Drug Design, Structural Dynamics and Physiological Functions of G Protein-Coupled Receptors (GPCRs)
Wüthrich, Kurt
There are 826 G protein-coupled receptors (GPCR) in the human proteome, which regulate key physiological processes and have therefore long been attractive as drug targets. With crystal structure determinations of more than 50 different human GPCRs during the last decade, an initial platform for structure-based rational design was established for drugs that target GPCRs. The principal method used for our own research is nuclear magnetic resonance (NMR) spectroscopy in solution, which is one of the key approaches for expanding the crystal structure platform with dynamics features of GPCRs at near-physiological conditions. In this context it is of special interest that NMR measurements can be performed without extensive modifications of the GPCRs, such as those used to facilitate crystallization, i.e., by amino acid replacements and/or fusion with other proteins. In my lecture I describe strategies for the use of NMR techniques with GPCRs, which will be illustrated with projects where results obtained with X-ray crystallography or cryo-electron microscopy (cryo-EM) have been complemented with NMR investigations in solution, and discuss the impact of this integrative approach on GPCR biology and drug discovery. References Liu, J.J., Horst, R., Katritch, V., Stevens, R.C. and Wüthrich, K. (2012) Science 335, 1106-1110. Biased signaling pathways in β2-adrenergic receptor characterized by 19F-NMR. Eddy, M., Lee, M.-Y., Gao, Z.-G., White, K.L., Didenko, T., Horst, R., Audet, M., Stanczak, P., McClary, K.M., Han, G.W., Jacobson, K.A., Stevens, R. and Wüthrich, K. (2018) Cell 172, 68–80. Allosteric coupling of drug binding and intracellular signaling in the A2A adenosine receptor.
 It's great to be here in Lindau again and I want to thank all of you, my colleagues and the students for all the input that I received so far this week. The title of my talk is focused on GPCRs, Geproton Coupled Receptors. This is not new for you, Professor Levkovic, who discovered GPCRs, gave you a marvelous survey of the history and the latest developments in the field. So I don't need to introduce the subject and what I want to do in this favorable situation is to give you an impression of what structural biology can do in a rapidly evolving field, which also finds an incredible breadth of practical applications, especially in drug discovery and drug design. So the problem that we as biophysicists face is that GPCRs, you see here a schematic drawing of the seven trans membrane helices and well I'm told that the screen should help me to point. Well, the yellow shape is a ligand in the so-called orthostatic binding site and that's where drugs bind on the outside of the cell membrane and they signal across the cell membrane over the distance of about 30 angstroms to the intracellular partner proteins. This is typically classified as an allosteric system. See now in analogy to an enzyme, the active site would be on the intracellular surface and the effector is 30 angstroms away and the signal is transmitted across this distance and that's what we are trying to understand. We try to understand how it is possible that this allosteric signal transmission occurs what the structural basis is for this. However, in contrast to Professor Levkovic, GPCR has not been my project for a very long time. I received, as was mentioned, I received my Nobel Prize for having used NMR to study proteins and nucleic acids. And the important point in contribution from our research is that we can get three-dimensional structures of proteins and nucleic acids at the atomic resolution by NMR in solution. Now you realize that our proteins are active in contact with aqueous solutions or they are even dissolved in aqueous media. And so the results that we can obtain with this approach can be very close to results that we would see on the physiological conditions. Now NMR stands for nuclear magnetic resonance. It could stand for no more research. It could also stand for no meaningful results. But it is nuclear magnetic resonance. I think at this point I want to digress a little bit to indicate to the students in what surroundings we started this work. The structural biology became an official high-level part of natural sciences when the Nobel Prize in 1962 was awarded to this group of people we see from left to right, Maurice Wilkins, Max Perout, Francis Crick, John Steinbeck, that's a literature laureate, Jim Watson and John Kendrew. On these, of course, you know this prize was given on the one hand for the discovery of the DNA double helix, on the other hand for the determination of the three-dimensional structure of a protein, namely hemoglobin and a related molecule, myoglobin. And Max Perout, who is the second person from the left on this picture, was really leading structural biology for decades after receiving the Nobel Prize in 1962. And the atmosphere is that was present and which we would start to develop another technique is well represented by this quotation by Max Perout of J. Bernal in 1936. The secret of life lies in the structure of proteins. And there is only one way of solving it and that is by X-ray crystallography. Now of course that asks for competition. You see it is very much easier to take up ideas from others and then compete against. If you have your own ideas you cannot compete against your ideas. That would be sort of stupid. But so you have to get competitive and competition has been part of my life early on. And you see in 2015 a nature article included this picture of myself training high jumping in Switzerland and actually in the Federal School of Sports in Nierbien. And you see so I started to collect material for an age review already when I was 17 years old. It is a structure of hemoglobin. It is a time when Perout and Kendra shared a Nobel Prize for this achievement looked like this. No individual atoms could be seen. And there was actually a lot of possibilities for us in message to get into it. And I am not going to talk about this today but my first major project was to study hemoglobin and it was immediately possible to use NMR in such ways that we would see individual atoms that were beyond the resolution of the crystal structure and I had very close relations with Max Peroutes ever since we did this in the late 1960s until well into 2000. It is also important to keep the competitive spirit over a long time. You see this picture is taken by Peter Bach in 2015 and I am still trying to keep the competitive spirit up. And I would like to illustrate to you with the next project that I am going to describe the importance of being on the ball for a long time in the life of a scientist and at the same time I want to illustrate for the first time, not this last time in this lecture, how important it is to integrate results from different techniques when you try to solve a particular problem. Now when Professor Levkovitz was just discovering GPCRs in the early 1970s, we were going to work on ligands to GPCRs without knowing that they are GPCRs. And I show you here a picture that comes from Sir Tom Blondell, a crystallographer who was able to determine the structure of one of the important ligands for a GPCR, namely glucagon. And unfortunately it seems that this cream doesn't work, so I cannot point. In the upper center you see a triangular structure. This is a crystal structure that was determined by Tom Blondell. And it consists of a trimeric aggregate of three molecules of glucagon. Having obtained this structure, Tom hypothesized that this trimeric form was indeed present in our body, that it would dissolve into monomers that would also be fully alpha helical, the same as the molecule was in the trimeric form, that it could then easily bind to the membrane surface as an alpha helix or it could dissolve into a random coil. At that point we started to study the same molecule with NMR. And the first thing was that we could show that when it is dissolved in water, it is not at all helical, that it is random coil and contains one short piece of spatial structures that is well