Drug Research Academy

Drug Research Academy Projektforslag til post doc. stipendium Hermed fremsendes et projektforslag til et post doc. stipendium under Drug Research Academy. Projektet fokuserer på den ionotrope glutamate receptor GluR6 og sigter mod kloning, ekspression, oprensning og strukturbestemmelse af fuldlængde GluR6. GluR6 er en membranbundet ionkanal og det er i dag en stor udfordring at bestemme 3D-dimensionelle strukturer af membranbundne proteiner. Hvis det lykkes, vil det få uvurderlig betydning for de involverede forskningsgrupper og ikke mindst medvirke til at bringe forskningsniveauet på DFU i frontlinje. Stipendiet ønskes gerne besat med en udenlandsk statsborger. 1. Fokusområde: Strukturbaseret lægemiddelforskning 2. Titel: 3D-structure of the full-length GluR6 kainate receptor 3. Institutter involveret: og Institut for Farmakologi 4. Seniorforskere involveret: Darryl Pickering, lektor, Institut for Farmakologi; picker@dfuni.dk Arne Schousboe, professor, Institut for Farmakologi; as@dfuni.dk Jette Sandholm Kastrup, lektor, ; jsk@dfuni.dk Michael Gajhede, professor, ; mig@dfuni.dk Ovenstående to forskningsgrupper har gennem årene opbygget ekspertise inden for farmakologi af glutamat receptor ligander og inden for strukturbestemmelser af AMPA og kainat receptorers ligand-bindende domæne. I løbet af de seneste to år er et stærkt integreret samarbejde blevet etableret og afgørende fremskridt opnået. Vi har i fælleskab bl.a. fornyligt publiseret et større studie, hvor vi viser, at der eksisterer en excellent korrelation ved binding af en række strukturelt meget forskellige agonister mellem domænelukning i den ligand-bindende del af GluR2 og efficacy af fuld-længde GluR2 (Frandsen et al. (2005) Mol. Pharm. 67:703-713). Et stort gennembrud i 2004 har været strukturbestemmelsen af den ligand-bindende del af kainat receptoren GluR5 (Naur et al. (2005) FEBS Lett. 579: 1154-1160). Med denne struktur er der åbnet for selektivitetsstudier mellem AMPA receptorer (GluR1-GluR4) og kainat receptorer (GluR5-GluR7 og KA1-KA2). Forskningsarbejdet vedrørende ionotrope glutamat receptorer har også været udført i tæt samarbejde med NEME-gruppen ved, DFU samt med lægemiddelindustrien (Lundbeck A/S og NeuroSearch A/S). 1

5. Projektets formål: It is the main objective of this proposed study to re-examine the functional properties of agonist efficacy, affinity and desensitization at ionotropic glutamate receptors (iglurs) in relation to structural changes occurring within the channel pore region upon ligand binding and to determine precisely how the energy of ligand binding is transduced into conformational changes in the channel leading to pore opening. Therefore, the goal is to elucidate the 3D-structure of an intact, tetrameric ionotropic glutamate receptor ion channel, GluR6, for the structural determination of: (i) the ligand-binding domain, (ii) non-competitive modulatory sites, (iii) intersubunit binding domains and (iv) the channel pore. Our proven expertise in the cloning, expression, purification, crystallization and structural characterization of GluR2 and GluR5 ligand-binding core constructs provides a strong foundation from which to implement this project. 6. Projektbeskrivelse: 3D-structure of the full-length GluR6 kainate receptor. BACKGROUND Binding of (S)-glutamate to ionotropic glutamate receptors (iglurs) is a key step in the mechanism of rapid excitatory synaptic transmission among nerve cells within the mammalian central nervous system. iglurs are important in the development and function of the central nervous system and are implicated in learning and memory formation. Furthermore, iglurs also seem to be associated with certain neurological and psychiatric diseases and are therefore considered as potential drug targets. Detailed structural information at the molecular level may greatly facilitate the process of designing new iglur agonists and antagonists with enhanced selectivity and/or novel pharmacological profiles. The iglurs, which take the form of tetrameric ligand-gated ion channels, have been divided into three different classes on the basis of protein sequence identity and ligand selectivity: the AMPA, kainic acid (KA) and NMDA receptors. iglurs couple the energy of agonist binding to the opening of a transmembrane ion pore, allowing flow of Na +, K + or Ca 2+ ions. Two extracellular segments, S1 and S2, of the receptor protein have been shown to constitute the ligand-binding core of the receptors and previous studies have shown that this ligand-binding core is necessary and sufficient for achieving binding properties similar to that of the full-length, membrane-bound receptor. Recombinant, soluble constructs of the ligand-binding core of the AMPA-R (GluR2-S1S2J), of the NMDA- R (NR1-S1S2) and, recently, of the KA-Rs (GluR5-S1S2 and GluR6-S1S2) have been produced and structures of several agonists and antagonists in complex with these constructs have been reported by others and us. Ligands bind within a cleft formed by two domains (D1 and D2), where D1 is mainly composed of residues from segment S1, and D2 is composed mainly of residues from segment S2. Others and we have shown that AMPA-R agonists with different efficacy induce distinct conformations of GluR2-S1S2J by inducing differing degrees of D1-D2 domain closure. A recent study of a series of ligand-binding core complexes of willardiine analogues has demonstrated that the size of a single substituent could control the degree of domain closure. This leads to a range of liganddependent conformational states of the ligand-binding core, which are proposed to give rise to differential opening of the receptor channel pore. A non-conserved tyrosine residue in AMPA-Rs, (Tyr702 in GluR2-S1S2J), was originally identified by us as being the main contributor to the selectivity of the agonist (S)-Br-HIBO for GluR1 over GluR3. We have recently utilized a combination of X-ray crystallographic and pharmacological techniques to further investigate, at the molecular level, the role of Tyr702 in agonist binding affinity, D1-D2 domain closure, agonist efficacy and iglur selectivity (Frandsen et al. (2005) Mol. Pharm. 67:703-713). The structures of the selective partial agonist (S)-CPW399 and the non-selective partial agonist kainate in complex with GluR2- S1S2J and with (Y702F)GluR2-S1S2J were all compared. Interestingly, it was observed that it is 2

