Structural Biology (Grzesiek)Head of Research Unit Prof. Dr.Stephan GrzesiekOverviewMembersPublicationsProjects & CollaborationsProjects & Collaborations OverviewMembersPublicationsProjects & Collaborations Projects & Collaborations 13 foundShow per page10 10 20 50 Structure, dynamics and function of biological macromolecules by NMR Research Project | 2 Project MembersNo Description available Structure, dynamics and function of CCR5-arrestin interactions Research Project | 3 Project MembersThe human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in general inflammatory processes by recruiting and activating leukocytes. CCR5 is also the principal HIV coreceptor, is involved in the pathology of both cancer and neuroinflammation, and has been im plicated in the inflammatory complications of COVID-19. Binding of ligands to GPCRs results in the activation of G protein-, arrestin-mediated and other signaling pathways. The Grzesiek lab has recently solved the cryo-EM structure of an agonist chemokine CCL5-CCR5-G protein complex, which delineates the G protein activation pathway triggered by chemokine agonists within CCR5. While meanwhile a number GPCR-G protein complexes have been solved, much less is known on the structural basis of GPCR arrestin signaling. In particular, no structure of a chemokine receptor-arrestin complex exists. The Shukla is one of the world leading labs in the structural and functional analysis of arrestins. We propose here to combine the expertises and capabilities of these two groups to (A) solve the structure of a CCR5-ß arrestin 1 complex, (B) investigate the dynamics of interactions between CCR5-derived peptides and arrestin by NMR and other biophysical techniques, as well as (C) characterize the CCR5-ß arrestin 1 interactions by cell-based assays and develop new synthetic antibody fragments to stabilize the CCR5-ß arrestin 1 complex. The structural and functional insights from these combined experiments should decipher key elements of chemokine-induced CCR5-arrestin signaling and reveal differences to the G protein signaling pathway. This may pave the way for the development of directed therapeutics targeting either pathway. Due to the homology of CCR5 to a number of other chemokine receptors, the results obtained may serve as a paradigm for other chemokine receptor/ligand systems. Structures, dynamics and interactions of disease-relevant proteins and new developments in NMR technology Research Project | 6 Project MembersBiological function results from time-dependent interactions between biomolecules. NMR spectroscopy is the only experimental method, which yields both structural and dynamical information on biomolecules at atomic resolution with minimal invasiveness and at close to natural conditions. However, it is limited in the molecular size and sensitivity. In the last years we have combined NMR, cryo-EM, single-molecule FRET and other biophysical techniques to derive mechanistic insights at the atomic level into the function and interactions of a number of disease-relevant protein systems. In the next years we want to further develop and conclude several of these projects, which address (1) G protein-coupled receptors (GPCRs), (2) Abelson kinase, (3) c-di-GMP binding proteins, and (4) NMR technology. Subproject (1) addresses the further dynamical characterization by NMR and other techniques of the function of the beta1-adrenergic receptor (b1AR), which is one of the main targets of beta-blockers, as well as the characterization of functional complexes by cryo-EM and NMR of the human chemokine receptor CCR5, which is also the coreceptor for HIV. Subproject (2) addresses the further dynamical characterization by single-molecule FRET and NMR of Abelson kinase (Abl), which is a large multi-domain protein and important leukemia drug target. We have now established labeling with fluorophore pairs at arbitrary sites by using unnatural amino acid technology. We want to use this development to study the dynamical behavior of single Abl molecules within (cancer) cells by FRET and combine the obtained information with high-resolution NMR. Subproject (3) addresses the interactions of the bacterial virulence factor cyclic di-guanosine-monophosphate (c-di-GMP). We have determined the NMR structure and dynamics of several complexes of c-di-GMP binding proteins and want to continue to develop a genetically encodable c-di-GMP fluorescence sensor based on our structural data. Subproject (4) foresees further developments in NMR technology addressing the rapid assignment of large proteins and the improvement of isotope labeling techniques in higher eukaryotes. Functional dry cavities in proteins Research Project | 1 Project MembersThe interior of experimentally determined protein structures often contains cavities, which are not filled by protein atoms. Such cavities are generally thought to be filled by water. However, in few proteins completely empty voids, devoid of any water molecules have been detected by X-ray crystallography or NMR spectroscopy. The most recent example is a 141 Å3-sized, dry pocket in thermolysin, which was unambiguously detected by high-resolution X-ray crystallography using an absolute-scale electron density map (Krimmer et al. JACS 2017, 139, 30, 10419-10431). This hydrophobic cavity could then be filled by noble gases as also detected by X-ray crystallography. These observations are limited to a few model systems, and it is unclear how abundant dry voids are in proteins, and whether such voids are connected to protein function and structural stability.Such a functional role may exist in the medically highly relevant G protein-coupled receptors (GPCRs), which constitute the most abundant class of membrane proteins in the human genome. Indeed, a recent study on the ß1-adrenergic receptor (ß1AR, Warne et al. Science 2019, 364, 775-778) showed that its extracellular ligand binding pocket shrinks by 20-40% upon activation by an intracellular G protein mimic. This compression of the ligand pocket explains the affinity increase of G protein-coupled receptors (GPCRs) for agonist ligands in their G protein-bound state. From the 2.8 Å-resolution crystal structure, it remained unclear to what extent the extracellular ligand binding cavity is filled by water. However, a recent pressure NMR study (Abiko et al. JACS 2019, 141, 42, 16663-16670) showed quantitatively that the same receptor experiences a volume reduction of ~100 Å3 upon transition to the active state. This shrinking can only be explained by the collapse of empty, non-hydrated cavities, but the location of these empty cavities remained unknown from the NMR study.We hypothesize that the external ligand pocket of ß1AR is at least partially empty and that this void plays a crucial role in GPCR activation. We propose to determine the location of these empty cavities by X-ray crystallography. In contrast to previous studies, we will incubate the crystals of ß1AR in different functional states at mild pressures with xenon. If empty hydrophobic cavities are present, they should incorporate xenon, which is then easily located in the electron density by its high scattering power.These experiments may reveal the first dry pocket in a membrane protein and connect its existence to a crucial function in GPCRs. More generally, such a proof of existence may establish dry voids as a new interaction principle in proteins. Thus, dry pockets may serve as structural motifs that confine protein movements during folding and function or which specifically recognize distinct hydrophobic moieties of a ligand. This may have major implications on protein folding research, the structure-based explanation of protein function, and the development of new drugs. Biomacromolecular structures, dynamics and interactions by NMR and new developments in NMR technology Research Project | 1 Project MembersBiological function results from time-dependent interactions between biomolecules. NMR spectroscopy is the only experimental method, which yields both structural and dynamical information on biomolecules at atomic resolution with minimal invasiveness and at close to natural conditions. As such it can provide unique information to understand the connection between structure, dynamics and function. It is the goal of this proposal to apply and further develop these strengths of NMR technology in two subprojects. Subproject A is directed towards the determination of structure, dynamics, and interactions in several medically important systems, for which we have made significant progress in recent years: (1) two G-protein coupled receptors (GPCRs), i.e. the beta1-adrenergic receptor (b1AR) and the chemokine receptor CCR5, which is also the coreceptor for HIV. For b1AR, we could show that it is possible to follow signal transduction by 1 H- 15 N backbone resonances. We now want to carry out a full NMR dynamics characterization comprising the binding of drug, G protein and arrestin ligands in different membrane environments. Beside solution NMR, this will also entail the study in full lipid bilayers by solid state NMR. Thus we hope to establish b1AR as a reference system for studies of GPCRs by NMR. We have recently obtained solution spectra for CCR5 of comparable quality to b1AR. We could also detect many resonances of the engineered chemokine ligand 5P12-RANTES in complex with CCR5 by solid state NMR. 5P12-RANTES is currently in clinical trials as an HIV entry inhibitor. We want to carry out a solution and solid state NMR analysis of this receptor with 5P12-RANTES and engineered RANTES ligands that elicit different arrestin and G-protein signaling activities with the aim to provide the structural and dynamical explanation for this differing signaling behavior. (2) Interactions of the bacterial virulence factor cyclic di-guanosine-monophosphate (c-di-GMP). We have solved the structures of several c-di-GMP protein complexes and c-di-GMP oligomers as well as determined the kinetics of c-di-GMP oligomer formation in solution. In particular, we could recently solve the structure of the c-di-GMP recognition region of the chemotactic protein CleD in complex with c-di-GMP. C-di-GMP binding to CleD induces formation of a ternary complex with the flagellar motor switch protein FliM thereby controlling bacterial flagellar motor function. We now want to solve the