Jung Hee Park

Crystal structure of opsin in its G-protein-interacting conformation

Opsin, the ligand-free form of the G-protein-coupled receptor rhodopsin, at low pH adopts a conformationally distinct, active G-protein-binding state known as Ops*. A synthetic peptide derived from the main binding site of the heterotrimeric G protein—the carboxy terminus of the α-subunit (GαCT)—stabilizes Ops*. Here we present the 3.2 Å crystal structure of the bovine Ops*–GαCT peptide complex. GαCT binds to a site in opsin that is opened by an outward tilt of transmembrane helix (TM) 6, a pairing of TM5 and TM6, and a restructured TM7–helix 8 kink. Contacts along the inner surface of TM5 and TM6 induce an α-helical conformation in GαCT with a C-terminal reverse turn. Main-chain carbonyl groups in the reverse turn constitute the centre of a hydrogen-bonded network, which links the two receptor regions containing the conserved E(D)RY and NPxxY(x)5,6F motifs. On the basis of the Ops*–GαCT structure and known conformational changes in Gα, we discuss signal transfer from the receptor to the G protein nucleotide-binding site.

Crystal structure of the ligand-free G-protein-coupled receptor opsin.

In the G-protein-coupled receptor (GPCR) rhodopsin, the inactivating ligand 11-cis-retinal is bound in the seven-transmembrane helix (TM) bundle and is cis/trans isomerized by light to form active metarhodopsin II. With metarhodopsin II decay, all-trans-retinal is released, and opsin is reloaded with new 11-cis-retinal. Here we present the crystal structure of ligand-free native opsin from bovine retinal rod cells at 2.9 ångström (Å) resolution. Compared to rhodopsin, opsin shows prominent structural changes in the conserved E(D)RY and NPxxY(x)5,6F regions and in TM5–TM7. At the cytoplasmic side, TM6 is tilted outwards by 6–7 Å, whereas the helix structure of TM5 is more elongated and close to TM6. These structural changes, some of which were attributed to an active GPCR state, reorganize the empty retinal-binding pocket to disclose two openings that may serve the entry and exit of retinal. The opsin structure sheds new light on ligand binding to GPCRs and on GPCR activation.

Crystal structure of metarhodopsin II.

G-protein-coupled receptors (GPCRs) are seven transmembrane helix (TM) proteins that transduce signals into living cells by binding extracellular ligands and coupling to intracellular heterotrimeric G proteins (Gαβγ). The photoreceptor rhodopsin couples to transducin and bears its ligand 11-cis-retinal covalently bound via a protonated Schiff base to the opsin apoprotein. Absorption of a photon causes retinal cis/trans isomerization and generates the agonist all-trans-retinal in situ. After early photoproducts, the active G-protein-binding intermediate metarhodopsin II (Meta II) is formed, in which the retinal Schiff base is still intact but deprotonated. Dissociation of the proton from the Schiff base breaks a major constraint in the protein and enables further activating steps, including an outward tilt of TM6 and formation of a large cytoplasmic crevice for uptake of the interacting C terminus of the Gα subunit. Owing to Schiff base hydrolysis, Meta II is short-lived and notoriously difficult to crystallize. We therefore soaked opsin crystals with all-trans-retinal to form Meta II, presuming that the crystal’s high concentration of opsin in an active conformation (Ops*) may facilitate all-trans-retinal uptake and Schiff base formation. Here we present the 3.0 Å and 2.85 Å crystal structures, respectively, of Meta II alone or in complex with an 11-amino-acid C-terminal fragment derived from Gα (GαCT2). GαCT2 binds in a large crevice at the cytoplasmic side, akin to the binding of a similar Gα-derived peptide to Ops* (ref. 7). In the Meta II structures, the electron density from the retinal ligand seamlessly continues into the Lys 296 side chain, reflecting proper formation of the Schiff base linkage. The retinal is in a relaxed conformation and almost undistorted compared with pure crystalline all-trans-retinal. By comparison with early photoproducts we propose how retinal translocation and rotation induce the gross conformational changes characteristic for Meta II. The structures can now serve as models for the large GPCR family.