defined and that is in the positions from 21 to 25. And you can see that there is a phenylalanine and a tryptophan and these form a sandwich and that's the only stable structure in the entire molecule. Next, we did study the molecule, this short polypeptide when it is bound to a lipid surface in the torsion micelles. And we found out that it is not helical as hypothesized on the basis of the crystal structure that there is only a short piece of helix again from residues 20 to 27 and the remainder of the molecule is in random coil form. And this is just a detail, this is the very first structure that was determined at high resolution by NMR. We finished this work in 1983. Now exactly 30 years later, I started a new project in my laboratory in Shanghai and we studied now the interaction of glucagon with glucagon receptor which is a GPCR. So this gives incredibly nice possibilities. We can study the response of the GPCR to G-brote and beta-rescuit to the interaction of the ligand with the orthosteric side, with the outside of the receptor and at the same time we can study what happens to the ligand, to the polypeptide ligand when it interacts with the receptor. Now we started to study this system in 2013 and this is still too early to talk about it. It takes a long time to develop such a system for structural biology. We have actually very encouraging results but for various reasons I'm not supposed to present these at the moment. So I will talk about two other receptors and one of these is a receptor that was a mainstay in Professor Levkovic's work, namely beta-2AR. And I use this scheme to indicate to you how we approached biophysical studies of these GPCRs. What you see here is a scheme that shows on the vertical axis the biological response against different ligands that are applied in increasing concentration from left to right on the horizontal axis. And you have agonists, you have the 8x4, that means the receptor without the ligand bound or with an antagonist bound that would inactivate the receptor and then you may have an inverse agonist. And so you see also the expression basal activity which in this scheme is 50 biological response units. Are you a response unit? Now we may have GPCRs that have a basal activity of zero. We may have GPCRs which have a basal activity that corresponds to 20 or 30 percent of the maximum activity that you can obtain with an agonist. And with this introduction I now want to describe to you our approach. You see here on the left the schematic drawing of beta-2AR, so the seven-trans membrane helix structure of this GPCR. Where it says C265 at the lower left end and it says C327 which is somewhere in the middle, at the bottom, we inserted labels that we could observe by NMR. These are fluorine labels. There is no fluorine in this whole system so that we can observe these probes without interference from the biological material. And we would then use a small library of ligands which are all drugs. And use, I mean, the Quenbuterol is the worst because it has been extensively used for doping in competitive sports, especially by weight lifters and so on. And you see that this includes inverse agonists. It includes a neutral antagonist. It includes agonists and it also includes biased agonists which were extensively discussed by Professor Levkovitz. So we would go, we would study these are, I think, 10 ligands and we would now study the response of those fluorine probes on the intracellular surface to the variable efficacy of the ligands that are bound 30 angstroms away. And these are the raw data which I like to show just to let you see that results can be subtle. What you can see is that for each of these ligands on the left for the position C327, that is one of the probes on the right for C265, that's the other probe, there are all these two hardly resolved peaks. And you can see even in this raw data that the relative intensity between the two peaks shifts from left to right, from right to left, depending on the efficacy of the ligand. And here now is the interpretation of the data. You have here in the center where it says Apo on the right, a schematic drawing of two of the helices in the GPCR, Helix 6 and Helix 7, and we assume based on data and the literature that signaling to the G protein goes primarily through Helix 6 and signaling to arresting goes primarily through Helix 7. And that's where our probes are located. Now when we look at carousel all, that's the drawing in the upper left and the spectrum is shown here. I mean you see always a red line and the blue line, this we call this deconvolution of the spectrum where we can determine the relative intensities of the two peaks that are overlapped. When we have an inverse agonist, we kill transmission to both signaling pathways. When we have a full agonist, we open the channels for both interactions and when we have a biased agonist, we close down the G protein entirely, beta-resting is opened and with a biased agonist, we open the beta-resting channel and reduce the transfer through the G protein channel. Now the important thing to be seen here, which cannot be seen by any other method, is that we have two conformations in equilibrium. They are here at the same time and never do we have 100% of the active like state or of the inactive like state. And it is so possible that additional interactions, for example with G protein, with beta-resting, can first shift the equilibrium to be near 100% activation or to be closer to inactivation. So we have here a highly dynamic system that can respond to the needs of the moment when the GPCR is functioning in our body. And that is before we look at many of the surrounding effectors that can also play a role in this game. Now I want to show to you how important it is that beta-2AR was studied not only by crystallography but also by NMR. In crystallography, the receptor was fused with a soluble protein T4L isosine. And you see that the T4L isosine, that's a blue shape, has almost the same size as the GPCR. What we now studied was how these function-related equilibria are affected by the fusion of this soluble protein into one of the intracellular loops. What happens once you fused T4L in the equilibrium is completely blocked and we have throughout essentially 100% active state going through the G protein. So this function-related conformational equilibrium is completely abolished with the preparation that was used for crystallography. I think it's a very convincing demonstration of the importance of integrative use of multiple techniques. Now I'm changing to the second GPCR that I want to discuss with you. And this is A2AAR, adenosine receptor. And this is new work. It's unpublished. That's why I mentioned the name of Lucas, Sujats and Tatiana Dedenko. What we did here was to engineer groups into the intracellular surface, where so-to-speak circles are. Again we used fluorine and observed the effect of variable ligands on these fluorine markers. Here is a library of ligands that we used, one of those you have probably enjoyed this morning. It's caffeine, which works as an antagonist for A2AAR. The important point that I want to make is that these different agonists and different antagonists have very different chemical structures, but they all give the same spectrum in our NMR experiment. On the left you have the antagonist spectrum, which has two conformations in equilibrium in the agonist spectrum. We have three conformations. Now we can take the A4 form, add an agonist, so we go from the inactive spectrum to the active-like spectrum, and then we can replace the agonist with a more strongly binding antagonist, and we go back to the inactive spectrum. The important thing here is that Y-type A2AAR has essentially zero base selectivity, and when you now look at the peak, which is between 12 and 14 ppm, then it's completely gone in the inactive form. See, this is a yellow peak, P4, showing that indeed we have it in hand. There is no residual activity that we can detect. Now we take a related GPCR with a single amino acid replacement of Cere91 with Alenine. This form is known to have significant base selectivity, and we immediately see