structure of the full c-di-GMP-CleD complex and study the formation of the ternary c-di-GMP-CleD-FliM complex. (3) Abl kinase, which is an important leukemia drug target. We have shown that binding of different classes of inhibitors to a large, multidomain Abl construct induces distinct domain rearrangements, which shed light on the mechanism of kinase regulation. We have now assessed the influence of a number of medically relevant point mutations and studied a larger class of inhibitors. In particular, the opening of the multidomain structure is correlated to the conformation of the activation loop in the catalytic domain. We now want to obtain an in-depth dynamic description of the catalytic domain, which is possible due to an advance in labeling techniques, as well as continue to develop suitable labeling of Abl for single molecule FRET studies. Subproject B is directed towards NMR technique development. Often proteins from higher eukaryotes cannot be expressed in functional form in E. coli , but only in higher eukaryotic expression systems. The difficulties of isotope labeling in such systems presents a bottleneck for the analysis by modern heteronuclear NMR. We have recently developed a robust method for isotope labeling in insect cells by feeding isotope-labeled yeast extract. The method currently yields 90 % 15 N and 13 C as well as >60 % 2 H labeling with very good yields. We want to improve this method by increasing the level of 2 H incorporation and cost efficiency for 13 C labeling as well as extend it to other eukaryotic systems. We also want to obtain an in-depth understanding of the often adverse effects of 2 H incorporation to cellular behavior by a proteomics analysis of the response to growth on 2 H 2 O for E. coli , yeast and higher eukaryotes. Elucidating allosteric signal transmission in the beta1-adrenergic receptor Research Project | 3 Project MembersG protein coupled receptors (GPCRs) are an important class of trans-membrane proteins that recognize a multitude of extracellular molecules and transmit their signal to the intracellular side. Despite recent achievements in X-ray crystallography of GPCRs, the high-resolution structures obtained do not capture their intrinsic dynamic properties, which are tightly associated with their function. NMR spectroscopy promises to provide such missing dynamic information. However so far, despite being valuable, only limited information has been obtained by NMR due to the difficult spectroscopic properties of this protein class. At this point, the potential of solution NMR analysis of GPCRs has not been fully realized. Recently, I have overcome many of the obstacles that hinder the application of solution NMR to study signal transduction in GPCRs. Using a stabilized form of the β 1 -adrenergic receptor and a selective isotope labeling method in the baculovirus-insect cell expression system, I was able to acquire well-resolved backbone amide proton-nitrogen correlation spectra. These spectra revealed numerous mechanisms within the receptor that are new, or had been postulated but never observed directly. Thus I have established a system that can now be used to study many more functional mechanisms of GPCRs at atomic resolution. In addition, we have developed an economic method to produce uniformly isotope-labeled (including deuteration) GPCRs in the insect cell systems. This will allow more advanced applications of NMR spectroscopy to GPCRs such as the study of their dynamics by relaxation measurements. In the present proposal I want to use this system to obtain detailed insights into the receptor's signal transmission mechanism with the aim to understand how the receptor recognizes ligands and passes this information to the G protein in order to modulate its activity. Using state-of-the-art methods of isothermal titration calorimetry, protein rigidity theory, coevolutionary sequence alignment, in vitro real-time observation of GDP/GTP exchange by fluorescence as well as solution NMR, I want to elucidate the underlying molecular mechanism of the thermodynamic behavior of ligand-receptor interactions, determine a high-resolution allosteric network model of signal transmission, and provide mechanical insights into how different agonists elicit varying levels of G protein activation. If successful, the results will have implications for the general understanding of GPCR function and the developed methods should be applicable to other GPCRs. Structural and dynamical basis of allosteric regulation and inhibition of abelson tyrosine kinase a drug target in the treatment of chronic mylogeneous leukaemia Research Project | 2 Project MembersAbelson tyrosine kinase (Abl) in its healthy state is a tightly regulated, human protein involved in many cellular processes. An abnormal rearrangement of chromosomes leads to the formation of the aberrant fusion protein Bcr-Abl. Bcr-Abl is highly active and causes uncontrolled production of immature blood cells, which ultimately results in the blood cancers chronic myelogenous leukemia (CML) or acute lymphoblastic leukemia (ALL). Drugs like imatinib (gleevec), nilotinib (tasigna), and dasatinib (sprycel) have been developed to bind to a specific location on Bcr-Abl (ATP-binding pocket) thereby blocking its abnormal activity. They are highly successful in the clinic. However, after prolonged treatment a fraction of the cancers become resistant to these drugs by spontaneous mutations in Bcr-Abl, making the treatment ineffective. Recently, a new class of inhibitors has been discovered that bind to a different location on Bcr-Abl. These so-called allosteric inhibitors have promise to overcome drug resistance in combination with conventional ATP-site inhibitors. The mode of action of this combination is unclear. Bcr-Abl consists of many subdomains and their relative movement and interplay is thought to be responsible for its regulation. We have recently been able to detect such movements at atomic resolution by Nuclear Magnetic Resonance (NMR) methods. We now want to build on these initial studies and determine Abl's molecular movements and interactions in response to various inhibitors and disease- or functionally relevant mutations by a combination of NMR, single molecule fluorescence resonance energy transfer (FRET) and other biophysical techniques. The results should provide an atomic-level understanding of the mechanism of Abl regulation and a rationale for the improvement of existing and the development of new therapeutic strategies for Bcr-Abl inhibition. Purchase of a 900 MHz High-Resolution NMR Instrument Research Project | 5 Project MembersBackground - Biological function results from time-dependent interactions between biomolecules. It is ultimately encoded in the primary chemical structures of molecules, which determine the positions and movements of their atoms in space. NMR spectroscopy is the only experimental method, which yields both structural and dynamical information on biomolecules at atomic resolution with minimal invasiveness and at close to natural conditions. As such, it can provide unique information to understand the connection between chemical structure, three-dimensional structure, dynamics and ultimately function. The application of NMR spectroscopy to large interesting biomolecular systems is limited by sensitivity and resolution, both of which increase strongly with magnetic field strength. The planned new building for the Biozentrum of the University Basel provides the unique opportunity to upgrade the present 800 MHz NMR instrument to a state-of-the-art 900 MHz instrument. This will substantially improve the scientific capacities of the Biozentrum's high-field NMR center. The new installation will benefit from the strong technical and biological expertise of the principal applicants and the high demand for NMR characterization of exciting biological projects inside and outside of the University of Basel. Research proposal - Expertise, maintenance, running, building and a large part of the total purchase costs will be provided by the Biozentrum and the University of Basel. The core users will use the instrument to A) study structures and interactions of disease-relevant biomolecules and to further develop high-resolution NMR methods (Grzesiek, principal applicant); B) characterize structure, function and folding mechanisms of large biomacromolecules and their complexes with a focus on integral membrane proteins (Hiller); C) investigate proteins, interaction and functional mechanisms in cyclic-di-GMP signaling (Schirmer, Jenal); D) study large protein complexes (Maier, Hiller). Further research projects comprise 1) interactions of the uropathogenic E. coli protein FimH with glycoproteins from the urothelial cell surface (Ernst); 2) interactions of bioactive natural products with target proteins (Hamburger); 3) the catalytic activity of artificial metalloenzymes (Ward, Häussinger); 4) dynamics and interaction of G-protein coupled receptors (Schertler); 5) interactions and dynamics of histone-binding proteins and their role in heterochromatin silencing (Bühler); 6) alterations in cellular metabolism of eukaryotic cells upon infection with pathogens (Bumann). Expected value - Structures and motions of biomolecules need to be determined at atomic resolution to understand their mechanisms from first principles. This information can be provided by NMR analysis. This is a fundamental prerequisite to unravel the connection between biomolecular structure, dynamics and function, as well as for the rational intervention into biological processes such as drug design. Justification of the needed equipment - The new instrument will provide significant increases in sensitivity and resolution for the NMR analysis of structures and motions of biomolecules. The reduced required concentrations and the larger achievable molecular sizes will further extend its applicability to highly challenging biomolecular systems. The instrument will satisfy the high demand for such NMR characterizations within the University of Basel and from outside collaborations. The foreseen research projects comprise several medically relevant G-protein coupled receptors (GPCRs), cancer drug targets, pathogenic bacterial systems, large molecular machines such as membrane protein assembly and polypeptide translocation complexes, as well as