A G protein-coupled receptor at work: the rhodopsin model

G protein-coupled receptors (GPCRs) are ubiquitous signal transducers in cell membranes, as well as important drug targets. Interaction with extracellular agonists turns the seven transmembrane helix (7TM) scaffold of a GPCR into a catalyst for GDP and GTP exchange in heterotrimeric Galphabetagamma proteins. Activation of the model GPCR, rhodopsin, is triggered by photoisomerization of its retinal ligand. From the augmentation of biochemical and biophysical studies by recent high-resolution 3D structures, its activation intermediates can now be interpreted as the stepwise engagement of protein domains. Rearrangement of TM5-TM6 opens a crevice at the cytoplasmic side of the receptor into which the C terminus of the Galpha subunit can bind. The Galpha C-terminal helix is used as a transmission rod to the nucleotide binding site. The mechanism relies on dynamic interactions between conserved residues and could therefore be common to other GPCRs.

A ligand channel through the G protein coupled receptor opsin.

The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM) structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal. The crystal structure of opsin in its active Ops* conformation provides the basis for computational modeling of retinal release and uptake. The ligand-free 7TM bundle of opsin opens into the hydrophobic membrane layer through openings A (between TM1 and 7), and B (between TM5 and 6), respectively. Using skeleton search and molecular docking, we find a continuous channel through the protein that connects these two openings and comprises in its central part the retinal binding pocket. The channel traverses the receptor over a distance of ca. 70 Å and is between 11.6 and 3.2 Å wide. Both openings are lined with aromatic residues, while the central part is highly polar. Four constrictions within the channel are so narrow that they must stretch to allow passage of the retinal β-ionone-ring. Constrictions are at openings A and B, respectively, and at Trp265 and Lys296 within the retinal pocket. The lysine enforces a 90° elbow-like kink in the channel which limits retinal passage. With a favorable Lys side chain conformation, 11-cis-retinal can take the turn, whereas passage of the all-trans isomer would require more global conformational changes. We discuss possible scenarios for the uptake of 11-cis- and release of all-trans-retinal. If the uptake gate of 11-cis-retinal is assigned to opening B, all-trans is likely to leave through the same gate. The unidirectional passage proposed previously requires uptake of 11-cis-retinal through A and release of photolyzed all-trans-retinal through B.

Opsin, a Structural Model for Olfactory Receptors?

Receptor-ligand interaction: Olfactory receptors (ORs) are G-protein-coupled receptors (GPCRs), which detect signaling molecules such as hormones and odorants. The structure of opsin, the GPCR employed in vision, with a detergent molecule bound deep in its orthosteric ligand-binding pocket provides a template for OR homology modeling, thus enabling investigation of the structural basis of the mechanism of odorant-receptor recognition.

Structural and kinetic modeling of an activating helix switch in the rhodopsin-transducin interface

Extracellular signals prompt G protein-coupled receptors (GPCRs) to adopt an active conformation (R*) and catalyze GDP/GTP exchange in the α-subunit of intracellular G proteins (Gαβγ). Kinetic analysis of transducin (Gtαβγ) activation shows that an intermediary R*·Gtαβγ·GDP complex is formed that precedes GDP release and formation of the nucleotide-free R*·G protein complex. Based on this reaction sequence, we explore the dynamic interface between the proteins during formation of these complexes. We start from the R* conformation stabilized by a Gtα C-terminal peptide (GαCT) obtained from crystal structures of the GPCR opsin. Molecular modeling allows reconstruction of the fully elongated C-terminal α-helix of Gtα (α5) and shows how α5 can be docked to the open binding site of R*. Two modes of interaction are found. One of them – termed stable or S-interaction – matches the position of the GαCT peptide in the crystal structure and reproduces the hydrogen-bonding networks between the C-terminal reverse turn of GαCT and conserved E(D)RY and NPxxY(x)5,6F regions of the GPCR. The alternative fit – termed intermediary or I-interaction – is distinguished by a tilt (42°) and rotation (90°) of α5 relative to the S-interaction and shows different α5 contacts with the NPxxY(x)5,6F region and the second cytoplasmic loop of R*. From the 2 α5 interactions, we derive a “helix switch” mechanism for the transition of R*·Gtαβγ·GDP to the nucleotide-free R*·G protein complex that illustrates how α5 might act as a transmission rod to propagate the conformational change from the receptor-G protein interface to the nucleotide binding site.