that P4 is present in what is supposed to be the active form. So again, we have here a subtle equilibrium between different inactive and different active forms. And of course we want to know what the exchange is between the two. Here I show just as an illustration the spectrum of the active-like form, and you see that rate of exchange between P1 and P2, this is a peak from the active-like form. P2 from the inactive-like form is slow, it's about 20, sorry, it's relatively fast, around 20 per second. Whereas the exchange between P1 from the inactive form and P4 from the active form is very slow, less than one per second. So we have a very high activation barrier between the ensemble of active-like shapes, states, and an ensemble of inactive-like states that is intrinsic to the GPCR before it interacts with partner proteins that are of course important in the course of the follow-up reactions. Now, we have introduced these fluorine labels and that gives us only a few points to observe. Now we have succeeded in expressing this particular GPCR in Pisa pastoris, four years of work for two people. And at this point now we are able to first to show that it has a full activity of the material that was expressed with the traditional method from the Lefkowitz Laboratory with SF9, and we can now produce a milligram quantities of these receptors and many large number of variant forms of the receptor. And in Pisa in the yeast we can now label the proteins with nitrogen 15 isotope and with deuterium. And as a result, using a technique that we introduced about 20 years ago, drosy, transverse relaxation, optimized spectroscopy, we can now get the spectrum with hundreds of resolved lines. And we can use this in two ways, we can use the whole spectrum to check that the global structure of the molecule is always preserved when we generate variants. And then we have well resolved lines in the two squares that are outlined. The one hand, these are the tryptophan, indole, NH groups, on the other hand the backbone, glycine, AMH groups, and then we replace one and one each tryptophan with tyrosine, and you can see, you best see it on the right side with the cross sections. The peak that is marked by an asterisk disappears after replacing tryptophan with tyrosine, and so we have one by one assigned the peaks, we did the same for eight glycines, and now we have observation points across the entire molecule, from the intracellular surface through the center of the molecule to the extracellular surface. And I want to point out two residues, one is D52, the spastic acid 52, which is known to be the allosteric clock in the molecule. So we will destroy this allosteric center and check what happens. And then we have tryptophan 246, which has been referred to as Goggleswitch, tryptophan and has been found, has been reported to be of particular importance during the activation process of the receptor. So what comes out, when we look at the response to variable drug efficacy, then we get no response out to the extracellular surface, and we get a structural response all over the receptor, all the way from the ligand binding side to the intracellular surface. Here are just a few examples of how clear-cut these responses are when we vary the efficacy of the ligand in the allosteric side. And here, the biggest effect we see indeed for this togle-switch tryptophan, where we get the chemical shift difference, again identified by the stars on the right of almost one part per million, and we know what happens there. I think I forgot, we know exactly what happens there with the arrangement of the aromatic grains in the center of the receptor. But now, it's very last, my time is up, I take an extra minute to describe to you what we did with regard to the studies on the structural basis of the allosteric signaling from the extracellular to the intracellular surface. On the extreme left, you have the functional GPCR, where you bind the ligand to get G-protein signaling. Then we replace the center of the allosteric switch, which is a negatively charged residue with its neutral analog, and we get no signaling through. So the question is, why do we not get signaling through? And this question, to answer this question, is important because 86% of all class HGPCRs contain aspartic acid in this position. So when we make this replacement, we again get a huge change in the dogle-switch resonance, and just look at the left side, what happens is that when we destroy the allosteric switch, we get no signaling down to the intracellular surface. All the signaling is pushed back, and structural changes are seen only above W246 and also way up into the extracellular surface. So what we have shown is that the signaling through the allosteric switch is killed when we replace the negatively charged residue, and the signal is actually pushed back to the outside of the cell, and all the dynamics of the residues on the surface, on the intracellular surface is stopped. And we see only a single rigid surface structure, whereas in the active side, we have dynamic equilibria between all the sides that we can observe. So in summary, we have been able to show that activity of GPCRs cannot be understood simply by looking at limiting structures of the active or the inactive forms. We have local conformational polymorphism, which make it a highly dynamic system that can adopt to the needs in any given situation. We can show that ligand efficacy is related not to rigid structural changes, but to shifts in conformational equilibria. We are able, and now this is in contrast to the results obtained from CRIOEM, which also shows that there are multiple different conformations, but we can measure the exchange between these conformations. We don't just sample views of different structures. And of course, continuing on, we can get information on all of these properties from interactions with extracellular ligands and with intracellular reaction partners. Thank you.
 It's great to be here in Lindau again and I want to thank all of you, my colleagues and  the students for all the input that I received so far this week.  The title of my talk is focused on GPCRs, Geproton Coupled Receptors.  This is not new for you, Professor Levkovic, who discovered GPCRs, gave you a marvelous  survey of the history and the latest developments in the field.  So I don't need to introduce the subject and what I want to do in this favorable situation  is to give you an impression of what structural biology can do in a rapidly evolving field,  which also finds an incredible breadth of practical applications, especially in drug  discovery and drug design.  So the problem that we as biophysicists face is that GPCRs, you see here a schematic drawing  of the seven trans membrane helices and well I'm told that the screen should help me to  point.  Well, the yellow shape is a ligand in the so-called orthostatic binding site and that's where  drugs bind on the outside of the cell membrane and they signal across the cell membrane over  the distance of about 30 angstroms to the intracellular partner proteins.  This is typically classified as an allosteric system.  See now in analogy to an enzyme, the active site would be on the intracellular surface  and the effector is 30 angstroms away and the signal is transmitted across this distance  and that's what we are trying to understand.  We try to understand how it is possible that this allosteric signal transmission occurs  what the structural basis is for this.  However, in contrast to Professor Levkovic, GPCR has not been my project for a very long  time.  I received, as was mentioned, I received my Nobel Prize for having used NMR to study  proteins and nucleic acids.  And the important point in contribution from our research is that we can get three-dimensional  structures of proteins and nucleic acids at the atomic resolution by NMR in solution.  Now you realize that our proteins are active in contact with aqueous solutions or they  are even dissolved in aqueous media.  And so the results that we can obtain with this approach can be very close to results  that we would see on the physiological conditions.  