general questions of protein folding. The new instrument will ensure that this research can be carried out at an internationally competitive level. Biomacromolecular structures, dynamics and interactions by NMR and new developments in NMR technology Research Project | 1 Project MembersBiological function results from time-dependent interactions between biomolecules. It is ultimately encoded within the primary chemical structure of molecules resulting in defined three-dimensional atom positions and movements. NMR spectroscopy is the only experimental method, which yields both structural and dynamical information on biomolecules at atomic resolution with minimal invasiveness and at close to natural conditions. As such it can provide unique information to understand the connection between primary structure, tertiary structure, dynamics and function. It is the goal of this proposal to apply and further develop these strengths of NMR technology with the aim to reveal general principles of protein structure function relations. The proposal is divided into two subprojects: Subproject A is directed towards the determination of structure, dynamics, and interactions in four medically important systems, for which we have made significant progress in recent years: (1) Abl kinase, which is an important leukemia drug target. We have obtained assignment of a large, multidomain Abl construct that is the minimal autoregulatory fragment. Our data show that binding of different classes of inhibitors induces distinct domain rearrangements, which shed light on the mechanism of kinase regulation. We now want to reveal the atomic causes of these allosteric rearrangements, investigate several medically and functionally relevant mutants, and extend the studies to single molecule FRET. (2) the HIV-1 coreceptor and G-protein coupled receptor (GPCR) CCR5. We have obtained large-scale functional expression of CCR5 in insect cells and E. coli, which permits effective isotope labeling. We seek to apply new solid-state and solution NMR techniques for structure determination and to develop general strategies for GPCR analysis by a comparison to other available GPCRs. (3) Interactions of the bacterial virulence factor cyclic di-guanosine-monophosphate (c-di-GMP). We have determined the structure of the PilZ homolog PA4608 in complex with c-di-GMP and the kinetics of c-di-GMP oligomer formation in solution. We now want to solve the structures of a complex c-di-GMP with the chemotactic protein CC3300 and of higher c-di-GMP oligomers. (4) Lipopolysaccharide (LPS), the causative agent of endotoxic shock. We have developed a method to study LPS by solution NMR and obtained expression of the lipopolysaccharide binding protein, which is the first receptor of the endotoxic recognition cascade. We intend to study its interaction with LPS. Subproject B is directed towards the NMR characterization of unfolded states of proteins and their relation to the folded structure with an emphasis on pressure denaturation and structural modeling of unfolded ensembles. The overarching goal is to rationalize protein folding by correlating high resolution experimental data on unfolded states with primary, secondary and tertiary structure information. (1) We have obtained unique data on the pressure/cold-denatured state of ubiquitin, which shows similar subpopulations of partial native structures as an alcohol-denatured state. By addition of non-denaturing concentrations of alcohol, full pressure-induced unfolding can be achieved at room temperature in a completely reversible way. This allows following the unfolding transition at very high resolution. We want to extend this analysis to a larger set of about 10 proteins representing different folds and sequences with the aim to correlate unfolding behavior with sequence or structure and to understand the nature of pressure unfolding. (2) We have developed an effective method to calculate structural ensembles of unfolded proteins from large sets of NMR data. We want to continue these efforts with the aim to include FRET and chemical shift data in order to obtain highly defined quantitative models for larger sets of unfolded proteins. NMR studies of GPCRs: Structure, dynamics and interactions with ligands and signaling proteins Research Project | 1 Project MembersG protein-coupled receptors (GPCRs) are integral membrane proteins that transmit extracellular neural, endocrine, olfactory and visual signals across the plasma membrane. When activated by light or a ligand GPCRs undergo a conformational change leading to activation of a G protein, arrestin and other signaling pathways. Over 30% of compounds used in medicine today modulate the activity of GPCRs. Some GPCRs can bind a range of ligands, some of which, instead of fully activating the receptor, rather bias the signal transduction cascade toward distinct intracellular pathways. The currently available X-ray structures of several GPCRs and of a 2-adrenoreceptor/Gs signaling complex provide a frozen snapshot of a signal transduction event, but do not fully explain how the ligand selectivity is achieved, and how the ligand binding results in preferential binding of a specific signaling protein such as Gi, Gs or arrestin. Understanding the precise structural and dynamic nature of these phenomena is critical to knowing the mechanism of GPCR activation and will help further development of pharmaceuticals with desired pharmacological properties. We propose here to provide the missing dynamical information on GPCR function by Nuclear magnetic resonance spectroscopy (NMR), which shall determine the conformational changes and dynamics of the receptors and signaling complexes in solution. This should then a yield a comprehensive view of GPCR activation of and signal transduction. We will initially focus on rhodopsin and 1-adrenoreceptor ( 1AR), as well as their signaling complexes with G proteins and arrestin, and later apply developed expertise to the vasopressin receptor V2R and its signaling complexes. In particular, we propose: a) to label GPCRs for NMR analysis; b) to obtain a dynamic view of the conformational changes in GPCRs induced by a range of ligands with different activities including agonists, antagonists and biased ligands; c) to investigate the effect of G proteins and arrestin binding on the receptors; d) to identify the molecular basis for signaling selectivity via a comparative NMR and biophysical analysis of receptor binding to the various ligands, G proteins and arrestin. Due to the high sequence conservation among G protein-coupled receptor proteins our results are expected to have wide implications for signal transduction by GPCRs in general. 12 12 OverviewMembersPublicationsProjects & Collaborations
Projects & Collaborations 13 foundShow per page10 10 20 50 Structure, dynamics and function of biological macromolecules by NMR Research Project | 2 Project MembersNo Description available Structure, dynamics and function of CCR5-arrestin interactions Research Project | 3 Project MembersThe human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in general inflammatory processes by recruiting and activating leukocytes. CCR5 is also the principal HIV coreceptor, is involved in the pathology of both cancer and neuroinflammation, and has been im plicated in the inflammatory complications of COVID-19. Binding of ligands to GPCRs results in the activation of G protein-, arrestin-mediated and other signaling pathways. The Grzesiek lab has recently solved the cryo-EM structure of an agonist chemokine CCL5-CCR5-G protein complex, which delineates the G protein activation pathway triggered by chemokine agonists within CCR5. While meanwhile a number GPCR-G protein complexes have been solved, much less is known on the structural basis of GPCR arrestin signaling. In particular, no structure of a chemokine receptor-arrestin complex exists. The Shukla is one of the world leading labs in the structural and functional analysis of arrestins. We propose here to combine the expertises and capabilities of these two groups to (A) solve the structure of a CCR5-ß arrestin 1 complex, (B) investigate the dynamics of interactions between CCR5-derived peptides and arrestin by NMR and other biophysical techniques, as well as (C) characterize the CCR5-ß arrestin 1 interactions by cell-based assays and develop new synthetic antibody fragments to stabilize the CCR5-ß arrestin 1 complex. The structural and functional insights from these combined experiments should decipher key elements of chemokine-induced CCR5-arrestin signaling and reveal differences to the G protein signaling pathway. This may pave the way for the development of directed therapeutics targeting either pathway. Due to the homology of CCR5 to a number of other chemokine receptors, the results obtained may serve as a paradigm for other chemokine receptor/ligand systems. Structures, dynamics and interactions of disease-relevant proteins and new developments in NMR technology Research Project | 6 Project MembersBiological function results from time-dependent interactions between biomolecules. NMR spectroscopy is the only experimental method, which yields both structural and dynamical information on biomolecules at atomic resolution with minimal invasiveness and at close to natural conditions. However, it is limited in the molecular size and sensitivity. In the last years we have combined NMR, cryo-EM, single-molecule FRET and other biophysical techniques to derive mechanistic insights at the atomic level into the function and interactions of a number of disease-relevant protein systems. In the next years we want to further develop and conclude several of these projects, which address (1) G protein-coupled receptors (GPCRs), (2) Abelson kinase, (3) c-di-GMP binding proteins, and (4) NMR technology. Subproject (1) addresses the further dynamical characterization by NMR and other techniques of the function of the beta1-adrenergic receptor (b1AR), which is one of the main targets of beta-blockers, as well as the characterization of functional complexes by cryo-EM and NMR of the human chemokine receptor CCR5, which is also the coreceptor for HIV. Subproject (2) addresses the further dynamical characterization by single-molecule FRET and NMR of Abelson kinase (Abl), which is a large multi-domain protein and important leukemia