not only the specific chemical structure of agonists that induces a variety of receptor conformational states but that changes in the protein binding site itself (i.e. the presence of Tyr702 or Phe702 in the binding cleft) can give rise to a similar conformational state of the ligand-binding core for chemically distinct agonists. In relation to this, while a strong correlation between agonist efficacy and D1-D2 domain closure was observed for the GluR2 receptor subtype, no such correlation was seen for GluR3. This suggests that amino acid residues outside of the binding site also influence the transduction of the conformational changes induced by ligand binding to channel pore opening. PROJECT PERSPECTIVES Overall information on intact iglurs was recently gained from electron microscopy studies. As yet, no detailed structural information is available for any iglur with regards to the N-terminal domain and the pore of the channel. In order to understand the functioning of these channels at a molecular level and to relate this to the observed electrophysiological properties of the channel it is of paramount importance to resolve the 3D structure of an intact receptor. In addition to such fundamental channel pore properties as ion selectivity, gating, voltage-dependence, channel conductance and desensitization there are numerous polyamine, voltage-dependent, uncompetitive channel blockers, both naturally occurring (e.g. wasp and spider toxins) and many synthetically produced here at DFU in the Dept. of Medicinal Chemistry, which bind to unknown site(s) within the pore region. These types of antagonists could prove to have a therapeutic value in, for example, stroke and epilepsy. To date, the only detailed structural data for the iglurs has come from the soluble binding site constructs. It is now proposed to extend this structure database by expressing, purifying and crystallizing a member of the kainic acid class of iglurs, GluR6. Moreover, to investigate the current model by which ligand binding energy is transduced into conformational changes leading to channel pore opening; it is proposed to determine the structure of the full-length tetrameric GluR6 ion channel. While crystallization of an intact, intrinsic membrane protein ion channel is a challenging prospect; it has been successfully accomplished for the potassium channel KvAP. AIM It is the main objective of this proposed study to re-examine the functional properties of agonist efficacy, affinity and desensitization at iglurs in relation to structural changes occurring within the channel pore region upon ligand binding and to determine precisely how the energy of ligand binding is transduced into conformational changes in the channel leading to pore opening. Therefore, the goal is to elucidate the 3D-structure of an intact, tetrameric ionotropic glutamate receptor ion channel, GluR6, for the structural determination of: (i) the ligand-binding domain, (ii) non-competitive modulatory sites, (iii) intersubunit binding domains and (iv) the channel pore. Our proven expertise in the cloning, expression, purification, crystallization and structural characterization of GluR2 and GluR5 ligand-binding core constructs provides a strong foundation from which to implement this project. RESEARCH PLAN Year 1. Protein expression and purification. We have observed that GluR6 is very well expressed using the Sf9-baculoviral insect cell expression system (DSP). This is the primary consideration for the choice of GluR6 in this project. To facilitate purification of the expressed protein we shall at first need to design and produce an appropriate baculovirus transfer vector for the creation of a GluR6 fusion protein containing both C-terminal and N-terminal affinity tags which can be subsequently removed by specific protease cleavage steps. Starting from the commercially available vector pacghlt (Pharmingen), we will use standard PCR mutagenesis methods for creation of the construct as diagrammed in Fig. 1. By inclusion of both N-terminal and C-terminal affinity tags it will be possible to purify only full-size GluR6 and avoid partially degraded protein molecules. Using a strategy similar to the one successfully applied to the purification of other proteins, we shall incorporate both N-terminal (GST) and C-terminal (6-His) proteolytically cleavable, affinity purifi- 3