Now NMR stands for nuclear magnetic resonance.  It could stand for no more research.  It could also stand for no meaningful results.  But it is nuclear magnetic resonance.  I think at this point I want to digress a little bit to indicate to the students in  what surroundings we started this work.  The structural biology became an official high-level part of natural sciences when the  Nobel Prize in 1962 was awarded to this group of people we see from left to right, Maurice  Wilkins, Max Perout, Francis Crick, John Steinbeck, that's a literature laureate,  Jim Watson and John Kendrew.  On these, of course, you know this prize was given on the one hand for the discovery of  the DNA double helix, on the other hand for the determination of the three-dimensional  structure of a protein, namely hemoglobin and a related molecule, myoglobin.  And Max Perout, who is the second person from the left on this picture, was really leading  structural biology for decades after receiving the Nobel Prize in 1962.  And the atmosphere is that was present and which we would start to develop another technique  is well represented by this quotation by Max Perout of J. Bernal in 1936.  The secret of life lies in the structure of proteins.  And there is only one way of solving it and that is by X-ray crystallography.  Now of course that asks for competition.  You see it is very much easier to take up ideas from others and then compete against.  If you have your own ideas you cannot compete against your ideas.  That would be sort of stupid.  But so you have to get competitive and competition has been part of my life early on.  And you see in 2015 a nature article included this picture of myself training high jumping  in Switzerland and actually in the Federal School of Sports in Nierbien.  And you see so I started to collect material for an age review already when I was 17 years  old.  It is a structure of hemoglobin.  It is a time when Perout and Kendra shared a Nobel Prize for this achievement looked  like this.  No individual atoms could be seen.  And there was actually a lot of possibilities for us in message to get into it.  And I am not going to talk about this today but my first major project was to study hemoglobin  and it was immediately possible to use NMR in such ways that we would see individual atoms  that were beyond the resolution of the crystal structure and I had very close relations with  Max Peroutes ever since we did this in the late 1960s until well into 2000.  It is also important to keep the competitive spirit over a long time.  You see this picture is taken by Peter Bach in 2015 and I am still trying to keep the  competitive spirit up.  And I would like to illustrate to you with the next project that I am going to describe  the importance of being on the ball for a long time in the life of a scientist and at  the same time I want to illustrate for the first time, not this last time in this lecture,  how important it is to integrate results from different techniques when you try to solve  a particular problem.  Now when Professor Levkovitz was just discovering GPCRs in the early 1970s, we were going to  work on ligands to GPCRs without knowing that they are GPCRs.  And I show you here a picture that comes from Sir Tom Blondell, a crystallographer who was  able to determine the structure of one of the important ligands for a GPCR, namely glucagon.  And unfortunately it seems that this cream doesn't work, so I cannot point.  In the upper center you see a triangular structure.  This is a crystal structure that was determined by Tom Blondell.  And it consists of a trimeric aggregate of three molecules of glucagon.  Having obtained this structure, Tom hypothesized that this trimeric form was indeed present  in our body, that it would dissolve into monomers that would also be fully alpha helical, the  same as the molecule was in the trimeric form, that it could then easily bind to the membrane  surface as an alpha helix or it could dissolve into a random coil.  At that point we started to study the same molecule with NMR.  And the first thing was that we could show that when it is dissolved in water, it is  not at all helical, that it is random coil and contains one short piece of spatial structures  that is well defined and that is in the positions from 21 to 25.  And you can see that there is a phenylalanine and a tryptophan and these form a sandwich  and that's the only stable structure in the entire molecule.  Next, we did study the molecule, this short polypeptide when it is bound to a lipid surface  in the torsion micelles.  And we found out that it is not helical as hypothesized on the basis of the crystal structure  that there is only a short piece of helix again from residues 20 to 27 and the remainder  of the molecule is in random coil form.  And this is just a detail, this is the very first structure that was determined at high  resolution by NMR.  We finished this work in 1983.  Now exactly 30 years later, I started a new project in my laboratory in Shanghai and we  studied now the interaction of glucagon with glucagon receptor which is a GPCR.  So this gives incredibly nice possibilities.  We can study the response of the GPCR to G-brote and beta-rescuit to the interaction of the  ligand with the orthosteric side, with the outside of the receptor and at the same time  we can study what happens to the ligand, to the polypeptide ligand when it interacts  with the receptor.  Now we started to study this system in 2013 and this is still too early to talk about  it.  It takes a long time to develop such a system for structural biology.  We have actually very encouraging results but for various reasons I'm not supposed to  present these at the moment.  So I will talk about two other receptors and one of these is a receptor that was a mainstay  in Professor Levkovic's work, namely beta-2AR.  And I use this scheme to indicate to you how we approached biophysical studies of these  GPCRs.  What you see here is a scheme that shows on the vertical axis the biological response  against different ligands that are applied in increasing concentration from left to right  on the horizontal axis.  And you have agonists, you have the 8x4, that means the receptor without the ligand bound  or with an antagonist bound that would inactivate the receptor and then you may have an inverse  agonist.  And so you see also the expression basal activity which in this scheme is 50 biological  response units.  Are you a response unit?  Now we may have GPCRs that have a basal activity of zero.  We may have GPCRs which have a basal activity that corresponds to 20 or 30 percent of the  maximum activity that you can obtain with an agonist.  And with this introduction I now want to describe to you our approach.  You see here on the left the schematic drawing of beta-2AR, so the seven-trans membrane helix  structure of this GPCR.  Where it says C265 at the lower left end and it says C327 which is somewhere in the middle,  at the bottom, we inserted labels that we could observe by NMR.  These are fluorine labels.  There is no fluorine in this whole system so that we can observe these probes without  interference from the biological material.  And we would then use a small library of ligands which are all drugs.  And use, I mean, the Quenbuterol is the worst because it has been extensively used for  doping in competitive sports, especially by weight lifters and so on.  And you see that this includes inverse agonists.  It includes a neutral antagonist.  It includes agonists and it also includes biased agonists which were extensively discussed  by Professor Levkovitz.  So we would go, we would study these are, I think, 10 ligands and we would now study  the response of those fluorine probes on the intracellular surface to the variable efficacy  of the ligands that are bound 30 angstroms away.  And these are the raw data which I like to show just to let you see that results can  be subtle.  What you can see is that for each of these ligands on the left for the position C327,  that is one of the probes on the right for C265, that's the other probe, there are all  these two hardly resolved peaks.  