drug target. We have now established labeling with fluorophore pairs at arbitrary sites by using unnatural amino acid technology. We want to use this development to study the dynamical behavior of single Abl molecules within (cancer) cells by FRET and combine the obtained information with high-resolution NMR. Subproject (3) addresses the interactions of the bacterial virulence factor cyclic di-guanosine-monophosphate (c-di-GMP). We have determined the NMR structure and dynamics of several complexes of c-di-GMP binding proteins and want to continue to develop a genetically encodable c-di-GMP fluorescence sensor based on our structural data. Subproject (4) foresees further developments in NMR technology addressing the rapid assignment of large proteins and the improvement of isotope labeling techniques in higher eukaryotes. Functional dry cavities in proteins Research Project | 1 Project MembersThe interior of experimentally determined protein structures often contains cavities, which are not filled by protein atoms. Such cavities are generally thought to be filled by water. However, in few proteins completely empty voids, devoid of any water molecules have been detected by X-ray crystallography or NMR spectroscopy. The most recent example is a 141 Å3-sized, dry pocket in thermolysin, which was unambiguously detected by high-resolution X-ray crystallography using an absolute-scale electron density map (Krimmer et al. JACS 2017, 139, 30, 10419-10431). This hydrophobic cavity could then be filled by noble gases as also detected by X-ray crystallography. These observations are limited to a few model systems, and it is unclear how abundant dry voids are in proteins, and whether such voids are connected to protein function and structural stability.Such a functional role may exist in the medically highly relevant G protein-coupled receptors (GPCRs), which constitute the most abundant class of membrane proteins in the human genome. Indeed, a recent study on the ß1-adrenergic receptor (ß1AR, Warne et al. Science 2019, 364, 775-778) showed that its extracellular ligand binding pocket shrinks by 20-40% upon activation by an intracellular G protein mimic. This compression of the ligand pocket explains the affinity increase of G protein-coupled receptors (GPCRs) for agonist ligands in their G protein-bound state. From the 2.8 Å-resolution crystal structure, it remained unclear to what extent the extracellular ligand binding cavity is filled by water. However, a recent pressure NMR study (Abiko et al. JACS 2019, 141, 42, 16663-16670) showed quantitatively that the same receptor experiences a volume reduction of ~100 Å3 upon transition to the active state. This shrinking can only be explained by the collapse of empty, non-hydrated cavities, but the location of these empty cavities remained unknown from the NMR study.We hypothesize that the external ligand pocket of ß1AR is at least partially empty and that this void plays a crucial role in GPCR activation. We propose to determine the location of these empty cavities by X-ray crystallography. In contrast to previous studies, we will incubate the crystals of ß1AR in different functional states at mild pressures with xenon. If empty hydrophobic cavities are present, they should incorporate xenon, which is then easily located in the electron density by its high scattering power.These experiments may reveal the first dry pocket in a membrane protein and connect its existence to a crucial function in GPCRs. More generally, such a proof of existence may establish dry voids as a new interaction principle in proteins. Thus, dry pockets may serve as structural motifs that confine protein movements during folding and function or which specifically recognize distinct hydrophobic moieties of a ligand. This may have major implications on protein folding research, the structure-based explanation of protein function, and the development of new drugs. Biomacromolecular structures, dynamics and interactions by NMR and new developments in NMR technology Research Project | 1 Project MembersBiological function results from time-dependent interactions between biomolecules. NMR spectroscopy is the only experimental method, which yields both structural and dynamical information on biomolecules at atomic resolution with minimal invasiveness and at close to natural conditions. As such it can provide unique information to understand the connection between structure, dynamics and function. It is the goal of this proposal to apply and further develop these strengths of NMR technology in two subprojects. Subproject A is directed towards the determination of structure, dynamics, and interactions in several medically important systems, for which we have made significant progress in recent years: (1) two G-protein coupled receptors (GPCRs), i.e. the beta1-adrenergic receptor (b1AR) and the chemokine receptor CCR5, which is also the coreceptor for HIV. For b1AR, we could show that it is possible to follow signal transduction by 1 H- 15 N backbone resonances. We now want to carry out a full NMR dynamics characterization comprising the binding of drug, G protein and arrestin ligands in different membrane environments. Beside solution NMR, this will also entail the study in full lipid bilayers by solid state NMR. Thus we hope to establish b1AR as a reference system for studies of GPCRs by NMR. We have recently obtained solution spectra for CCR5 of comparable quality to b1AR. We could also detect many resonances of the engineered chemokine ligand 5P12-RANTES in complex with CCR5 by solid state NMR. 5P12-RANTES is currently in clinical trials as an HIV entry inhibitor. We want to carry out a solution and solid state NMR analysis of this receptor with 5P12-RANTES and engineered RANTES ligands that elicit different arrestin and G-protein signaling activities with the aim to provide the structural and dynamical explanation for this differing signaling behavior. (2) Interactions of the bacterial virulence factor cyclic di-guanosine-monophosphate (c-di-GMP). We have solved the structures of several c-di-GMP protein complexes and c-di-GMP oligomers as well as determined the kinetics of c-di-GMP oligomer formation in solution. In particular, we could recently solve the structure of the c-di-GMP recognition region of the chemotactic protein CleD in complex with c-di-GMP. C-di-GMP binding to CleD induces formation of a ternary complex with the flagellar motor switch protein FliM thereby controlling bacterial flagellar motor function. We now want to solve the structure of the full c-di-GMP-CleD complex and study the formation of the ternary c-di-GMP-CleD-FliM complex. (3) Abl kinase, which is an important leukemia drug target. We have shown that binding of different classes of inhibitors to a large, multidomain Abl construct induces distinct domain rearrangements, which shed light on the mechanism of kinase regulation. We have now assessed the influence of a number of medically relevant point mutations and studied a larger class of inhibitors. In particular, the opening of the multidomain structure is correlated to the conformation of the activation loop in the catalytic domain. We now want to obtain an in-depth dynamic description of the catalytic domain, which is possible due to an advance in labeling techniques, as well as continue to develop suitable labeling of Abl for single molecule FRET studies. Subproject B is directed towards NMR technique development. Often proteins from higher eukaryotes cannot be expressed in functional form in E. coli , but only in higher eukaryotic expression systems. The difficulties of isotope labeling in such systems presents a bottleneck for the analysis by modern heteronuclear NMR. We have recently developed a robust method for isotope labeling in insect cells by feeding isotope-labeled yeast extract. The method currently yields 90 % 15 N and 13 C as well as >60 % 2 H labeling with very good yields. We want to improve this method by increasing the level of 2 H incorporation and cost efficiency for 13 C labeling as well as extend it to other eukaryotic systems. We also want to obtain an in-depth understanding of the often adverse effects of 2 H incorporation to cellular behavior by a proteomics analysis of the response to growth on 2 H 2 O for E. coli , yeast and higher eukaryotes. Elucidating allosteric signal transmission in the beta1-adrenergic receptor Research Project | 3 Project MembersG protein coupled receptors (GPCRs) are an important class of trans-membrane proteins that recognize a multitude of extracellular molecules and transmit their signal to the intracellular side. Despite recent achievements in X-ray crystallography of GPCRs, the high-resolution structures obtained do not capture their intrinsic dynamic properties, which are tightly associated with their function. NMR spectroscopy promises to provide such missing dynamic information. However so far, despite being valuable, only limited information has been obtained by NMR due to the difficult spectroscopic properties of this protein class. At this point, the potential of solution NMR analysis of GPCRs has not been fully realized. Recently, I have overcome many of the obstacles that hinder the application of solution NMR to study signal transduction in GPCRs. Using a stabilized form of the β 1 -adrenergic receptor and a selective isotope labeling method in the baculovirus-insect cell expression system, I was able to acquire well-resolved backbone amide proton-nitrogen correlation spectra. These spectra revealed numerous mechanisms within the receptor that are new, or had been postulated but never observed directly. Thus I have established a system that can now be used to study many more functional mechanisms of GPCRs at atomic resolution. In addition, we have developed an economic method to produce uniformly isotope-labeled (including deuteration) GPCRs in the insect cell systems. This will allow more advanced applications of NMR spectroscopy to GPCRs such as the study of their dynamics by relaxation measurements. In the present proposal I want to use this system to obtain detailed insights into the receptor's signal transmission mechanism with the aim to understand how the receptor recognizes ligands and passes this information to the G protein in order to modulate its activity. Using state-of-the-art methods of