we increase the tension we modulate the radius and as we modulate the radius you can see here that the protein is sorting there is no protein as you increasing the tension you are modulating decreasing the radius and then the protein sorting is started. So relative enrichment calculated 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 of the correlation doesn't go very well and we plotted our protein enrichment versus curvature and it depend on the protein density on the surface first and also it 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 to the curved surface lipid is flowing from outside so there is a mixing and this allows the reduction in burning energy due to the spontaneous curvature that follows and after the fitting our curve with the model we deduced the curvature of the protein around inverse of 6 nanometer. Initially we presume that the protein is cylindrical but it is not cylindrical it has a spontaneous curvature and this goes well with the crystallite 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. Let us take an example of the open conformation here I am not I pull the tube and I waited for 15 minute I am not changing any tension I am not modulating my radius protein itself modulating it sorts it reaches to the curved surface when you provide and it remodulate the radius and it reaches up to 30 nanometer so this almost enrichment to the tube is almost 30 time and it automatically modulate from 100 nanometer 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 here the protein is not sorting from inside and here our curvature for this one is 30 nanometer from the previous cryo E M image they came up with the radius of 15 nanometer so you have to keep in mind that this radius which we are calculating which is this trans membrane domain and interplay of the lipid bilayer this is coming from the extracellular domain also so we have to keep in mind there might be protein-protein interaction we are not ruling out because we have a huge we do not know is it cluster or not and there is a crowding effect because we have almost 50 time enrichment in the tube. Now let us take an example of ATP AVC transporter dynamics where in presence of ATP you can see when it is open there is a one curvature when it is 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 it 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 and this is you can see with the and this decreases with the time and then it goes to the study 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 are in a dynamic state inside molecules are open always. So and when I add usually this has when they are in the tube outside protein having is in the wrong side it does not have a preferred curvature so they move to the and the good part is it is impossible to check in this giant enamel vascals the activity of the protein so here we we shown that the protein is active first. In conclusion I want to say that the ABC transporter has a dynamic which is the closed conformation has a membrane curvature around inverse of 90 nanometer it has a conical shape it is not a cylindrical shape. Apoform modulates the membrane curvature by itself and it reaches almost to 30 nanometer my protein is active and it is in dynamic state because of the flexibility and negative curvature preference they move out to the surface and this is so BMRA in my experiment with I propose that the BMRA stay longer in a post hydrodetic conformation during the cyclic state and our observation is orthogonal to the rest of the experiments they propose is that the BMRA ABC transporter use will stay in open conformation longer time. Thanks to Patricia Vasero to our groups and the funding agencies and also I want to mention the Daniel who is preparing the proto liposome and also the other collaborator Maxim down. Thank you very much. So what would happen if you drive the ATP hydrolysis inside of the GV by let's say optical methods right and catch ATP. So you are adding the ATP from inside. So one is a technical part when we are okay when I am going to add the ATP inside then the protein which are going to cycle 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 if they will start pumping out some there is a physics also there is a they will cluster first 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 derive to have a cluster. So we will have a two sort of clusters 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 questions. All right. I like that. Is there any evidence that in the cell you have some sorting based on the conformational state of the protein? Okay 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 but as such there is no direct evidence that the transporter are clustering. It's just a theoretical model proposed in 86 and that says that if the protein are curved they will cluster. So that's a logical conclusion in physics that if they are curved they will cluster. Did you have a question? Oh yeah. So around the same line so what is it the implication for in vivo because there is not what's the density of this protein in normal cells and there will be other normal cells. So what can you think about what's the physiological importance of this if there is an 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 or 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 this ABC transporter or the lipid flip is also. 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 people propose is that lipid need to be flipped they do these functions properly so this is but I'm not correlating the concentration in vitro with the in vivo.