And you can see even in this raw data that the relative intensity between the two peaks  shifts from left to right, from right to left, depending on the efficacy of the ligand.  And here now is the interpretation of the data.  You have here in the center where it says Apo on the right, a schematic drawing of two  of the helices in the GPCR, Helix 6 and Helix 7, and we assume based on data and the literature  that signaling to the G protein goes primarily through Helix 6 and signaling to arresting  goes primarily through Helix 7.  And that's where our probes are located.  Now when we look at carousel all, that's the drawing in the upper left and the spectrum  is shown here.  I mean you see always a red line and the blue line, this we call this deconvolution of the  spectrum where we can determine the relative intensities of the two peaks that are overlapped.  When we have an inverse agonist, we kill transmission to both signaling pathways.  When we have a full agonist, we open the channels for both interactions and when we have a  biased agonist, we close down the G protein entirely, beta-resting is opened and with  a biased agonist, we open the beta-resting channel and reduce the transfer through the  G protein channel.  Now the important thing to be seen here, which cannot be seen by any other method, is that  we have two conformations in equilibrium.  They are here at the same time and never do we have 100% of the active like state or  of the inactive like state.  And it is so possible that additional interactions, for example with G protein, with beta-resting,  can first shift the equilibrium to be near 100% activation or to be closer to inactivation.  So we have here a highly dynamic system that can respond to the needs of the moment when  the GPCR is functioning in our body.  And that is before we look at many of the surrounding effectors that can also play a  role in this game.  Now I want to show to you how important it is that beta-2AR was studied not only by crystallography  but also by NMR.  In crystallography, the receptor was fused with a soluble protein T4L isosine.  And you see that the T4L isosine, that's a blue shape, has almost the same size as  the GPCR.  What we now studied was how these function-related equilibria are affected by the fusion of  this soluble protein into one of the intracellular loops.  What happens once you fused T4L in the equilibrium is completely blocked and we have throughout  essentially 100% active state going through the G protein.  So this function-related conformational equilibrium is completely abolished with the preparation  that was used for crystallography.  I think it's a very convincing demonstration of the importance of integrative use of multiple  techniques.  Now I'm changing to the second GPCR that I want to discuss with you.  And this is A2AAR, adenosine receptor.  And this is new work.  It's unpublished.  That's why I mentioned the name of Lucas, Sujats and Tatiana Dedenko.  What we did here was to engineer groups into the intracellular surface, where so-to-speak  circles are.  Again we used fluorine and observed the effect of variable ligands on these fluorine markers.  Here is a library of ligands that we used, one of those you have probably enjoyed this  morning.  It's caffeine, which works as an antagonist for A2AAR.  The important point that I want to make is that these different agonists and different  antagonists have very different chemical structures, but they all give the same spectrum in our  NMR experiment.  On the left you have the antagonist spectrum, which has two conformations in equilibrium  in the agonist spectrum.  We have three conformations.  Now we can take the A4 form, add an agonist, so we go from the inactive spectrum to the  active-like spectrum, and then we can replace the agonist with a more strongly binding antagonist,  and we go back to the inactive spectrum.  The important thing here is that Y-type A2AAR has essentially zero base selectivity, and  when you now look at the peak, which is between 12 and 14 ppm, then it's completely gone  in the inactive form.  See, this is a yellow peak, P4, showing that indeed we have it in hand.  There is no residual activity that we can detect.  Now we take a related GPCR with a single amino acid replacement of Cere91 with Alenine.  This form is known to have significant base selectivity, and we immediately see that P4  is present in what is supposed to be the active form.  So again, we have here a subtle equilibrium between different inactive and different active  forms.  And of course we want to know what the exchange is between the two.  Here I show just as an illustration the spectrum of the active-like form, and you see that  rate of exchange between P1 and P2, this is a peak from the active-like form.  P2 from the inactive-like form is slow, it's about 20, sorry, it's relatively fast, around  20 per second.  Whereas the exchange between P1 from the inactive form and P4 from the active form is very slow,  less than one per second.  So we have a very high activation barrier between the ensemble of active-like shapes,  states, and an ensemble of inactive-like states that is intrinsic to the GPCR before it interacts  with partner proteins that are of course important in the course of the follow-up reactions.  Now, we have introduced these fluorine labels and that gives us only a few points to observe.  Now we have succeeded in expressing this particular GPCR in Pisa pastoris, four years of work  for two people.  And at this point now we are able to first to show that it has a full activity of the  material that was expressed with the traditional method from the Lefkowitz Laboratory with  SF9, and we can now produce a milligram quantities of these receptors and many large number of  variant forms of the receptor.  And in Pisa in the yeast we can now label the proteins with nitrogen 15 isotope and with  deuterium.  And as a result, using a technique that we introduced about 20 years ago, drosy, transverse  relaxation, optimized spectroscopy, we can now get the spectrum with hundreds of resolved  lines.  And we can use this in two ways, we can use the whole spectrum to check that the global  structure of the molecule is always preserved when we generate variants.  And then we have well resolved lines in the two squares that are outlined.  The one hand, these are the tryptophan, indole, NH groups, on the other hand the backbone,  glycine, AMH groups, and then we replace one and one each tryptophan with tyrosine,  and you can see, you best see it on the right side with the cross sections.  The peak that is marked by an asterisk disappears after replacing tryptophan with tyrosine,  and so we have one by one assigned the peaks, we did the same for eight glycines, and now  we have observation points across the entire molecule, from the intracellular surface through  the center of the molecule to the extracellular surface.  And I want to point out two residues, one is D52, the spastic acid 52, which is known  to be the allosteric clock in the molecule.  So we will destroy this allosteric center and check what happens.  And then we have tryptophan 246, which has been referred to as Goggleswitch, tryptophan  and has been found, has been reported to be of particular importance during the activation  process of the receptor.  So what comes out, when we look at the response to variable drug efficacy, then we get no  response out to the extracellular surface, and we get a structural response all over  the receptor, all the way from the ligand binding side to the intracellular surface.  Here are just a few examples of how clear-cut these responses are when we vary the efficacy  of the ligand in the allosteric side.  And here, the biggest effect we see indeed for this togle-switch tryptophan, where we  get the chemical shift difference, again identified by the stars on the right of almost one part  per million, and we know what happens there.  I think I forgot, we know exactly what happens there with the arrangement of the aromatic  grains in the center of the receptor.  