isothermal titration calorimetry, protein rigidity theory, coevolutionary sequence alignment, in vitro real-time observation of GDP/GTP exchange by fluorescence as well as solution NMR, I want to elucidate the underlying molecular mechanism of the thermodynamic behavior of ligand-receptor interactions, determine a high-resolution allosteric network model of signal transmission, and provide mechanical insights into how different agonists elicit varying levels of G protein activation. If successful, the results will have implications for the general understanding of GPCR function and the developed methods should be applicable to other GPCRs. Structural and dynamical basis of allosteric regulation and inhibition of abelson tyrosine kinase a drug target in the treatment of chronic mylogeneous leukaemia Research Project | 2 Project MembersAbelson tyrosine kinase (Abl) in its healthy state is a tightly regulated, human protein involved in many cellular processes. An abnormal rearrangement of chromosomes leads to the formation of the aberrant fusion protein Bcr-Abl. Bcr-Abl is highly active and causes uncontrolled production of immature blood cells, which ultimately results in the blood cancers chronic myelogenous leukemia (CML) or acute lymphoblastic leukemia (ALL). Drugs like imatinib (gleevec), nilotinib (tasigna), and dasatinib (sprycel) have been developed to bind to a specific location on Bcr-Abl (ATP-binding pocket) thereby blocking its abnormal activity. They are highly successful in the clinic. However, after prolonged treatment a fraction of the cancers become resistant to these drugs by spontaneous mutations in Bcr-Abl, making the treatment ineffective. Recently, a new class of inhibitors has been discovered that bind to a different location on Bcr-Abl. These so-called allosteric inhibitors have promise to overcome drug resistance in combination with conventional ATP-site inhibitors. The mode of action of this combination is unclear. Bcr-Abl consists of many subdomains and their relative movement and interplay is thought to be responsible for its regulation. We have recently been able to detect such movements at atomic resolution by Nuclear Magnetic Resonance (NMR) methods. We now want to build on these initial studies and determine Abl's molecular movements and interactions in response to various inhibitors and disease- or functionally relevant mutations by a combination of NMR, single molecule fluorescence resonance energy transfer (FRET) and other biophysical techniques. The results should provide an atomic-level understanding of the mechanism of Abl regulation and a rationale for the improvement of existing and the development of new therapeutic strategies for Bcr-Abl inhibition. Purchase of a 900 MHz High-Resolution NMR Instrument Research Project | 5 Project MembersBackground - Biological function results from time-dependent interactions between biomolecules. It is ultimately encoded in the primary chemical structures of molecules, which determine the positions and movements of their atoms in space. NMR spectroscopy is the only experimental method, which yields both structural and dynamical information on biomolecules at atomic resolution with minimal invasiveness and at close to natural conditions. As such, it can provide unique information to understand the connection between chemical structure, three-dimensional structure, dynamics and ultimately function. The application of NMR spectroscopy to large interesting biomolecular systems is limited by sensitivity and resolution, both of which increase strongly with magnetic field strength. The planned new building for the Biozentrum of the University Basel provides the unique opportunity to upgrade the present 800 MHz NMR instrument to a state-of-the-art 900 MHz instrument. This will substantially improve the scientific capacities of the Biozentrum's high-field NMR center. The new installation will benefit from the strong technical and biological expertise of the principal applicants and the high demand for NMR characterization of exciting biological projects inside and outside of the University of Basel. Research proposal - Expertise, maintenance, running, building and a large part of the total purchase costs will be provided by the Biozentrum and the University of Basel. The core users will use the instrument to A) study structures and interactions of disease-relevant biomolecules and to further develop high-resolution NMR methods (Grzesiek, principal applicant); B) characterize structure, function and folding mechanisms of large biomacromolecules and their complexes with a focus on integral membrane proteins (Hiller); C) investigate proteins, interaction and functional mechanisms in cyclic-di-GMP signaling (Schirmer, Jenal); D) study large protein complexes (Maier, Hiller). Further research projects comprise 1) interactions of the uropathogenic E. coli protein FimH with glycoproteins from the urothelial cell surface (Ernst); 2) interactions of bioactive natural products with target proteins (Hamburger); 3) the catalytic activity of artificial metalloenzymes (Ward, Häussinger); 4) dynamics and interaction of G-protein coupled receptors (Schertler); 5) interactions and dynamics of histone-binding proteins and their role in heterochromatin silencing (Bühler); 6) alterations in cellular metabolism of eukaryotic cells upon infection with pathogens (Bumann). Expected value - Structures and motions of biomolecules need to be determined at atomic resolution to understand their mechanisms from first principles. This information can be provided by NMR analysis. This is a fundamental prerequisite to unravel the connection between biomolecular structure, dynamics and function, as well as for the rational intervention into biological processes such as drug design. Justification of the needed equipment - The new instrument will provide significant increases in sensitivity and resolution for the NMR analysis of structures and motions of biomolecules. The reduced required concentrations and the larger achievable molecular sizes will further extend its applicability to highly challenging biomolecular systems. The instrument will satisfy the high demand for such NMR characterizations within the University of Basel and from outside collaborations. The foreseen research projects comprise several medically relevant G-protein coupled receptors (GPCRs), cancer drug targets, pathogenic bacterial systems, large molecular machines such as membrane protein assembly and polypeptide translocation complexes, as well as general questions of protein folding. The new instrument will ensure that this research can be carried out at an internationally competitive level. Biomacromolecular structures, dynamics and interactions by NMR and new developments in NMR technology Research Project | 1 Project MembersBiological function results from time-dependent interactions between biomolecules. It is ultimately encoded within the primary chemical structure of molecules resulting in defined three-dimensional atom positions and movements. NMR spectroscopy is the only experimental method, which yields both structural and dynamical information on biomolecules at atomic resolution with minimal invasiveness and at close to natural conditions. As such it can provide unique information to understand the connection between primary structure, tertiary structure, dynamics and function. It is the goal of this proposal to apply and further develop these strengths of NMR technology with the aim to reveal general principles of protein structure function relations. The proposal is divided into two subprojects: Subproject A is directed towards the determination of structure, dynamics, and interactions in four medically important systems, for which we have made significant progress in recent years: (1) Abl kinase, which is an important leukemia drug target. We have obtained assignment of a large, multidomain Abl construct that is the minimal autoregulatory fragment. Our data show that binding of different classes of inhibitors induces distinct domain rearrangements, which shed light on the mechanism of kinase regulation. We now want to reveal the atomic causes of these allosteric rearrangements, investigate several medically and functionally relevant mutants, and extend the studies to single molecule FRET. (2) the HIV-1 coreceptor and G-protein coupled receptor (GPCR) CCR5. We have obtained large-scale functional expression of CCR5 in insect cells and E. coli, which permits effective isotope labeling. We seek to apply new solid-state and solution NMR techniques for structure determination and to develop general strategies for GPCR analysis by a comparison to other available GPCRs. (3) Interactions of the bacterial virulence factor cyclic di-guanosine-monophosphate (c-di-GMP). We have determined the structure of the PilZ homolog PA4608 in complex with c-di-GMP and the kinetics of c-di-GMP oligomer formation in solution. We now want to solve the structures of a complex c-di-GMP with the chemotactic protein CC3300 and of higher c-di-GMP oligomers. (4) Lipopolysaccharide (LPS), the causative agent of endotoxic shock. We have developed a method to study LPS by solution NMR and obtained expression of the lipopolysaccharide binding protein, which is the first receptor of the endotoxic recognition cascade. We intend to study its interaction with LPS. Subproject B is directed towards the NMR characterization of unfolded states of proteins and their relation to the folded structure with an emphasis on pressure denaturation and structural modeling of unfolded ensembles. The overarching goal is to rationalize protein folding by correlating high resolution experimental data on unfolded states with primary, secondary and tertiary structure information. (1) We have obtained unique data on the pressure/cold-denatured state of ubiquitin, which shows similar subpopulations of partial native structures as an alcohol-denatured state. By addition of non-denaturing concentrations of alcohol, full pressure-induced unfolding can be achieved at room temperature in a completely reversible way. This allows following the unfolding transition at very high resolution. We want to extend this analysis to a larger set of about 10 proteins representing different folds and sequences with the aim to correlate unfolding behavior with sequence