 Thanks for selecting me for a short talk.  I'm a post-doc researcher in Institute Curie working with Patricia Becero.  I'm working in a collaboration with Daniel Levy and Maxim Dom from Institute Curie.  I'm going to introduce you my ongoing work on conformational dynamics related distribution  on membrane.  It's a cross talk between conformational dynamic of transmembrane protein and biophysical  properties of the membrane such as curvature or tensors.  As we all know that cell membranes are two-dimensional pseudo-seat in which transmembrane proteins  are embedded or peripheral membrane proteins.  Some are transmembrane proteins such as ABC transporters are the measure class of among  the measure class of the transporters involved in transporter activity, lipid flip-ins activity.  Especially they are involved in a multi-drug resistance in a cancer cell, especially also  they 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.  ATP binds, closes and then the drug translocated outside the cell.  So it has two shape, 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 is conveyed to my bilayer.  So bilayer curvature is also changing upon dynamics.  So what happens or what is a cross talk when the conformation changes happening to the  group membrane and what happens when we have a certain membrane biophysical fixed parameter  to the conformational dynamics.  So it's a both cross talks.  Recently in Daniel Levy group has shown that the open conformation which is a conical has  a spherical arrangement.  They form ring like structure whereas when you inhibit by ortho vanadate cycle get arrested  ATP cycle and you have a cylinder shape protein you usually end up getting a ribbon like structure.  So what happens here that whenever there is a protein inclusion which 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 curvature membrane strain.  Usually so the conformational change from conical cylindrical and I'm going to talk  what is on the effect of the membrane.  To study so bit about the physics membrane is a flat membrane you can have a bending  you can define the bending of the membrane.  When you have insertion of the protein then this spontaneous curvature induced by the  protein comes into the play and this bending energy you can reduce as effective spontaneous  curvature of protein is derived the recruitment of the protein in the curved surface to minimize  the bending energy of the membrane and that's dependent on that's how the curved protein  try to enrich into the curved membrane and that's derived the membrane curvature the  protein shortings of the curved molecules like conical transmembrane proteins or helical  insertions.  Our lab developed a tool to study how we can play around the membrane curvature and protein  sorting.  We grow a giant enololour vesicles this is a minimal in vitro reconstituted systems in  purified minimal components where you have lipid membrane 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 tensons 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 whereas the equiporin which is a cylindrical in shape doesn't have any preference for the  flat or curved surface.  But what happens that the protein has the both shape conical and it's going in a dynamic  state so I'm going to take open conformation, closed 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 closed conformation.  I want to remind here that when we do the reconstitutions by electro formation we usually having a both  leaflet or symmetric reconstitutions of the protein in both case so I'm going to use this  system symmetric reconstituted system.  Let's talk about the closed conformation which we got it in presence of ortho vanadette  this is the fixed conformation we reconstituted this protein.  We pull the tube and we started increasing the tension you can see from the movie.  When we increase the tension we modulate the radius and as we modulate the radius you can  see here that the protein is sorting there is no protein as you increasing the tension  you are modulating decreasing the radius and then the protein sorting is started.  So relative enrichment calculated 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 of the correlation doesn't go very well and we plotted our protein  enrichment versus curvature and it depend on the protein density on the surface first  and also it 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 to the curved surface lipid is flowing from  outside so there is a mixing and this allows the reduction in burning energy due to the  spontaneous curvature that follows and after the fitting our curve with the model we deduced  the curvature of the protein around inverse of 6 nanometer.  Initially we presume that the protein is cylindrical but it is not cylindrical it has a spontaneous  curvature and this goes well with the crystallite 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.  Let us take an example of the open conformation here I am not I pull the tube and I waited  for 15 minute I am not changing any tension I am not modulating my radius protein itself  modulating it sorts it reaches to the curved surface when you provide and it remodulate  the radius and it reaches up to 30 nanometer so this almost enrichment to the tube is almost  30 time and it automatically modulate from 100 nanometer 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 here the protein  is not sorting from inside and here our curvature for this one is 30 nanometer from the previous  cryo E M image they came up with the radius of 15 nanometer so you have to keep in mind  that this radius which we are calculating which is this trans membrane domain and interplay  of the lipid bilayer this is coming from the extracellular domain also so we have to keep  in mind there might be protein-protein interaction we are not ruling out because we have a huge  we do not know is it cluster or not and there is a crowding effect because we have almost  50 time enrichment in the tube.  Now let us take an example of ATP AVC transporter dynamics where in presence of ATP you can  see when it is open there is a one curvature when it is 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 it 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  and this is you can see with the and this decreases with the time and then it goes to  the study 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 are in a dynamic state inside molecules are  open always.  So and when I add usually this has when they are in the tube outside protein having is  in the wrong side it does not have a preferred curvature so they move to the and the good  part is it is impossible to check in this giant enamel vascals the activity of the protein  so here we we shown that the protein is active first.  In conclusion I want to say that the ABC transporter has a dynamic which is the closed conformation  has a membrane curvature around inverse of 90 nanometer it has a conical shape it is not  a cylindrical shape.  Apoform modulates the membrane curvature by itself and it reaches almost to 30 nanometer  my protein is active and it is in dynamic state because of the flexibility and negative  curvature preference they move out to the surface and this is so BMRA in my experiment  with I propose that the BMRA stay longer in a post hydrodetic conformation during the  cyclic state and our observation is orthogonal to the rest of the experiments they propose  is that the BMRA ABC transporter use will stay in open conformation longer time.  Thanks to Patricia Vasero to our groups and the funding agencies and also I want to mention  the Daniel who is preparing the proto liposome and also the other collaborator Maxim down.  Thank you very much.  So what would happen if you drive the ATP hydrolysis inside of the GV by let's say optical methods  right and catch ATP.  So you are adding the ATP from inside.  So one is a technical part when we are okay when I am going to add the ATP inside then  the protein which are going to cycle 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 if they will start pumping out some there is  a physics also there is a they will cluster first 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 derive  to have a cluster.  So we will have a two sort of clusters 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 questions.  All right.  I like that.  Is there any evidence that in the cell you have some sorting based on the conformational  state of the protein?  Okay 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 but as such there is no direct evidence that the transporter are clustering.  It's just a theoretical model proposed in 86 and that says that if the protein are curved  they will cluster.  So that's a logical conclusion in physics that if they are curved they will cluster.  Did you have a question?  Oh yeah.  So around the same line so what is it the implication for in vivo because there is not  what's the density of this protein in normal cells and there will be other normal cells.  So what can you think about what's the physiological importance of this if there is an 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 or 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 this ABC transporter or the lipid flip is also.  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 people propose is that lipid need to be flipped they  do these functions properly so this is but I'm not correlating the concentration in  vitro with the in vivo.
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