But now, it's very last, my time is up, I take an extra minute to describe to you what  we did with regard to the studies on the structural basis of the allosteric signaling from the  extracellular to the intracellular surface.  On the extreme left, you have the functional GPCR, where you bind the ligand to get G-protein  signaling.  Then we replace the center of the allosteric switch, which is a negatively charged residue  with its neutral analog, and we get no signaling through.  So the question is, why do we not get signaling through?  And this question, to answer this question, is important because 86% of all class HGPCRs  contain aspartic acid in this position.  So when we make this replacement, we again get a huge change in the dogle-switch resonance,  and just look at the left side, what happens is that when we destroy the allosteric switch,  we get no signaling down to the intracellular surface.  All the signaling is pushed back, and structural changes are seen only above W246 and also  way up into the extracellular surface.  So what we have shown is that the signaling through the allosteric switch is killed when  we replace the negatively charged residue, and the signal is actually pushed back to  the outside of the cell, and all the dynamics of the residues on the surface, on the intracellular  surface is stopped.  And we see only a single rigid surface structure, whereas in the active side, we have dynamic  equilibria between all the sides that we can observe.  So in summary, we have been able to show that activity of GPCRs cannot be understood simply  by looking at limiting structures of the active or the inactive forms.  We have local conformational polymorphism, which make it a highly dynamic system that  can adopt to the needs in any given situation.  We can show that ligand efficacy is related not to rigid structural changes, but to shifts  in conformational equilibria.  We are able, and now this is in contrast to the results obtained from CRIOEM, which also  shows that there are multiple different conformations, but we can measure the exchange between these  conformations.  We don't just sample views of different structures.  And of course, continuing on, we can get information on all of these properties from interactions  with extracellular ligands and with intracellular reaction partners.  Thank you.
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It's great to be here in Lindau again and I want to thank all of you, my colleagues and the students for all the input that I received so far this week. The title of my talk is focused on GPCRs, Geproton Coupled Receptors. This is not new for you, Professor Levkovic, who discovered GPCRs, gave you a marvelous survey of the history and the latest developments in the field. So I don't need to introduce the subject and what I want to do in this favorable situation is to give you an impression of what structural biology can do in a rapidly evolving field, which also finds an incredible breadth of practical applications, especially in drug discovery and drug design. So the problem that we as biophysicists face is that GPCRs, you see here a schematic drawing of the seven trans membrane helices and well I'm told that the screen should help me to point. Well, the yellow shape is a ligand in the so-called orthostatic binding site and that's where drugs bind on the outside of the cell membrane and they signal across the cell membrane over the distance of about 30 angstroms to the intracellular partner proteins. This is typically classified as an allosteric system. See now in analogy to an enzyme, the active site would be on the intracellular surface and the effector is 30 angstroms away and the signal is transmitted across this distance and that's what we are trying to understand. We try to understand how it is possible that this allosteric signal transmission occurs what the structural basis is for this. However, in contrast to Professor Levkovic, GPCR has not been my project for a very long time. I received, as was mentioned, I received my Nobel Prize for having used NMR to study proteins and nucleic acids. And the important point in contribution from our research is that we can get three-dimensional structures of proteins and nucleic acids at the atomic resolution by NMR in solution. Now you realize that our proteins are active in contact with aqueous solutions or they are even dissolved in aqueous media. And so the results that we can obtain with this approach can be very close to results that we would see on the physiological conditions. Now NMR stands for nuclear magnetic resonance. It could stand for no more research. It could also stand for no meaningful results. But it is nuclear magnetic resonance. I think at this point I want to digress a little bit to indicate to the students in what surroundings we started this work. The structural biology became an official high-level part of natural sciences when the Nobel Prize in 1962 was awarded to this group of people we see from left to right, Maurice Wilkins, Max Perout, Francis Crick, John Steinbeck, that's a literature laureate, Jim Watson and John Kendrew. On these, of course, you know this prize was given on the one hand for the discovery of the DNA double helix, on the other hand for the determination of the three-dimensional structure of a protein, namely hemoglobin and a related molecule, myoglobin. And Max Perout, who is the second person from the left on this picture, was really leading structural biology for decades after receiving the Nobel Prize in 1962. And the atmosphere is that was present and which we would start to develop another technique is well represented by this quotation by Max Perout of J. Bernal in 1936. The secret of life lies in the structure of proteins. And there is only one way of solving it and that is by X-ray crystallography. Now of course that asks for competition. You see it is very much easier to take up ideas from others and then compete against. If you have your own ideas you cannot compete against your ideas. That would be sort of stupid. But so you have to get competitive and competition has been part of my life early on. And you see in 2015 a nature article included this picture of myself training high jumping in Switzerland and actually in the Federal School of Sports in Nierbien. And you see so I started to collect material for an age review already when I was 17 years old. It is a structure of hemoglobin. It is a time when Perout and Kendra shared a Nobel Prize for this achievement looked like this. No individual atoms could be seen. And there was actually a lot of possibilities for us in message to get into it. And I am not going to talk about this today but my first major project was to study hemoglobin and it was immediately possible to use NMR in such ways that we would see individual atoms that were beyond the resolution of the crystal structure and I had very close relations with Max Peroutes ever since we did this in the late 1960s until well into 2000. It is also important to keep the competitive spirit over a long time. You see this picture is taken by Peter Bach in 2015 and I am still trying to keep the competitive spirit up. And I would like to illustrate to you with the next project that I am going to describe the importance of being on the ball for a long time in the life of a scientist and at the same time I want to illustrate for the first time, not this last time in this lecture, how important it is to integrate results from different techniques when you try to solve a particular problem. Now when Professor Levkovitz was just discovering GPCRs in the early 1970s, we were going to work on ligands to GPCRs without knowing that they are GPCRs. And I show you here a picture that comes from Sir Tom Blondell, a crystallographer who was able to determine the structure of one of the important ligands for a GPCR, namely glucagon. And unfortunately it seems that this cream doesn't work, so I cannot point. In the upper center you see a triangular structure. This is a crystal structure that was determined by Tom Blondell. And it consists of a trimeric aggregate of three molecules of glucagon. Having obtained this structure, Tom hypothesized that this trimeric form was indeed present in our body, that