Laboratory of Biomodelling

Head: Postdocs: PhD students: Undergraduate students:

Slawomir Filipek, PhD, DSc Krzysztof Jozwiak, PhD Anna Modzelewska, MSc Michal Kolinski Krystiana Krzysko, MSc Aleksander Debinski, BSc

Current research and projects

1. Oligomerization of G Protein Coupled Receptors based on the rhodopsin case
G-protein-coupled receptors (GPCRs) constitute a large superfamily of receptor proteins responsible for signal transduction. These receptors mediate recognition of environmental stimuli like light, odor, and taste, but also involve responses to peptides, hormones, proteases, chemokines and other ligands across plasma membranes. They are also important targets for pharmacological intervention via activating or blocking their action. The current state of research is that GPCRs exist and act as dimers. However, a couple of years ago the monomeric state of GPCR was commonly accepted and a few cases of dimerization (like GABAB receptors) were treated as an exception. Gradually, due to growing evidence of experimental data the hypothesis of dimerization became a dominant one.
Based on distances between rhodopsin (Rh) monomers, measured by Atomic Force Microscopy (AFM), we built a model of rhodopsin oligomer. The model was subsequently enhanced by simulation in membranes specific to native rod outer segment (ROS) discs. Such membranes contain unsaturated lipids (docosahexaenoyl chains) in high concentrations.
Recently, our model of rhodopsin oligomer was experimentally confirmed by Guo et al. PNAS 2005 (Fig. 1). They used cysteine mutants of amino acids from the putative interface between two GPCR molecules and found that only amino acid residues from TM-4 and TM-5 were able to crosslink. Additionally, they discovered that after binding of an agonist to the receptor (they used D2 dopamine receptor) the interface is changing and involves another face of TM-4. This unexpected problem makes oligomerization research still advantageous and we will try to reveal how the change of interface is possible in the oligomeric structure (rotation of TM-4, mutual movement of the adjacent receptor molecules etc.) and also how it affects activation and inactivation processes GPCRs are involved in.

2. Transduction of the signal from GPCR to G protein
The existence of oligomeric assemblies of GPCRs has been confirmed by biochemical and biophysical studies including direct AFM imaging of rhodopsin in native membranes. The present question is how they work together to transduce the signal into the cell and, further on, how the oligomeric state influences all aspects of their biological function. Crystallographic structure of rhodopsin revealed that its cytoplasmic surface is too small to bind the whole trimeric G protein and accommodate all interactions predicted from crosslinking data.
According to our modelling, transducin (Gt) binds to rhodopsin tetramer. After dissociation of the Gtβγ subunit, the remaining alpha subunit can bind to a second molecule of transducin and facilitate docking to the adjacent rhodopsin tetramer, providing positive cooperativity for binding of another trimer Gtαβγ. Currently, we continue to elucidate the model and the role of posttranslational modifications on binding of Gt to rhodopsin.

3. Quenching of GPCRs – phosphorylation and binding to arrestin
Deactivation of GPCRs is associated with binding of arrestin. But before this process can start it is necessary that receptor become phosphorylated. We built a model of a complex of rhodopsin dimer and then, subsequently, tetramer with rhodopsin kinase. The kinase domain of RK binds and phosphorylates the C-terminus of activated rhodopsin while the second domain of RK (RGS homology domain) is in contact with another, inactive rhodopsin molecule.
During a formation of the arrestin - rhodopsin dimer complex the long range electrostatic forces pull the two interacting parts together since there is a strict complementarity of the electrostatic potentials between binding surfaces of both arrestin and the rhodopsin dimer (Fig. 2). Our modelling revealed that arrestin, composed of two stiff lobes, can undergo a 40° rotation which is a maximal value because the hinge region of arrestin is maximally stretched in this conformation (Fig. 3). In the rotated conformation arrestin can bind the rhodopsin dimmer very firmly and, what was also found by the modelling, recognizes the active state of rhodopsin. The activated rhodopsin structure is characterized by displaced transmembrane helix VI. Arrestin can recognize the new position of this helix and binds only when rhodopsin is activated (Fig. 4).

4. The structure of the γ-secretase complex and explaining the role of Alzheimer’s disease mutations
Familial form of Alzheimer’s disease (FAD) is associated with mutations in several genes. Most of them were found in presenilin-1 (PS-1), a protein involved in formation of γ-secretase. This membrane-embedded complex is a key player in AD development generating toxic β-amyloid peptide. The core of this complex consists of four membrane proteins: PS-1, APH-1, PEN-2 and NCT. To explain why some mutations of PS-1 are harmful and some neutral, and to predict which amino acid mutations may be potentially dangerous, we created a molecular model of PS-1. It was known that mutations are forming linear patterns on the putative transmembrane helices of PS-1. We confirmed this idea based on much bigger database of AD mutations and extended these patterns to areas spanning even to three faces of hydrophobic regions (HRs) of PS-1. The complementary areas of residues free of AD mutations were identified based on location of silent polymorphism and PS-1 vs. PS-2 amino acid discordances. The created model of PS-1, although preliminary, properly classifies different faces of HRs based on the contact to adjacent HRs of PS-1 or to putative locations of other membrane proteins from γ-secretase complex.

5. Investigation of molecular interactions that stabilize membrane proteins
Using Atomic Force Microscopy cantilever in single-molecule force spectroscopy (SMFS) method (Fig. 5), it was possible to probe molecular interactions within native bovine rhodopsin and discover structural segments of well-defined mechanical stability. Such structural segments stabilize secondary structure elements of the native protein. They also position and hold the highly conserved residues at functionally important environments. The changes in unfolding pattern may underlie dysfunctions associated with particular mutations. Theoretical investigations that we started recently in our lab focus on the pulling of individual helices of these proteins out of the membrane and observing the changes that occur during this process. This also involves investigations of the membraneprotein interface.

Figure 1. Models of the rhodopsin dimer interface. a. The interface involving TM-4 and TM-5 found by us based on AFM measurements for bovine Rh. b. The interface involving TM-4 deduced by Guo et al. from ECM (electron cryomicroscopy) of squid Rh. c. TM-4 of Rh. Solid circles denote positions of amino acids for which the corresponding cysteine mutants were crosslinked. Colors mark particular interfaces. d. Homology model of D2R. Residues belonging to AFM predicted interface are shown in yellow, belonging to ECM one are shown in red. Residue 4.56 not belonging to any of these interfaces is shown in green.

Figure 2. The complementarity of electrostatic potentials of arrestin and cytoplasmic part of rhodopsin dimer. Colour red denotes negative potential, blue – positive.

Figure 3. Superimposition of arrestin in basal (green) and with lobes 40° rotated (orange) conformations. Hinge region before (purple) and after (magenta) rotation.

Figure 4. Arrestin recognizes active rhodopsin. View rotated 180°. Arrestin’s strand from C-terminal lobe (magenta) interacts with rhodopsin TM-VI and goes down more only if this helix is moved away in activated rhodopsin (Rh*).

Figure 5. Secondary structure of Rh mapped with structurally stable segments observed by SMFS. Different colours mark each structural segment of Rh.

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