or structure and to understand the nature of pressure unfolding. (2) We have developed an effective method to calculate structural ensembles of unfolded proteins from large sets of NMR data. We want to continue these efforts with the aim to include FRET and chemical shift data in order to obtain highly defined quantitative models for larger sets of unfolded proteins. NMR studies of GPCRs: Structure, dynamics and interactions with ligands and signaling proteins Research Project | 1 Project MembersG protein-coupled receptors (GPCRs) are integral membrane proteins that transmit extracellular neural, endocrine, olfactory and visual signals across the plasma membrane. When activated by light or a ligand GPCRs undergo a conformational change leading to activation of a G protein, arrestin and other signaling pathways. Over 30% of compounds used in medicine today modulate the activity of GPCRs. Some GPCRs can bind a range of ligands, some of which, instead of fully activating the receptor, rather bias the signal transduction cascade toward distinct intracellular pathways. The currently available X-ray structures of several GPCRs and of a 2-adrenoreceptor/Gs signaling complex provide a frozen snapshot of a signal transduction event, but do not fully explain how the ligand selectivity is achieved, and how the ligand binding results in preferential binding of a specific signaling protein such as Gi, Gs or arrestin. Understanding the precise structural and dynamic nature of these phenomena is critical to knowing the mechanism of GPCR activation and will help further development of pharmaceuticals with desired pharmacological properties. We propose here to provide the missing dynamical information on GPCR function by Nuclear magnetic resonance spectroscopy (NMR), which shall determine the conformational changes and dynamics of the receptors and signaling complexes in solution. This should then a yield a comprehensive view of GPCR activation of and signal transduction. We will initially focus on rhodopsin and 1-adrenoreceptor ( 1AR), as well as their signaling complexes with G proteins and arrestin, and later apply developed expertise to the vasopressin receptor V2R and its signaling complexes. In particular, we propose: a) to label GPCRs for NMR analysis; b) to obtain a dynamic view of the conformational changes in GPCRs induced by a range of ligands with different activities including agonists, antagonists and biased ligands; c) to investigate the effect of G proteins and arrestin binding on the receptors; d) to identify the molecular basis for signaling selectivity via a comparative NMR and biophysical analysis of receptor binding to the various ligands, G proteins and arrestin. Due to the high sequence conservation among G protein-coupled receptor proteins our results are expected to have wide implications for signal transduction by GPCRs in general. 12 12
Structure, dynamics and function of biological macromolecules by NMR Research Project | 2 Project MembersNo Description available
Structure, dynamics and function of CCR5-arrestin interactions Research Project | 3 Project MembersThe human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in general inflammatory processes by recruiting and activating leukocytes. CCR5 is also the principal HIV coreceptor, is involved in the pathology of both cancer and neuroinflammation, and has been im plicated in the inflammatory complications of COVID-19. Binding of ligands to GPCRs results in the activation of G protein-, arrestin-mediated and other signaling pathways. The Grzesiek lab has recently solved the cryo-EM structure of an agonist chemokine CCL5-CCR5-G protein complex, which delineates the G protein activation pathway triggered by chemokine agonists within CCR5. While meanwhile a number GPCR-G protein complexes have been solved, much less is known on the structural basis of GPCR arrestin signaling. In particular, no structure of a chemokine receptor-arrestin complex exists. The Shukla is one of the world leading labs in the structural and functional analysis of arrestins. We propose here to combine the expertises and capabilities of these two groups to (A) solve the structure of a CCR5-ß arrestin 1 complex, (B) investigate the dynamics of interactions between CCR5-derived peptides and arrestin by NMR and other biophysical techniques, as well as (C) characterize the CCR5-ß arrestin 1 interactions by cell-based assays and develop new synthetic antibody fragments to stabilize the CCR5-ß arrestin 1 complex. The structural and functional insights from these combined experiments should decipher key elements of chemokine-induced CCR5-arrestin signaling and reveal differences to the G protein signaling pathway. This may pave the way for the development of directed therapeutics targeting either pathway. Due to the homology of CCR5 to a number of other chemokine receptors, the results obtained may serve as a paradigm for other chemokine receptor/ligand systems.
Structures, dynamics and interactions of disease-relevant proteins and new developments in NMR technology Research Project | 6 Project MembersBiological function results from time-dependent interactions between biomolecules. NMR spectroscopy is the only experimental method, which yields both structural and dynamical information on biomolecules at atomic resolution with minimal invasiveness and at close to natural conditions. However, it is limited in the molecular size and sensitivity. In the last years we have combined NMR, cryo-EM, single-molecule FRET and other biophysical techniques to derive mechanistic insights at the atomic level into the function and interactions of a number of disease-relevant protein systems. In the next years we want to further develop and conclude several of these projects, which address (1) G protein-coupled receptors (GPCRs), (2) Abelson kinase, (3) c-di-GMP binding proteins, and (4) NMR technology. Subproject (1) addresses the further dynamical characterization by NMR and other techniques of the function of the beta1-adrenergic receptor (b1AR), which is one of the main targets of beta-blockers, as well as the characterization of functional complexes by cryo-EM and NMR of the human chemokine receptor CCR5, which is also the coreceptor for HIV. Subproject (2) addresses the further dynamical characterization by single-molecule FRET and NMR of Abelson kinase (Abl), which is a large multi-domain protein and important leukemia drug target. We have now established labeling with fluorophore pairs at arbitrary sites by using unnatural amino acid technology. We want to use this development to study the dynamical behavior of single Abl molecules within (cancer) cells by FRET and combine the obtained information with high-resolution NMR. Subproject (3) addresses the interactions of the bacterial virulence factor cyclic di-guanosine-monophosphate (c-di-GMP). We have determined the NMR structure and dynamics of several complexes of c-di-GMP binding proteins and want to continue to develop a genetically encodable c-di-GMP fluorescence sensor based on our structural data. Subproject (4) foresees further developments in NMR technology addressing the rapid assignment of large proteins and the improvement of isotope labeling techniques in higher eukaryotes.
Functional dry cavities in proteins Research Project | 1 Project MembersThe interior of experimentally determined protein structures often contains cavities, which are not filled by protein atoms. Such cavities are generally thought to be filled by water. However, in few proteins completely empty voids, devoid of any water molecules have been detected by X-ray crystallography or NMR spectroscopy. The most recent example is a 141 Å3-sized, dry pocket in thermolysin, which was unambiguously detected by high-resolution X-ray crystallography using an absolute-scale electron density map (Krimmer et al. JACS 2017, 139, 30, 10419-10431). This hydrophobic cavity could then be filled by noble gases as also detected by X-ray crystallography. These observations are limited to a few model systems, and it is unclear how abundant dry voids are in proteins, and whether such voids are connected to protein function and structural stability.Such a functional role may exist in the medically highly relevant G protein-coupled receptors (GPCRs), which constitute the most abundant class of membrane proteins in the human genome. Indeed, a recent study on the ß1-adrenergic receptor (ß1AR, Warne et al. Science 2019, 364, 775-778) showed that its extracellular ligand binding pocket shrinks by 20-40% upon activation by an intracellular G protein mimic. This compression of the ligand pocket explains the affinity increase of G protein-coupled receptors (GPCRs) for agonist ligands in their G protein-bound state. From the 2.8 Å-resolution crystal structure, it remained unclear to what extent the extracellular ligand binding cavity is filled by water. However, a recent pressure NMR study (Abiko et al. JACS 2019, 141, 42, 16663-16670) showed quantitatively that the same receptor experiences a volume reduction of ~100 Å3 upon transition to the active state. This shrinking can only be explained by the collapse of empty, non-hydrated cavities, but the location of these empty cavities remained unknown from the NMR study.We hypothesize that the external ligand pocket of ß1AR is at least partially empty and that this void plays a crucial role in GPCR activation. We propose to determine the location of these empty cavities by X-ray crystallography. In contrast to previous studies, we will incubate the crystals of ß1AR in different functional states at mild pressures with xenon. If empty hydrophobic cavities are present, they should incorporate xenon, which is then easily located in the electron density by its high scattering power.These experiments may reveal the first dry pocket in a membrane protein and connect its existence to a crucial function in GPCRs. More generally, such a proof of existence may establish dry voids as a new interaction principle in proteins. Thus, dry pockets may serve as structural motifs that confine protein movements during folding and function or which specifically recognize distinct hydrophobic moieties of a ligand. This may have major implications on protein folding research, the structure-based explanation of protein function, and the development of new drugs.