it would dissolve into monomers that would also be fully alpha helical, the same as the molecule was in the trimeric form, that it could then easily bind to the membrane surface as an alpha helix or it could dissolve into a random coil. At that point we started to study the same molecule with NMR. And the first thing was that we could show that when it is dissolved in water, it is not at all helical, that it is random coil and contains one short piece of spatial structures that is well defined and that is in the positions from 21 to 25. And you can see that there is a phenylalanine and a tryptophan and these form a sandwich and that's the only stable structure in the entire molecule. Next, we did study the molecule, this short polypeptide when it is bound to a lipid surface in the torsion micelles. And we found out that it is not helical as hypothesized on the basis of the crystal structure that there is only a short piece of helix again from residues 20 to 27 and the remainder of the molecule is in random coil form. And this is just a detail, this is the very first structure that was determined at high resolution by NMR. We finished this work in 1983. Now exactly 30 years later, I started a new project in my laboratory in Shanghai and we studied now the interaction of glucagon with glucagon receptor which is a GPCR. So this gives incredibly nice possibilities. We can study the response of the GPCR to G-brote and beta-rescuit to the interaction of the ligand with the orthosteric side, with the outside of the receptor and at the same time we can study what happens to the ligand, to the polypeptide ligand when it interacts with the receptor. Now we started to study this system in 2013 and this is still too early to talk about it. It takes a long time to develop such a system for structural biology. We have actually very encouraging results but for various reasons I'm not supposed to present these at the moment. So I will talk about two other receptors and one of these is a receptor that was a mainstay in Professor Levkovic's work, namely beta-2AR. And I use this scheme to indicate to you how we approached biophysical studies of these GPCRs. What you see here is a scheme that shows on the vertical axis the biological response against different ligands that are applied in increasing concentration from left to right on the horizontal axis. And you have agonists, you have the 8x4, that means the receptor without the ligand bound or with an antagonist bound that would inactivate the receptor and then you may have an inverse agonist. And so you see also the expression basal activity which in this scheme is 50 biological response units. Are you a response unit? Now we may have GPCRs that have a basal activity of zero. We may have GPCRs which have a basal activity that corresponds to 20 or 30 percent of the maximum activity that you can obtain with an agonist. And with this introduction I now want to describe to you our approach. You see here on the left the schematic drawing of beta-2AR, so the seven-trans membrane helix structure of this GPCR. Where it says C265 at the lower left end and it says C327 which is somewhere in the middle, at the bottom, we inserted labels that we could observe by NMR. These are fluorine labels. There is no fluorine in this whole system so that we can observe these probes without interference from the biological material. And we would then use a small library of ligands which are all drugs. And use, I mean, the Quenbuterol is the worst because it has been extensively used for doping in competitive sports, especially by weight lifters and so on. And you see that this includes inverse agonists. It includes a neutral antagonist. It includes agonists and it also includes biased agonists which were extensively discussed by Professor Levkovitz. So we would go, we would study these are, I think, 10 ligands and we would now study the response of those fluorine probes on the intracellular surface to the variable efficacy of the ligands that are bound 30 angstroms away. And these are the raw data which I like to show just to let you see that results can be subtle. What you can see is that for each of these ligands on the left for the position C327, that is one of the probes on the right for C265, that's the other probe, there are all these two hardly resolved peaks. And you can see even in this raw data that the relative intensity between the two peaks shifts from left to right, from right to left, depending on the efficacy of the ligand. And here now is the interpretation of the data. You have here in the center where it says Apo on the right, a schematic drawing of two of the helices in the GPCR, Helix 6 and Helix 7, and we assume based on data and the literature that signaling to the G protein goes primarily through Helix 6 and signaling to arresting goes primarily through Helix 7. And that's where our probes are located. Now when we look at carousel all, that's the drawing in the upper left and the spectrum is shown here. I mean you see always a red line and the blue line, this we call this deconvolution of the spectrum where we can determine the relative intensities of the two peaks that are overlapped. When we have an inverse agonist, we kill transmission to both signaling pathways. When we have a full agonist, we open the channels for both interactions and when we have a biased agonist, we close down the G protein entirely, beta-resting is opened and with a biased agonist, we open the beta-resting channel and reduce the transfer through the G protein channel. Now the important thing to be seen here, which cannot be seen by any other method, is that we have two conformations in equilibrium. They are here at the same time and never do we have 100% of the active like state or of the inactive like state. And it is so possible that additional interactions, for example with G protein, with beta-resting, can first shift the equilibrium to be near 100% activation or to be closer to inactivation. So we have here a highly dynamic system that can respond to the needs of the moment when the GPCR is functioning in our body. And that is before we look at many of the surrounding effectors that can also play a role in this game. Now I want to show to you how important it is that beta-2AR was studied not only by crystallography but also by NMR. In crystallography, the receptor was fused with a soluble protein T4L isosine. And you see that the T4L isosine, that's a blue shape, has almost the same size as the GPCR. What we now studied was how these function-related equilibria are affected by the fusion of this soluble protein into one of the intracellular loops. What happens once you fused T4L in the equilibrium is completely blocked and we have throughout essentially 100% active state going through the G protein. So this function-related conformational equilibrium is completely abolished with the preparation that was used for crystallography. I think it's a very convincing demonstration of the importance of integrative use of multiple techniques. Now I'm changing to the second GPCR that I want to discuss with you. And this is A2AAR, adenosine receptor. And this is new work. It's unpublished. That's why I mentioned the name of Lucas, Sujats and Tatiana Dedenko. What we did here was to engineer groups into the intracellular surface, where so-to-speak circles are. Again we used fluorine and observed the effect of variable ligands on these fluorine markers. Here is a library of ligands that we used, one of those you have probably enjoyed this morning. It's caffeine, which works as an antagonist for A2AAR. The important point that I want to make is that these different agonists and different antagonists have very different chemical structures, but they all give the same spectrum in our NMR experiment. On the left you have the antagonist spectrum, which has two conformations in equilibrium in the agonist spectrum. We have three conformations. Now we can take the A4 form, add an agonist, so we go from the inactive spectrum to the active-like spectrum, and then we can replace the agonist with a more strongly binding antagonist, and we go back to the inactive