Light-Induced Conformational Changes of the Chromophore and the Protein in Phytochromes: Bacterial Phytochromes as Model Systems

Recombinant phytochromes Agp1 and Agp2 from Agrobacterium tumefaciens are used as model phytochromes for biochemical and biophysical studies. In biliverdin binding phytochromes the site for covalent attachment of the chromophore lies in the N-terminal region of the protein, different from plant phytochromes. The issue which stereochemistry the chromophore adopts in the so-called Pr and Pfr forms is addressed by using a series of locked chromophores which form spectrally characteristic adducts with Agp1 and Agp2. Studies on light-induced conformational changes of Agp1 give an insight into how the intrinsic histidine kinase is modulated by light. Comparison of the crystal structure of an Agp1 fragment with other phytochrome crystal structures supports the idea that a light induced rearrangement of subunits within the homodimer modulates the activity of the kinase.

The Mycobacterium avium subsp. Paratuberculosis protein MAP1305 modulates dendritic cell-mediated T cell proliferation through Toll-like receptor-4

In this study, we show that Mycobacterium avium subsp. Paratuberculosis MAP1305 induces the maturation of bone marrow-derived dendritic cells (BMDCs), a representative antigen presenting cell (APC). MAP1305 protein induces DC maturation and the production of pro-inflammatory cytokines (Interleukin (IL)-6), tumor necrosis factor (TNF)-α, and IL-1β) through Toll like receptor-4 (TLR-4) signaling by directly binding with TLR4. MAP1305 activates the phosphorylation of MAPKs, such as ERK, p38MAPK, and JNK, which is essential for DC maturation. Furthermore, MAP1305-treated DCs transform naïve T cells to polarized CD4+ and CD8+ T cells, thus indicating a key role for this protein in the Th1 polarization of the resulting immune response. Taken together, M. avium subsp. Paratuberculosis MAP1305 is important for the regulation of innate immune response through DC-mediated proliferation of CD4+ and CD8+ T cells. [BMB Reports 2014; 47(2): 115-120]

A potential protein adjuvant derived from Mycobacterium tuberculosis Rv0652 enhances dendritic cells-based tumor immunotherapy.

A key factor in dendritic cell (DC)-based tumor immunotherapy is the identification of an immunoadjuvant capable of inducing DC maturation to enhance cellular immunity. The efficacy of a 50S ribosomal protein L7/L12 (rplL) from Mycobacterium tuberculosis Rv0652, as an immunoadjuvant for DC-based tumor immunotherapy, and its capacity for inducing DC maturation was investigated. In this study, we showed that Rv0652 is recognized by Toll-like receptor 4 (TLR4) to induce DC maturation, and pro-inflammatory cytokine production (TNF-alpha, IL-1beta, and IL-6) that is partially modulated by both MyD88 and TRIF signaling pathways. Rv0652-activated DCs could activate naïve T cells, effectively polarize CD4+ and CD8+ T cells to secrete IFN-gamma, and induce T cell-mediated-cytotoxicity. Immunization of mice with Rv0652-stimulated ovalbumin (OVA)-pulsed DCs resulted in induction of a potent OVA-specific CD8+ T cell response, slowed tumor growth, and promoted long-term survival in a murine OVA-expressing E.G7 thymoma model. These findings suggest that Rv0652 enhances the polarization of T effector cells toward a Th1 phenotype through DC maturation, and that Rv0652 may be an effective adjuvant for enhancing the therapeutic response to DC-based tumor immunotherapy.