Biomacromolecular structures, dynamics and interactions by NMR and new developments in NMR technology Research Project | 1 Project MembersBiological function results from time-dependent interactions between biomolecules. NMR spectroscopy is the only experimental method, which yields both structural and dynamical information on biomolecules at atomic resolution with minimal invasiveness and at close to natural conditions. As such it can provide unique information to understand the connection between structure, dynamics and function. It is the goal of this proposal to apply and further develop these strengths of NMR technology in two subprojects. Subproject A is directed towards the determination of structure, dynamics, and interactions in several medically important systems, for which we have made significant progress in recent years: (1) two G-protein coupled receptors (GPCRs), i.e. the beta1-adrenergic receptor (b1AR) and the chemokine receptor CCR5, which is also the coreceptor for HIV. For b1AR, we could show that it is possible to follow signal transduction by 1 H- 15 N backbone resonances. We now want to carry out a full NMR dynamics characterization comprising the binding of drug, G protein and arrestin ligands in different membrane environments. Beside solution NMR, this will also entail the study in full lipid bilayers by solid state NMR. Thus we hope to establish b1AR as a reference system for studies of GPCRs by NMR. We have recently obtained solution spectra for CCR5 of comparable quality to b1AR. We could also detect many resonances of the engineered chemokine ligand 5P12-RANTES in complex with CCR5 by solid state NMR. 5P12-RANTES is currently in clinical trials as an HIV entry inhibitor. We want to carry out a solution and solid state NMR analysis of this receptor with 5P12-RANTES and engineered RANTES ligands that elicit different arrestin and G-protein signaling activities with the aim to provide the structural and dynamical explanation for this differing signaling behavior. (2) Interactions of the bacterial virulence factor cyclic di-guanosine-monophosphate (c-di-GMP). We have solved the structures of several c-di-GMP protein complexes and c-di-GMP oligomers as well as determined the kinetics of c-di-GMP oligomer formation in solution. In particular, we could recently solve the structure of the c-di-GMP recognition region of the chemotactic protein CleD in complex with c-di-GMP. C-di-GMP binding to CleD induces formation of a ternary complex with the flagellar motor switch protein FliM thereby controlling bacterial flagellar motor function. We now want to solve the structure of the full c-di-GMP-CleD complex and study the formation of the ternary c-di-GMP-CleD-FliM complex. (3) Abl kinase, which is an important leukemia drug target. We have shown that binding of different classes of inhibitors to a large, multidomain Abl construct induces distinct domain rearrangements, which shed light on the mechanism of kinase regulation. We have now assessed the influence of a number of medically relevant point mutations and studied a larger class of inhibitors. In particular, the opening of the multidomain structure is correlated to the conformation of the activation loop in the catalytic domain. We now want to obtain an in-depth dynamic description of the catalytic domain, which is possible due to an advance in labeling techniques, as well as continue to develop suitable labeling of Abl for single molecule FRET studies. Subproject B is directed towards NMR technique development. Often proteins from higher eukaryotes cannot be expressed in functional form in E. coli , but only in higher eukaryotic expression systems. The difficulties of isotope labeling in such systems presents a bottleneck for the analysis by modern heteronuclear NMR. We have recently developed a robust method for isotope labeling in insect cells by feeding isotope-labeled yeast extract. The method currently yields 90 % 15 N and 13 C as well as >60 % 2 H labeling with very good yields. We want to improve this method by increasing the level of 2 H incorporation and cost efficiency for 13 C labeling as well as extend it to other eukaryotic systems. We also want to obtain an in-depth understanding of the often adverse effects of 2 H incorporation to cellular behavior by a proteomics analysis of the response to growth on 2 H 2 O for E. coli , yeast and higher eukaryotes.
Elucidating allosteric signal transmission in the beta1-adrenergic receptor Research Project | 3 Project MembersG protein coupled receptors (GPCRs) are an important class of trans-membrane proteins that recognize a multitude of extracellular molecules and transmit their signal to the intracellular side. Despite recent achievements in X-ray crystallography of GPCRs, the high-resolution structures obtained do not capture their intrinsic dynamic properties, which are tightly associated with their function. NMR spectroscopy promises to provide such missing dynamic information. However so far, despite being valuable, only limited information has been obtained by NMR due to the difficult spectroscopic properties of this protein class. At this point, the potential of solution NMR analysis of GPCRs has not been fully realized. Recently, I have overcome many of the obstacles that hinder the application of solution NMR to study signal transduction in GPCRs. Using a stabilized form of the β 1 -adrenergic receptor and a selective isotope labeling method in the baculovirus-insect cell expression system, I was able to acquire well-resolved backbone amide proton-nitrogen correlation spectra. These spectra revealed numerous mechanisms within the receptor that are new, or had been postulated but never observed directly. Thus I have established a system that can now be used to study many more functional mechanisms of GPCRs at atomic resolution. In addition, we have developed an economic method to produce uniformly isotope-labeled (including deuteration) GPCRs in the insect cell systems. This will allow more advanced applications of NMR spectroscopy to GPCRs such as the study of their dynamics by relaxation measurements. In the present proposal I want to use this system to obtain detailed insights into the receptor's signal transmission mechanism with the aim to understand how the receptor recognizes ligands and passes this information to the G protein in order to modulate its activity. Using state-of-the-art methods of isothermal titration calorimetry, protein rigidity theory, coevolutionary sequence alignment, in vitro real-time observation of GDP/GTP exchange by fluorescence as well as solution NMR, I want to elucidate the underlying molecular mechanism of the thermodynamic behavior of ligand-receptor interactions, determine a high-resolution allosteric network model of signal transmission, and provide mechanical insights into how different agonists elicit varying levels of G protein activation. If successful, the results will have implications for the general understanding of GPCR function and the developed methods should be applicable to other GPCRs.
Structural and dynamical basis of allosteric regulation and inhibition of abelson tyrosine kinase a drug target in the treatment of chronic mylogeneous leukaemia Research Project | 2 Project MembersAbelson tyrosine kinase (Abl) in its healthy state is a tightly regulated, human protein involved in many cellular processes. An abnormal rearrangement of chromosomes leads to the formation of the aberrant fusion protein Bcr-Abl. Bcr-Abl is highly active and causes uncontrolled production of immature blood cells, which ultimately results in the blood cancers chronic myelogenous leukemia (CML) or acute lymphoblastic leukemia (ALL). Drugs like imatinib (gleevec), nilotinib (tasigna), and dasatinib (sprycel) have been developed to bind to a specific location on Bcr-Abl (ATP-binding pocket) thereby blocking its abnormal activity. They are highly successful in the clinic. However, after prolonged treatment a fraction of the cancers become resistant to these drugs by spontaneous mutations in Bcr-Abl, making the treatment ineffective. Recently, a new class of inhibitors has been discovered that bind to a different location on Bcr-Abl. These so-called allosteric inhibitors have promise to overcome drug resistance in combination with conventional ATP-site inhibitors. The mode of action of this combination is unclear. Bcr-Abl consists of many subdomains and their relative movement and interplay is thought to be responsible for its regulation. We have recently been able to detect such movements at atomic resolution by Nuclear Magnetic Resonance (NMR) methods. We now want to build on these initial studies and determine Abl's molecular movements and interactions in response to various inhibitors and disease- or functionally relevant mutations by a combination of NMR, single molecule fluorescence resonance energy transfer (FRET) and other biophysical techniques. The results should provide an atomic-level understanding of the mechanism of Abl regulation and a rationale for the improvement of existing and the development of new therapeutic strategies for Bcr-Abl inhibition.