spectrum. The important thing here is that Y-type A2AAR has essentially zero base selectivity, and when you now look at the peak, which is between 12 and 14 ppm, then it's completely gone in the inactive form. See, this is a yellow peak, P4, showing that indeed we have it in hand. There is no residual activity that we can detect. Now we take a related GPCR with a single amino acid replacement of Cere91 with Alenine. This form is known to have significant base selectivity, and we immediately see that P4 is present in what is supposed to be the active form. So again, we have here a subtle equilibrium between different inactive and different active forms. And of course we want to know what the exchange is between the two. Here I show just as an illustration the spectrum of the active-like form, and you see that rate of exchange between P1 and P2, this is a peak from the active-like form. P2 from the inactive-like form is slow, it's about 20, sorry, it's relatively fast, around 20 per second. Whereas the exchange between P1 from the inactive form and P4 from the active form is very slow, less than one per second. So we have a very high activation barrier between the ensemble of active-like shapes, states, and an ensemble of inactive-like states that is intrinsic to the GPCR before it interacts with partner proteins that are of course important in the course of the follow-up reactions. Now, we have introduced these fluorine labels and that gives us only a few points to observe. Now we have succeeded in expressing this particular GPCR in Pisa pastoris, four years of work for two people. And at this point now we are able to first to show that it has a full activity of the material that was expressed with the traditional method from the Lefkowitz Laboratory with SF9, and we can now produce a milligram quantities of these receptors and many large number of variant forms of the receptor. And in Pisa in the yeast we can now label the proteins with nitrogen 15 isotope and with deuterium. And as a result, using a technique that we introduced about 20 years ago, drosy, transverse relaxation, optimized spectroscopy, we can now get the spectrum with hundreds of resolved lines. And we can use this in two ways, we can use the whole spectrum to check that the global structure of the molecule is always preserved when we generate variants. And then we have well resolved lines in the two squares that are outlined. The one hand, these are the tryptophan, indole, NH groups, on the other hand the backbone, glycine, AMH groups, and then we replace one and one each tryptophan with tyrosine, and you can see, you best see it on the right side with the cross sections. The peak that is marked by an asterisk disappears after replacing tryptophan with tyrosine, and so we have one by one assigned the peaks, we did the same for eight glycines, and now we have observation points across the entire molecule, from the intracellular surface through the center of the molecule to the extracellular surface. And I want to point out two residues, one is D52, the spastic acid 52, which is known to be the allosteric clock in the molecule. So we will destroy this allosteric center and check what happens. And then we have tryptophan 246, which has been referred to as Goggleswitch, tryptophan and has been found, has been reported to be of particular importance during the activation process of the receptor. So what comes out, when we look at the response to variable drug efficacy, then we get no response out to the extracellular surface, and we get a structural response all over the receptor, all the way from the ligand binding side to the intracellular surface. Here are just a few examples of how clear-cut these responses are when we vary the efficacy of the ligand in the allosteric side. And here, the biggest effect we see indeed for this togle-switch tryptophan, where we get the chemical shift difference, again identified by the stars on the right of almost one part per million, and we know what happens there. I think I forgot, we know exactly what happens there with the arrangement of the aromatic grains in the center of the receptor. But now, it's very last, my time is up, I take an extra minute to describe to you what we did with regard to the studies on the structural basis of the allosteric signaling from the extracellular to the intracellular surface. On the extreme left, you have the functional GPCR, where you bind the ligand to get G-protein signaling. Then we replace the center of the allosteric switch, which is a negatively charged residue with its neutral analog, and we get no signaling through. So the question is, why do we not get signaling through? And this question, to answer this question, is important because 86% of all class HGPCRs contain aspartic acid in this position. So when we make this replacement, we again get a huge change in the dogle-switch resonance, and just look at the left side, what happens is that when we destroy the allosteric switch, we get no signaling down to the intracellular surface. All the signaling is pushed back, and structural changes are seen only above W246 and also way up into the extracellular surface. So what we have shown is that the signaling through the allosteric switch is killed when we replace the negatively charged residue, and the signal is actually pushed back to the outside of the cell, and all the dynamics of the residues on the surface, on the intracellular surface is stopped. And we see only a single rigid surface structure, whereas in the active side, we have dynamic equilibria between all the sides that we can observe. So in summary, we have been able to show that activity of GPCRs cannot be understood simply by looking at limiting structures of the active or the inactive forms. We have local conformational polymorphism, which make it a highly dynamic system that can adopt to the needs in any given situation. We can show that ligand efficacy is related not to rigid structural changes, but to shifts in conformational equilibria. We are able, and now this is in contrast to the results obtained from CRIOEM, which also shows that there are multiple different conformations, but we can measure the exchange between these conformations. We don't just sample views of different structures. And of course, continuing on, we can get information on all of these properties from interactions with extracellular ligands and with intracellular reaction partners. Thank you.
especially in drug discovery and drug design. So orthostatic binding site and that's where drugs resolution by NMR in solution. Now you realize that our proteins are active in contact with obtain with this approach can be very close to results that we would see on the physiological conditions. Now NMR stands for nuclear magnetic resonance. It could stand for no more research. But it is nuclear magnetic resonance. I think at started this work. The structural biology became hand for the determination of the and that is by X-ray crystallography. Now of this last time in this lecture, how important it is to integrate results from different techniques crystal structure that was determined by Tom as hypothesized on the basis of the crystal receptor which is a GPCR. So this gives about two other receptors and one of these is a receptor without the ligand bound or with an activity which in this scheme is 50 biological transmission to both signaling pathways. When we interactions, for example with G protein, with affected by the fusion of this soluble protein preparation that was used for crystallography. I importance of integrative use of multiple that I want to discuss with you. And this is A2AAR, adenosine receptor. And this is new work. used, one of those you have probably enjoyed this have it in hand. There is no residual activity with a single amino acid replacement of Cere91 expressed with the traditional method from the allosteric clock in the molecule. So we will particular importance during the activation my time is up, I take an extra minute to describe from the extracellular to the intracellular been able to show that activity of GPCRs cannot this is in contrast to the results obtained from