Fig 1. New baculoviral transfer vector. N-terminal GST fusion tag with baculoviral signal peptide, FLAG-tag, PKA phosphorylation site, thrombin cleavage site, Multiple Cloning Site, TEV protease cleavage site, C-terminal 6x-Histidine affinity tag, Stop codon. 7. Projektbeskrivelse til WEB-site: cation tags into GluR6 for a two-step, rapid purification procedure. This should facilitate production of a very pure, homogenous sample from which crystallization should be possible. Addition of a baculoviral signal peptide is hoped to increase the yield of translated product to as high a level as possible. The necessary affinity resins, antibodies and reagents are all commercially available. Year 2. Protein crystallization. Attempts at crystallization of the purified protein will proceed. The full-length GluR6 receptor will be attempted to be crystallized using standard crystallization techniques, e.g. the hanging or sitting drop vapour diffusion methods. Critical for crystallization of a membranebound receptor protein are the choice of detergent for solubilisation and the presence of lipids or other additives. Initial crystallization trials will be set-up using various crystallization screens, including detergent screens to empirically determine the optimal conditions. 3D-Structure of the full-length GluR6 kainate receptor The binding of the neurotransmitter (S)-glutamate to ionotropic glutamate receptors (iglurs) is a key step in the mechanism of rapid excitatory synaptic transmission among nerve cells within the mammalian central nervous system (CNS). iglurs are important in the development and function of the CNS and are implicated in learning and in memory formation. Furthermore, iglurs also seem to be associated with certain neurological and psychiatric diseases, e.g. Alzheimer-type diseases, Parkinson s disease, epilepsy, stroke, ALS and schizophrenia. Hence, the iglurs are considered potential targets for the development of new therapeutic drugs. Detailed structural information at the molecular level will greatly facilitate the process of designing new iglur drugs with enhanced selectivity, potency and/or novel pharmacological profiles. The aim of this project is therefore to express, purify and crystallize one member of the iglur family (GluR6) in its entirety to gain 3D structural information about this ion channel, particularly the pore region. The 3D-structure will provide greater understanding of its function and of how certain drugs can activate or block its functioning. 8. Udbytte af projektet: Forskningsgrupperne på DFU: Forskningsgrupperne forventer at opnå en unik ekspertise indenfor både strukturel biologi og elektrofysiologi samt kombinationen af disse teknikker på fuldlængde receptorer. Forskningsgrupperne arbejder allerede med den ligand-bindende del af flere ionotrope glutamat receptorer og vil gerne udbygge den videnskabelige basis for dette projekt. Igennem flere år har vi i stadigt højere grad fokuseret på at kombinere strukturelle og elektrofysiologiske data for at styrke ekspertisen og positionen indenfor forskningsområdet ionkanaler. Forståelsen af fuldlængde iglurs på molekylært plan er i dag yderst begrænset. Fokusområdet: Indenfor fokusområdet Strukturbaseret lægemiddelforskning er opklaring af målmolekylernes struktur et vigtigt udgangspunkt for design af nye aktive forbindelser og anvendes som en komplementær metode til f.eks. screening af naturprodukter, stofbiblioteker mv. En af de helt store udfordringer i dag er strukturbestemmelse af membranbundne receptorer. Hvis projektet lykkes, vil det bringe fokusområdet i international frontlinje. Postdoc en: 4

Gennem projektet forventes postdoc en at opnå en stor og specifik viden om ionotrope glutamat receptorers struktur, funktion og farmakologi. Kandidaten vil opnå en unik ekspertise indenfor strukturel biologi af fuldlængde membranbundne receptorer. Postdoc en vil efter det 2- årige forløb besidde unik og eftertragtet ekspertise. 9. Relevante journaler for annoncering: Nature, CCP4 bulletin (free) 10. Underskrifter fra seniorforskere: Lektor Darryl Pickering Institut for Farmakologi E-mail: picker@dfuni.dk Professor Arne Schousboe E-mail: as@dfuni.dk Lektor Jette Sandholm Kastrup E-mail: jsk@dfuni.dk Professor Michael Gajhede E-mail: mig@dfuni.dk 11. Anbefaling fra institutter: Bilag 1: Institut fra Farmakologi Bilag 2: 5