Purchase of a 900 MHz High-Resolution NMR Instrument Research Project | 5 Project MembersBackground - Biological function results from time-dependent interactions between biomolecules. It is ultimately encoded in the primary chemical structures of molecules, which determine the positions and movements of their atoms in space. NMR spectroscopy is the only experimental method, which yields both structural and dynamical information on biomolecules at atomic resolution with minimal invasiveness and at close to natural conditions. As such, it can provide unique information to understand the connection between chemical structure, three-dimensional structure, dynamics and ultimately function. The application of NMR spectroscopy to large interesting biomolecular systems is limited by sensitivity and resolution, both of which increase strongly with magnetic field strength. The planned new building for the Biozentrum of the University Basel provides the unique opportunity to upgrade the present 800 MHz NMR instrument to a state-of-the-art 900 MHz instrument. This will substantially improve the scientific capacities of the Biozentrum's high-field NMR center. The new installation will benefit from the strong technical and biological expertise of the principal applicants and the high demand for NMR characterization of exciting biological projects inside and outside of the University of Basel. Research proposal - Expertise, maintenance, running, building and a large part of the total purchase costs will be provided by the Biozentrum and the University of Basel. The core users will use the instrument to A) study structures and interactions of disease-relevant biomolecules and to further develop high-resolution NMR methods (Grzesiek, principal applicant); B) characterize structure, function and folding mechanisms of large biomacromolecules and their complexes with a focus on integral membrane proteins (Hiller); C) investigate proteins, interaction and functional mechanisms in cyclic-di-GMP signaling (Schirmer, Jenal); D) study large protein complexes (Maier, Hiller). Further research projects comprise 1) interactions of the uropathogenic E. coli protein FimH with glycoproteins from the urothelial cell surface (Ernst); 2) interactions of bioactive natural products with target proteins (Hamburger); 3) the catalytic activity of artificial metalloenzymes (Ward, Häussinger); 4) dynamics and interaction of G-protein coupled receptors (Schertler); 5) interactions and dynamics of histone-binding proteins and their role in heterochromatin silencing (Bühler); 6) alterations in cellular metabolism of eukaryotic cells upon infection with pathogens (Bumann). Expected value - Structures and motions of biomolecules need to be determined at atomic resolution to understand their mechanisms from first principles. This information can be provided by NMR analysis. This is a fundamental prerequisite to unravel the connection between biomolecular structure, dynamics and function, as well as for the rational intervention into biological processes such as drug design. Justification of the needed equipment - The new instrument will provide significant increases in sensitivity and resolution for the NMR analysis of structures and motions of biomolecules. The reduced required concentrations and the larger achievable molecular sizes will further extend its applicability to highly challenging biomolecular systems. The instrument will satisfy the high demand for such NMR characterizations within the University of Basel and from outside collaborations. The foreseen research projects comprise several medically relevant G-protein coupled receptors (GPCRs), cancer drug targets, pathogenic bacterial systems, large molecular machines such as membrane protein assembly and polypeptide translocation complexes, as well as general questions of protein folding. The new instrument will ensure that this research can be carried out at an internationally competitive level.
Biomacromolecular structures, dynamics and interactions by NMR and new developments in NMR technology Research Project | 1 Project MembersBiological function results from time-dependent interactions between biomolecules. It is ultimately encoded within the primary chemical structure of molecules resulting in defined three-dimensional atom positions and movements. NMR spectroscopy is the only experimental method, which yields both structural and dynamical information on biomolecules at atomic resolution with minimal invasiveness and at close to natural conditions. As such it can provide unique information to understand the connection between primary structure, tertiary structure, dynamics and function. It is the goal of this proposal to apply and further develop these strengths of NMR technology with the aim to reveal general principles of protein structure function relations. The proposal is divided into two subprojects: Subproject A is directed towards the determination of structure, dynamics, and interactions in four medically important systems, for which we have made significant progress in recent years: (1) Abl kinase, which is an important leukemia drug target. We have obtained assignment of a large, multidomain Abl construct that is the minimal autoregulatory fragment. Our data show that binding of different classes of inhibitors induces distinct domain rearrangements, which shed light on the mechanism of kinase regulation. We now want to reveal the atomic causes of these allosteric rearrangements, investigate several medically and functionally relevant mutants, and extend the studies to single molecule FRET. (2) the HIV-1 coreceptor and G-protein coupled receptor (GPCR) CCR5. We have obtained large-scale functional expression of CCR5 in insect cells and E. coli, which permits effective isotope labeling. We seek to apply new solid-state and solution NMR techniques for structure determination and to develop general strategies for GPCR analysis by a comparison to other available GPCRs. (3) Interactions of the bacterial virulence factor cyclic di-guanosine-monophosphate (c-di-GMP). We have determined the structure of the PilZ homolog PA4608 in complex with c-di-GMP and the kinetics of c-di-GMP oligomer formation in solution. We now want to solve the structures of a complex c-di-GMP with the chemotactic protein CC3300 and of higher c-di-GMP oligomers. (4) Lipopolysaccharide (LPS), the causative agent of endotoxic shock. We have developed a method to study LPS by solution NMR and obtained expression of the lipopolysaccharide binding protein, which is the first receptor of the endotoxic recognition cascade. We intend to study its interaction with LPS. Subproject B is directed towards the NMR characterization of unfolded states of proteins and their relation to the folded structure with an emphasis on pressure denaturation and structural modeling of unfolded ensembles. The overarching goal is to rationalize protein folding by correlating high resolution experimental data on unfolded states with primary, secondary and tertiary structure information. (1) We have obtained unique data on the pressure/cold-denatured state of ubiquitin, which shows similar subpopulations of partial native structures as an alcohol-denatured state. By addition of non-denaturing concentrations of alcohol, full pressure-induced unfolding can be achieved at room temperature in a completely reversible way. This allows following the unfolding transition at very high resolution. We want to extend this analysis to a larger set of about 10 proteins representing different folds and sequences with the aim to correlate unfolding behavior with sequence or structure and to understand the nature of pressure unfolding. (2) We have developed an effective method to calculate structural ensembles of unfolded proteins from large sets of NMR data. We want to continue these efforts with the aim to include FRET and chemical shift data in order to obtain highly defined quantitative models for larger sets of unfolded proteins.
NMR studies of GPCRs: Structure, dynamics and interactions with ligands and signaling proteins Research Project | 1 Project MembersG protein-coupled receptors (GPCRs) are integral membrane proteins that transmit extracellular neural, endocrine, olfactory and visual signals across the plasma membrane. When activated by light or a ligand GPCRs undergo a conformational change leading to activation of a G protein, arrestin and other signaling pathways. Over 30% of compounds used in medicine today modulate the activity of GPCRs. Some GPCRs can bind a range of ligands, some of which, instead of fully activating the receptor, rather bias the signal transduction cascade toward distinct intracellular pathways. The currently available X-ray structures of several GPCRs and of a 2-adrenoreceptor/Gs signaling complex provide a frozen snapshot of a signal transduction event, but do not fully explain how the ligand selectivity is achieved, and how the ligand binding results in preferential binding of a specific signaling protein such as Gi, Gs or arrestin. Understanding the precise structural and dynamic nature of these phenomena is critical to knowing the mechanism of GPCR activation and will help further development of pharmaceuticals with desired pharmacological properties. We propose here to provide the missing dynamical information on GPCR function by Nuclear magnetic resonance spectroscopy (NMR), which shall determine the conformational changes and dynamics of the receptors and signaling complexes in solution. This should then a yield a comprehensive view of GPCR activation of and signal transduction. We will initially focus on rhodopsin and 1-adrenoreceptor ( 1AR), as well as their signaling complexes with G proteins and arrestin, and later apply developed expertise to the vasopressin receptor V2R and its signaling complexes. In particular, we propose: a) to label GPCRs for NMR analysis; b) to obtain a dynamic view of the conformational changes in GPCRs induced by a range of ligands with different activities including agonists, antagonists and biased ligands; c) to investigate the effect of G proteins and arrestin binding on the receptors; d) to identify the molecular basis for signaling selectivity via a comparative NMR and biophysical analysis of receptor binding to the various ligands, G proteins and arrestin. Due to the high sequence conservation among G protein-coupled receptor proteins our results are expected to have wide implications for signal transduction by GPCRs in general.
